register interest

Professor Derrick Crook

Research Area: Microbiology
Technology Exchange: Bioinformatics, Computational biology, Mass spectrometry, Medical statistics, SNP typing and Statistical genetics
Scientific Themes: Immunology & Infectious Disease
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The current programme of research carried out by Professor Derrick Crook's research consortium is largely focussed on translating new molecular technologies and advances in informatics into the investigation of microbial transmission, diagnosis of infectious disease and identifying novel outbreaks of communicable disease. This research is being undertaken through a joint programme of work funded by the Oxford BRC Infection Theme and the Modernising Medical Microbiology UK Clinical Research Consortium (UK-CRC), which consists of the Health Protection Agency (HPA), Wellcome Trust Sanger Institute and the University of Oxford combined with the ORH NHS Trust. In Oxford this involves joint working relationships with Professor Peter Donnelly, Department of Statistics and the Wellcome Trust Centre for Human Genetics (WTCHG), Professor Adrian Hill, Nuffield Department of Medicine, Professor Andrew Pollard, Department of Paediatrics and Professor David Mant and Professor Alastair Gray, Department of Public Health and Primary Care. The aims and objectives of this research is to translate deep sequencing of pathogens on an epidemiological scale for tracking hospital and locally acquired infections and it is focussed on four different major pathogens; Staphylococcus aureus (including MRSA), Clostridium difficile, Norovirus and Mycobacterium tuberculosis. A database linkage project that facilitates investigations of patterns of infectious disease among patients using the Oxford Hospitals and GPs, is also being carried out in tangent for the first three pathogens. Overall this joint programme of work is nationally leading the way in this area and is likely to radically transform the practice of microbiology and infectious disease as well as microbial research in the coming years.

Other research currently being carried out within Derrick Crook's group covers respiratory and gastrointestinal infections and diagnostics, Haemophilus genomics and capsular switch vaccine escape in Pneumococcus. Within the area of diagnostics a HTA funded project investigating the identification of C. difficile and other pathogens from patient stool samples with MassTag multiplex PCR and MLST is being carried out.

Name Department Institution Country
Christine McCartney Health Protection Agency (HPA) - London United Kingdom
Professor Julian Parkhill Wellcome Trust Sanger Institute United Kingdom
Professor Mark Wilcox Leeds University United Kingdom
Dr Grace Smith PHE West Midlands Public Health Laboratory, Birmingham United Kingdom
Dr Martin Llewelyn Brighton and Sussex Medical School United Kingdom
Professor Jenny Taylor Wellcome Trust Centre for Human Genetics University of Oxford United Kingdom
Stoesser N, Sheppard AE, Peirano G, Sebra R, Lynch T, Anson L, Kasarskis A, Motyl MR, Crook DW, Pitout JD. 2016. First report of blaIMP-14 on a plasmid harboring multiple drug resistance genes in Escherichia coli ST131. Antimicrob Agents Chemother, pp. AAC.00840-16-AAC.00840-16. | Show Abstract | Read more

The blaIMP-14 carbapenem resistance gene has largely previously been observed in Pseudomonas aeruginosa and Acinetobacter spp. As part of global surveillance and sequencing of carbapenem-resistant E. coli, we identified an ST131 strain harboring blaIMP-14 within a class 1 integron, itself nested within a ∼54kb multi-drug resistance region on an epidemic IncA/C2 plasmid. The emergence of blaIMP-14 in this context in the ST131 lineage is of potential clinical concern.

Leekitcharoenphon P, Hendriksen RS, Le Hello S, Weill FX, Baggesen DL, Jun SR, Ussery DW, Lund O, Crook DW, Wilson DJ, Aarestrup FM. 2016. Global Genomic Epidemiology of Salmonella enterica Serovar Typhimurium DT104. Appl Environ Microbiol, 82 (8), pp. 2516-2526. | Show Abstract | Read more

It has been 30 years since the initial emergence and subsequent rapid global spread of multidrug-resistantSalmonella entericaserovar Typhimurium DT104 (MDR DT104). Nonetheless, its origin and transmission route have never been revealed. We used whole-genome sequencing (WGS) and temporally structured sequence analysis within a Bayesian framework to reconstruct temporal and spatial phylogenetic trees and estimate the rates of mutation and divergence times of 315S Typhimurium DT104 isolates sampled from 1969 to 2012 from 21 countries on six continents. DT104 was estimated to have emerged initially as antimicrobial susceptible in ∼1948 (95% credible interval [CI], 1934 to 1962) and later became MDR DT104 in ∼1972 (95% CI, 1972 to 1988) through horizontal transfer of the 13-kbSalmonellagenomic island 1 (SGI1) MDR region into susceptible strains already containing SGI1. This was followed by multiple transmission events, initially from central Europe and later between several European countries. An independent transmission to the United States and another to Japan occurred, and from there MDR DT104 was probably transmitted to Taiwan and Canada. An independent acquisition of resistance genes took place in Thailand in ∼1975 (95% CI, 1975 to 1990). In Denmark, WGS analysis provided evidence for transmission of the organism between herds of animals. Interestingly, the demographic history of Danish MDR DT104 provided evidence for the success of the program to eradicateSalmonellafrom pig herds in Denmark from 1996 to 2000. The results from this study refute several hypotheses on the evolution of DT104 and suggest that WGS may be useful in monitoring emerging clones and devising strategies for prevention ofSalmonellainfections.

Didelot X, Walker AS, Peto TE, Crook DW, Wilson DJ. 2016. Within-host evolution of bacterial pathogens. Nat Rev Microbiol, 14 (3), pp. 150-162. | Show Abstract | Read more

Whole-genome sequencing has opened the way for investigating the dynamics and genomic evolution of bacterial pathogens during the colonization and infection of humans. The application of this technology to the longitudinal study of adaptation in an infected host--in particular, the evolution of drug resistance and host adaptation in patients who are chronically infected with opportunistic pathogens--has revealed remarkable patterns of convergent evolution, suggestive of an inherent repeatability of evolution. In this Review, we describe how these studies have advanced our understanding of the mechanisms and principles of within-host genome evolution, and we consider the consequences of findings such as a potent adaptive potential for pathogenicity. Finally, we discuss the possibility that genomics may be used in the future to predict the clinical progression of bacterial infections and to suggest the best option for treatment.

Stoesser N, Sheppard AE, Pankhurst L, De Maio N, Moore CE, Sebra R, Turner P, Anson LW et al. 2016. Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131. MBio, 7 (2), pp. e02162. | Show Abstract | Read more

UNLABELLED: Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE: Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

Sheppard AE, Stoesser N, Sebra R, Kasarskis A, Deikus G, Anson L, Walker AS, Peto TE, Crook DW, Mathers AJ. 2016. Complete Genome Sequence of KPC-Producing Klebsiella pneumoniae Strain CAV1193. Genome Announc, 4 (1), pp. e01649-15-e01649-15. | Show Abstract | Read more

Carbapenem resistance in Klebsiella pneumoniae, frequently conferred by the blaKPC gene, is a major public health threat. We sequenced a blaKPC-containing strain of K. pneumoniae belonging to the emergent lineage ST941, in order to better understand the evolution of blaKPC within this species.

Senn L, Clerc O, Zanetti G, Basset P, Prod'hom G, Gordon NC, Sheppard AE, Crook DW et al. 2016. The Stealthy Superbug: the Role of Asymptomatic Enteric Carriage in Maintaining a Long-Term Hospital Outbreak of ST228 Methicillin-Resistant Staphylococcus aureus. MBio, 7 (1), pp. e02039-e02015. | Show Abstract | Read more

UNLABELLED: Whole-genome sequencing (WGS) of 228 isolates was used to elucidate the origin and dynamics of a long-term outbreak of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 228 (ST228) SCCmec I that involved 1,600 patients in a tertiary care hospital between 2008 and 2012. Combining of the sequence data with detailed metadata on patient admission and movement confirmed that the outbreak was due to the transmission of a single clonal variant of ST228, rather than repeated introductions of this clone into the hospital. We note that this clone is significantly more frequently recovered from groin and rectal swabs than other clones (P < 0.0001) and is also significantly more transmissible between roommates (P < 0.01). Unrecognized MRSA carriers, together with movements of patients within the hospital, also seem to have played a major role. These atypical colonization and transmission dynamics can help explain how the outbreak was maintained over the long term. This "stealthy" asymptomatic colonization of the gut, combined with heightened transmissibility (potentially reflecting a role for environmental reservoirs), means the dynamics of this outbreak share some properties with enteric pathogens such as vancomycin-resistant enterococci or Clostridium difficile. IMPORTANCE: Using whole-genome sequencing, we showed that a large and prolonged outbreak of methicillin-resistant Staphylococcus aureus was due to the clonal spread of a specific strain with genetic elements adapted to the hospital environment. Unrecognized MRSA carriers, the movement of patients within the hospital, and the low detection with clinical specimens were also factors that played a role in this occurrence. The atypical colonization of the gut means the dynamics of this outbreak may share some properties with enteric pathogens.

Bradley P, Gordon NC, Walker TM, Dunn L, Heys S, Huang B, Earle S, Pankhurst LJ et al. 2015. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis. Nat Commun, 6 pp. 10063. | Show Abstract | Read more

The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

Laabei M, Uhlemann AC, Lowy FD, Austin ED, Yokoyama M, Ouadi K, Feil E, Thorpe HA et al. 2015. Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus. PLoS Biol, 13 (9), pp. e1002229. | Show Abstract | Read more

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.

Farzin A, Saha SK, Baqui AH, Choi Y, Ahmed NU, Simoes EA, El Arifeen S, Al-Emran HM et al. 2015. Population-based Incidence and Etiology of Community-acquired Neonatal Viral Infections in Bangladesh: A Community-based and Hospital-based Surveillance Study. Pediatr Infect Dis J, 34 (7), pp. 706-711. | Show Abstract | Read more

BACKGROUND: The etiology of >90% of cases of suspected neonatal infection remains unknown. We conducted community-based surveillance in conjunction with hospital-based surveillance in a rural region in Bangladesh from June 2006 to September 2007 to assess the incidence and etiology of community-acquired viral infections among neonates. METHODS: Community health workers (CHWs) assessed neonates at home on days 0, 2, 5 and 8 after birth and referred cases of suspected illness to the hospital (CHW surveillance). Among neonates with clinically suspected upper respiratory tract infection (URTI), pneumonia, sepsis and/or meningitis, virus identification studies were conducted on nasal wash, cerebrospinal fluid and/or blood specimens. In the hospital-based surveillance, similar screening was conducted among all neonates (referred by CHWs and self-referred) who were admitted to the hospital. RESULTS: CHW surveillance found an incidence rate of 15.6 neonatal viral infections per 1000 live births with 30% of infections identified on the day of birth. Among neonates with suspected sepsis, a viral etiology was identified in 36% of cases, with enterovirus accounting for two-thirds of those infections. Respiratory syncytial virus was the most common etiologic agent among those with viral pneumonia (91%) and URTI (68%). There was a low incidence (1.2%) of influenza in this rural population. CONCLUSION: Viral infections are commonly associated with acute newborn illness, even in the early neonatal period. The estimated incidence was 5-fold greater than reported previously for bacterial infections. Low-cost preventive measures for neonatal viral infections are urgently needed.

Stoesser N, Sheppard AE, Moore CE, Golubchik T, Parry CM, Nget P, Saroeun M, Day NP et al. 2015. Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses. J Clin Microbiol, 53 (7), pp. 2122-2131. | Show Abstract | Read more

Studies of the transmission epidemiology of antimicrobial-resistant Escherichia coli, such as strains harboring extended-spectrum beta-lactamase (ESBL) genes, frequently use selective culture of rectal surveillance swabs to identify isolates for molecular epidemiological investigation. Typically, only single colonies are evaluated, which risks underestimating species diversity and transmission events. We sequenced the genomes of 16 E. coli colonies from each of eight fecal samples (n = 127 genomes; one failure), taken from different individuals in Cambodia, a region of high ESBL-producing E. coli prevalence. Sequence data were used to characterize both the core chromosomal diversity of E. coli isolates and their resistance/virulence gene content as a proxy measure of accessory genome diversity. The 127 E. coli genomes represented 31 distinct sequence types (STs). Seven (88%) of eight subjects carried ESBL-positive isolates, all containing blaCTX-M variants. Diversity was substantial, with a median of four STs/individual (range, 1 to 10) and wide genetic divergence at the nucleotide level within some STs. In 2/8 (25%) individuals, the same blaCTX-M variant occurred in different clones, and/or different blaCTX-M variants occurred in the same clone. Patterns of other resistance genes and common virulence factors, representing differences in the accessory genome, were also diverse within and between clones. The substantial diversity among intestinally carried ESBL-positive E. coli bacteria suggests that fecal surveillance, particularly if based on single-colony subcultures, will likely underestimate transmission events, especially in high-prevalence settings.

Stoesser N, Xayaheuang S, Vongsouvath M, Phommasone K, Elliott I, Del Ojo Elias C, Crook DW, Newton PN, Buisson Y, Lee SJ, Dance DA. 2015. Colonization with Enterobacteriaceae producing ESBLs in children attending pre-school childcare facilities in the Lao People's Democratic Republic. J Antimicrob Chemother, 70 (6), pp. 1893-1897. | Show Abstract | Read more

OBJECTIVES: Intestinal carriage constitutes an important reservoir of antimicrobial-resistant bacteria, with some of the highest rates reported from Asia. Antibiotic resistance has been little studied in Laos, where some antibiotics are available without restriction, but others such as carbapenems are not available. PATIENTS AND METHODS: We collected stools from 397 healthy children in 12 randomly selected pre-school childcare facilities in and around Vientiane. Colonization with ESBL-producing Enterobacteriaceae (ESBLE) and carbapenemase-producing Enterobacteriaceae (CPE) was detected using a disc diffusion screening test and ESBLE were characterized using WGS. Risk factor data were collected by questionnaire. RESULTS: Ninety-two children (23%) were colonized with ESBLE, mainly Escherichia coli carrying blaCTX-M and Klebsiella pneumoniae carrying blaSHV or blaCTX-M, which were frequently resistant to multiple antibiotic classes. Although residence in Vientiane Capital, foreign travel, higher maternal level of education, antibiotic use in the preceding 3 months and attending a childcare facility with a 'good' level of hygiene were all associated with ESBLE colonization on univariable analysis, a significant association remained only for antibiotic use when a stepwise approach was used with a multivariate random-effects model. WGS analysis suggested transmission in both childcare facilities and community settings. CONCLUSIONS: The high prevalence of paediatric colonization with ESBLE in Laos, one of the highest reported in Asia, is probably the result of inappropriate antibiotic use. Paediatric colonization with CPE was not identified in this study, but it is important to continue to monitor the spread of antibiotic-resistant Enterobacteriaceae in Laos.

Ellis MK, Elliott KS, Rautanen A, Crook DW, Hill AV, Chapman SJ. 2015. Rare variants in MYD88, IRAK4 and IKBKG and susceptibility to invasive pneumococcal disease: a population-based case-control study. PLoS One, 10 (4), pp. e0123532. | Show Abstract | Read more

Although rare variants within the Toll-like receptor signalling pathway genes have been found to underlie human primary immunodeficiencies associated with selective predisposition to invasive pneumococcal disease (IPD), the contribution of variants in these genes to IPD susceptibility at the population level remains unknown. Complete re-sequencing of IRAK4, MYD88 and IKBKG genes was undertaken in 164 IPD cases from the UK and 164 geographically-matched population-based controls. 233 single-nucleotide variants (SNVs) were identified, of which ten were in coding regions. Four rare coding variants were predicted to be deleterious, two variants in MYD88 and two in IRAK4. The predicted deleterious variants in MYD88 were observed as two heterozygote cases but not seen in controls. Frequencies of predicted deleterious IRAK4 SNVs were the same in cases and controls. Our findings suggest that rare, functional variants in MYD88, IRAK4 or IKBKG do not significantly contribute to IPD susceptibility in adults at the population level.

Votintseva AA, Pankhurst LJ, Anson LW, Morgan MR, Gascoyne-Binzi D, Walker TM, Quan TP, Wyllie DH et al. 2015. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures. J Clin Microbiol, 53 (4), pp. 1137-1143. | Show Abstract | Read more

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

Hamaluba M, Kandasamy R, Ndimah S, Morton R, Caccamo M, Robinson H, Kelly S, Field A et al. 2015. A cross-sectional observational study of pneumococcal carriage in children, their parents, and older adults following the introduction of the 7-valent pneumococcal conjugate vaccine. Medicine (Baltimore), 94 (1), pp. e335. | Show Abstract | Read more

Using nasopharyngeal carriage as a marker of vaccine impact, pneumococcal colonization and its relation to invasive disease were examined in children, their parents, and older adults in the United Kingdom following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) and prior to 13-valent pneumococcal conjugate vaccine (PCV13).A cross-sectional observational study was conducted, collecting nasopharyngeal swabs from children aged 25 to 55 months who had previously received 3 doses of PCV7, their parents, and adults aged ≥65 years. Pneumococcal serotyping was conducted according to World Health Organization guidelines with nontypeable isolates further analyzed by molecular serotyping. A national invasive disease surveillance program was conducted throughout the corresponding period.Pneumococcus was isolated from 47% of children, 9% of parents, and 2.2% of older adults. For these groups, the percentage of serotypes covered by PCV7 were 1.5%, 0.0%, and 15.4%, with a further 20.1%, 44.4%, and 7.7% coverage added by those in PCV13. In each group, the percentage of disease due to serotypes covered by PCV7 were 1.0%, 7.4% and 5.1% with a further 65.3%, 42.1%, and 61.4% attributed to those in PCV13.The prevalence of carriage is the highest in children, with direct vaccine impact exemplified by low carriage and disease prevalence of PCV7 serotypes in vaccinated children, whereas the indirect effects of herd protection are implied by similar observations in unvaccinated parents and older adults.

Mathers AJ, Stoesser N, Sheppard AE, Pankhurst L, Giess A, Yeh AJ, Didelot X, Turner SD et al. 2015. Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing. Antimicrob Agents Chemother, 59 (3), pp. 1656-1663. | Show Abstract | Read more

The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination.

Stoesser N, Sheppard AE, Shakya M, Sthapit B, Thorson S, Giess A, Kelly D, Pollard AJ, Peto TE, Walker AS, Crook DW. 2015. Dynamics of MDR Enterobacter cloacae outbreaks in a neonatal unit in Nepal: insights using wider sampling frames and next-generation sequencing. J Antimicrob Chemother, 70 (4), pp. 1008-1015. | Show Abstract | Read more

OBJECTIVES: There are limited data on Enterobacter cloacae outbreaks and fewer describing these in association with NDM-1. With whole-genome sequencing, we tested the hypothesis that a cluster of 16 E. cloacae bacteraemia cases in a Nepali neonatal unit represented a single clonal outbreak, using a wider set of epidemiologically unrelated clinical E. cloacae isolates for comparison. METHODS: Forty-three isolates were analysed, including 23 E. cloacae and 3 Citrobacter sp. isolates obtained from blood cultures from 16 neonates over a 3 month period. These were compared with two contemporaneous community-associated drug-resistant isolates from adults, a unit soap dispenser isolate and a set of historical invasive isolates (n=14) from the same geographical locality. RESULTS: There were two clear neonatal outbreaks and one isolated case in the unit. One outbreak was associated with an NDM-1 plasmid also identified in a historical community-associated strain. The smaller, second outbreak was likely associated with a contaminated soap dispenser. The two community-acquired adult cases and three sets of historical hospital-associated neonatal isolates represented four additional genetic clusters. CONCLUSIONS: E. cloacae infections in this context represent several different transmission networks, operating at the community/hospital and host strain/plasmid levels. Wide sampling frames and high-resolution typing methods are needed to describe the complex molecular epidemiology of E. cloacae outbreaks, which is not appropriately reflected by routine susceptibility phenotypes. Soap dispensers may represent a reservoir for E. cloacae and bacterial strains and plasmids may persist in hospitals and in the community for long periods, sporadically being involved in outbreaks of disease.

Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. 2016. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis, 16 (3), pp. 285-286. | Read more

Weiss K, Louie T, Miller MA, Mullane K, Crook DW, Gorbach SL. 2015. Effects of proton pump inhibitors and histamine-2 receptor antagonists on response to fidaxomicin or vancomycin in patients with Clostridium difficile-associated diarrhoea. BMJ Open Gastroenterol, 2 (1), pp. e000028. | Show Abstract | Read more

OBJECTIVE: It has been established that use of proton pump inhibitors (PPIs) is associated with an increased risk of acquiring Clostridium difficile-associated diarrhoea (CDAD). However, it is not known whether the use of PPIs or histamine-2 receptor antagonists (H2RAs) concurrently with CDAD-targeted antibiotic treatment affects clinical response or recurrence rates. DESIGN: In two phase 3 trials, patients with toxin-positive CDAD were randomised to receive fidaxomicin 200 mg twice daily or vancomycin 125 mg four times daily for 10 days. Only inpatients with CDAD (due to complete medication record availability) were included in this post hoc analysis: 701 patients, of whom 446 (64%) used PPIs or H2RAs during study drug treatment or follow-up. Baseline factors that were statistically significant in univariate analyses were analysed in multivariate analyses of effects on clinical response and recurrence. RESULTS: Multivariate analysis showed that leukocytosis, elevated creatinine and hypoalbuminemia, but not PPI or H2RA use, were significant factors associated with poor clinical responses. Treatment group was the single significant predictor of recurrence; the probability of recurrence after fidaxomicin therapy was half that following vancomycin therapy. CONCLUSIONS: Acid-suppressing drugs, used by nearly two-thirds of inpatients with CDAD, did not worsen clinical response or recurrence when used concurrently with fidaxomicin or vancomycin. Therefore, development of CDAD does not require discontinuation of anti-acid treatment in patients who have an indication for continuing PPI or H2RA therapy, such as gastro-oesophageal reflux disease and risk of gastrointestinal bleed.

Wrightson JM, Wray JA, Street TL, Chapman SJ, Gleeson FV, Maskell NA, Peto TE, Rahman NM, Crook DW. 2015. Absence of Atypical Pathogens in Pleural Infection. Chest, 148 (3), pp. e102-e103. | Read more

Walker TM, Kohl TA, Omar SV, Hedge J, Del Ojo Elias C, Bradley P, Iqbal Z, Feuerriegel S et al. 2015. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study. Lancet Infect Dis, 15 (10), pp. 1193-1202. | Show Abstract | Read more

BACKGROUND: Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. METHODS: Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS: We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specificity (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. INTERPRETATION: A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early. FUNDING: Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Eyre DW, Tracey L, Elliott B, Slimings C, Huntington PG, Stuart RL, Korman TM, Kotsiou G et al. 2015. Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012 Euro surveillance : bulletin Européen sur les maladies transmissibles = European communicable disease bulletin, 20 (10), pp. 21059. | Show Abstract

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Bartels MD, Larner-Svensson H, Meiniche H, Kristoffersen K, Schonning K, Nielsen JB, Rohde SM, Christensen LB et al. 2015. Monitoring meticillin resistant Staphylococcus aureus and its spread in Copenhagen, Denmark, 2013, through routine whole genome sequencing. Euro Surveill, 20 (17), pp. 20-28. | Show Abstract

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n = 300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

Eyre DW, Tracey L, Elliott B, Slimings C, Huntington PG, Stuart RL, Korman TM, Kotsiou G et al. 2015. Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012. Euro Surveill, 20 (10), pp. 21059. | Show Abstract

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Westwood J, Burnett M, Spratt D, Ball M, Wilson DJ, Wellsteed S, Cleary D, Green A et al. 2014. The hospital microbiome project: meeting report for the UK science and innovation network UK-USA workshop ‘beating the superbugs: hospital microbiome studies for tackling antimicrobial resistance’, October 14th 2013 Standards in Genomic Sciences, 9 (1), pp. 12-12. | Show Abstract | Read more

© 2014 Westwood et al.; licensee BioMed Central Ltd.The UK Science and Innovation Network UK-USA workshop 'Beating the Superbugs: Hospital Microbiome Studies for tackling Antimicrobial Resistance' was held on October 14th 2013 at the UK Department of Health, London. The workshop was designed to promote US-UK collaboration on hospital microbiome studies to add a new facet to our collective understanding of antimicrobial resistance. The assembled researchers debated the importance of the hospital microbial community in transmission of disease and as a reservoir for antimicrobial resistance genes, and discussed methodologies, hypotheses, and priorities. A number of complementary approaches were explored, although the importance of the built environment microbiome in disease transmission was not universally accepted. Current whole genome epidemiological methods are being pioneered in the UK and the benefits of moving to community analysis are not necessarily obvious to the pioneers; however, rapid progress in other areas of microbiology suggest to some researchers that hospital microbiome studies will be exceptionally fruitful even in the short term. Collaborative studies will recombine different strengths to tackle the international problems of antimicrobial resistance and hospital and healthcare associated infections.

Yamaguchi Y, Anson L, Crook D, Knox K, Wyllie D. 2014. Evidence for control of Staphylococcus aureus nasal carriage by innate-like T lymphocyte populations in humans: a cross sectional study IMMUNOLOGY, 143 pp. 73-73.

Elliott B, Dingle KE, Didelot X, Crook DW, Riley TV. 2014. The complexity and diversity of the Pathogenicity Locus in Clostridium difficile clade 5. Genome Biol Evol, 6 (12), pp. 3159-3170. | Show Abstract | Read more

The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1-5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A(+)B(+) PaLoc variant and the other an A(-)B(+) variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A(-)B(+) PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.

Hart JD, Street T, Wrightson JM, Moore DP, Scott AG, Crook DW, Turner GD. 2014. Infectious Diseases and Tropical Disease Pathology: SC16-1 rRNA SEQUENCING IN MOLECULAR MICROBIOLOGICAL DIAGNOSIS OF BACTERIAL INFECTIONS IN THE AUTOPSY SETTING. Pathology, 46 Suppl 2 pp. S26. | Show Abstract | Read more

Diagnosing the aetiology of infectious diseases at autopsy, such as pneumonia, meningitis, sepsis or SUDI, is complicated due to issues including post mortem contamination, difficulty culturing fastidious organisms and subjective interpretation of polymicrobial cultures. Death of organisms may also occur post mortem, especially if antibiotics were given to the patient, but residual DNA from non-viable organisms, amenable to molecular detection, may remain. The 16S rRNA gene is present in all bacteria with conserved and hyper-variable regions along its length, allowing amplification and sequencing of all bacterial 16S sequences present in a sample. 16S sequencing offers potential advantages over culture-based diagnostics and is increasingly used in clinical practice. It has been used to identify bacteria in formalin fixed paraffin embedded (FFPE) surgical pathology specimens but its use has not been reported in autopsy diagnosis. This talk will summarise a study aimed to assess the utility of 16S sequencing as an adjunctive microbiological test in the autopsy. Our preliminary work has used post mortem lung tissue samples from children dying with pneumonia as part of the Pneumonia Etiology Research for Child Health (PERCH) project. The technique has identified known pathogens in some cases and provided additional diagnostic information in others. The presentation will discuss the technical aspects of 16S sequencing from FFPE and autopsy material, and the issues surrounding its application to diagnosis in comparison with standard culture based diagnostics on surgical/autopsy material.

Stoesser N, Giess A, Batty EM, Sheppard AE, Walker AS, Wilson DJ, Didelot X, Bashir A et al. 2014. Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting. Antimicrob Agents Chemother, 58 (12), pp. 7347-7357. | Show Abstract | Read more

NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying bla(NDM-1). The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although bla(NDM) variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.

Pankhurst L, Macfarlane-Smith L, Buchanan J, Anson L, Davies K, O'Connor L, Ashwin H, Pike G et al. 2014. Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study. Health Technol Assess, 18 (53), pp. 1-167. | Show Abstract | Read more

BACKGROUND: Every year approximately 5000-9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are 'blocked' by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients. OBJECTIVE: To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus. DESIGN: A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out. SETTING: MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts. MAIN OUTCOME MEASURES: Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus. RESULTS: Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance for S. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly for S. enterica, Giardia lamblia and Cryptosporidium spp. As the results indicated that S. enterica detection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag(®) gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). For Campylobacter spp., C. difficile and norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. For S. enterica, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients. CONCLUSIONS: Overall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen, S. enterica; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed. FUNDING: The Health Technology Assessment programme of the National Institute for Health Research.

Price JR, Golubchik T, Wilson DJ, Crook DW, Walker AS, Peto TE, Paul J, Llewelyn MJ. 2014. Reply to Mills and Linkin. Clin Infect Dis, 59 (5), pp. 752-753. | Read more

Buchanan J, Wordsworth S, O'Connor L, Pike G, Walker AS, Wilcox MH, Crook DW. 2015. Management of patients with suspected infectious diarrhoea in hospitals in England. J Hosp Infect, 90 (3), pp. 199-207. | Show Abstract | Read more

BACKGROUND: Advances in molecular and genomic testing for patients with suspected infectious diarrhoea are on the horizon. It is important to understand how infection control and microbiology departments currently operate with respect to the management of these patients in order to assess the implications of more widespread diagnostic testing. However, there are few data available on current practice in this context. AIM: To describe current infection control and microbiologist practice across England with respect to the management of patients with suspected infectious diarrhoea. METHODS: Hospitals in England completed three questionnaires on current testing practice in this context. Questionnaire design was informed by current practice within the Oxford University Hospitals group. FINDINGS: Forty-one percent of hospitals completed at least one questionnaire. A notable proportion of staff time was devoted to the management of patients with suspected infectious diarrhoea. Staff training was generally good, but compliance with policy documents was only 80%. Cleaning and isolation policies varied across hospitals, suggesting that either these were not evidence-based, or that the evidence base is weak. There was more agreement on outbreak definitions, management, and cohorting policies. Stool-testing decisions were mainly driven by patient characteristics, whereas strain typing was infrequently used (except to investigate Clostridium difficile infections). Multiple practical difficulties associated with patient management were identified, along with a clear appetite for more widespread genomic diagnostic testing. CONCLUSION: Managing patients with suspected infectious diarrhoea is a major burden in England. Advances in testing practice in this context could have significant clinical and economic impacts.

Miller RR, Walker AS, Godwin H, Fung R, Votintseva A, Bowden R, Mant D, Peto TE, Crook DW, Knox K. 2014. Dynamics of acquisition and loss of carriage of Staphylococcus aureus strains in the community: the effect of clonal complex. J Infect, 68 (5), pp. 426-439. | Show Abstract | Read more

BACKGROUND: Staphylococcus aureus nasal carriage increases infection risk. However, few studies have investigated S. aureus acquisition/loss over >1 year, and fewer still used molecular typing. METHODS: 1123 adults attending five Oxfordshire general practices had nasal swabs taken. 571 were re-swabbed after one month then every two months for median two years. All S. aureus isolates were spa-typed. Risk factors were collected from interviews and medical records. RESULTS: 32% carried S. aureus at recruitment (<1% MRSA). Rates of spa-type acquisition were similar in participants S. aureus positive (1.4%/month) and negative (1.8%/month, P = 0.13) at recruitment. Rates were faster in those carrying clonal complex (CC)15 (adjusted (a)P = 0.03) or CC8 (including USA300) (aP = 0.001) at recruitment versus other CCs. 157/274 (57%) participants S. aureus positive at recruitment returning ≥ 12 swabs carried S. aureus consistently, of whom 135 carried the same spa-type. CC22 (including EMRSA-15) was more prevalent in long-term than intermittent spa-type carriers (aP = 0.03). Antibiotics transiently reduced carriage, but no other modifiable risk factors were found. CONCLUSIONS: Both transient and longer-term carriage exist; however, the approximately constant rates of S. aureus gain and loss suggest that 'never' or truly 'persistent' carriage are rare. Long-term carriage varies by strain, offering new explanations for the success of certain S. aureus clones.

Moore CE, Paul J, Foster D, Mahar SA, Griffiths D, Knox K, Peto TE, Walker AS, Crook DW, Oxford Invasive Pneumococcal Surveillance Group. 2014. Reduction of invasive pneumococcal disease 3 years after the introduction of the 13-valent conjugate vaccine in the Oxfordshire region of England. J Infect Dis, 210 (7), pp. 1001-1011. | Show Abstract | Read more

BACKGROUND: The 7-valent pneumococcal conjugate (PCV7) vaccine's impact on invasive pneumococcal disease (IPD) is well described, but few reports exist on the additional impact of the 13-valent vaccine (PCV13). METHODS: We calculated the IPD incidence across all ages in a surveillance project following implementation of PCV7 (in September 2006) and PCV13 (in April 2010) in children aged <2 years (11 hospitals; 4935 cases). RESULTS: The overall incidence decreased from 10 cases/100 000 persons per year in 1996-1997 to 8 cases/100 000 persons per year in 2007-2008 and 7 cases/100 000 in 2012-2013. Declines were greater in children aged <2 years (from 37 cases/100 000 in 1996-1997 to 29 and 14 cases/100 000 in 2007-2008 and 2012-2013, respectively). The incidence of IPD due to PCV7 serotypes decreased in all ages after PCV7 introduction (P < .001), whereas the incidence of IPD due to the additional 6 serotypes in PCV13 and to nonvaccine types (NVTs) increased in children aged ≥2 years (P < .001 for both comparisons). The incidence of IPD due to the 6 additional serotypes in PCV13 declined significantly after PCV13 introduction in all ages (P ≤ .01), and the incidence of IPD due to NVTs declined significantly in children aged ≥2 years (P = .003). In 2011-2013, the overall incidences of IPD due to PCV7 serotypes, the 6 additional serotypes in PCV13, and NVTs were 0.3, 2.8, and 4.4 cases/100 000; the incidences among children aged <2 years were 0.9, 2.4, and 10.8 cases/100 000, respectively. CONCLUSIONS: The annual incidence of IPD due to vaccine serotypes (1-3 cases/100 000) among children aged <2 years and nontarget groups demonstrates the success of PCV7 and PCV13. A substantially higher incidence of IPD due to NVTs indicates the importance of ongoing surveillance and extension of vaccine polyvalency.

Price JR, Golubchik T, Cole K, Wilson DJ, Crook DW, Thwaites GE, Bowden R, Walker AS, Peto TE, Paul J, Llewelyn MJ. 2014. Whole-genome sequencing shows that patient-to-patient transmission rarely accounts for acquisition of Staphylococcus aureus in an intensive care unit. Clin Infect Dis, 58 (5), pp. 609-618. | Show Abstract | Read more

BACKGROUND:  Strategies to prevent Staphylococcus aureus infection in hospitals focus on patient-to-patient transmission. We used whole-genome sequencing to investigate the role of colonized patients as the source of new S. aureus acquisitions, and the reliability of identifying patient-to-patient transmission using the conventional approach of spa typing and overlapping patient stay. METHODS: Over 14 months, all unselected patients admitted to an adult intensive care unit (ICU) were serially screened for S. aureus. All available isolates (n = 275) were spa typed and underwent whole-genome sequencing to investigate their relatedness at high resolution. RESULTS: Staphylococcus aureus was carried by 185 of 1109 patients sampled within 24 hours of ICU admission (16.7%); 59 (5.3%) patients carried methicillin-resistant S. aureus (MRSA). Forty-four S. aureus (22 MRSA) acquisitions while on ICU were detected. Isolates were available for genetic analysis from 37 acquisitions. Whole-genome sequencing indicated that 7 of these 37 (18.9%) were transmissions from other colonized patients. Conventional methods (spa typing combined with overlapping patient stay) falsely identified 3 patient-to-patient transmissions (all MRSA) and failed to detect 2 acquisitions and 4 transmissions (2 MRSA). CONCLUSIONS: Only a minority of S. aureus acquisitions can be explained by patient-to-patient transmission. Whole-genome sequencing provides the resolution to disprove transmission events indicated by conventional methods and also to reveal otherwise unsuspected transmission events. Whole-genome sequencing should replace conventional methods for detection of nosocomial S. aureus transmission.

Gordon NC, Price JR, Cole K, Everitt R, Morgan M, Finney J, Kearns AM, Pichon B et al. 2014. Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing. J Clin Microbiol, 52 (4), pp. 1182-1191. | Show Abstract | Read more

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism's phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Votintseva AA, Miller RR, Fung R, Knox K, Godwin H, Peto TE, Crook DW, Bowden R, Walker AS. 2014. Multiple-strain colonization in nasal carriers of Staphylococcus aureus. J Clin Microbiol, 52 (4), pp. 1192-1200. | Show Abstract | Read more

Staphylococcus aureus is a commensal that can also cause invasive infection. Reports suggest that nasal cocolonization occurs rarely, but the resources required to sequence multiple colonies have precluded its large-scale investigation. A staged protocol was developed to maximize detection of mixed-spa-type colonization while minimizing laboratory resources using 3,197 S. aureus-positive samples from a longitudinal study of healthy individuals in Oxfordshire, United Kingdom. Initial typing of pooled material from each sample identified a single unambiguous strain in 89.6% of samples. Twelve single-colony isolates were typed from samples producing ambiguous initial results. All samples could be resolved into one or more spa types using the protocol. Cocolonization point prevalence was 3.4 to 5.8% over 24 months of follow-up in 360 recruitment-positives. However, 18% were cocolonized at least once, most only transiently. Cocolonizing spa types were completely unrelated in 56% of samples. Of 272 recruitment-positives returning ≥12 swabs, 166 (61%) carried S. aureus continuously but only 106 (39%) carried the same single spa type without any cocolonization; 31 (11%) switched spa type and 29 (11%) had transient cocarriage. S. aureus colonization is dynamic even in long-term carriers. New unrelated cocolonizing strains could increase invasive disease risk, and ongoing within-host evolution could increase invasive potential, possibilities that future studies should explore.

Buchanan R, Stoesser N, Crook D, Bowler IC. 2014. Multidrug-resistant Escherichia coli soft tissue infection investigated with bacterial whole genome sequencing. BMJ Case Rep, 2014 (oct19 1), pp. bcr2014207200-bcr2014207200. | Show Abstract | Read more

A 45-year-old man with dilated cardiomyopathy presented with acute leg pain and erythema suggestive of necrotising fasciitis. Initial surgical exploration revealed no necrosis and treatment for a soft tissue infection was started. Blood and tissue cultures unexpectedly grew a Gram-negative bacillus, subsequently identified by an automated broth microdilution phenotyping system as an extended-spectrum β-lactamase producing Escherichia coli. The patient was treated with a 3-week course of antibiotics (ertapenem followed by ciprofloxacin) and debridement for small areas of necrosis, followed by skin grafting. The presence of E. coli triggered investigation of both host and pathogen. The patient was found to have previously undiagnosed liver disease, a risk factor for E. coli soft tissue infection. Whole genome sequencing of isolates from all specimens confirmed they were clonal, of sequence type ST131 and associated with a likely plasmid-associated AmpC (CMY-2), several other resistance genes and a number of virulence factors.

Everitt RG, Didelot X, Batty EM, Miller RR, Knox K, Young BC, Bowden R, Auton A et al. 2014. Mobile elements drive recombination hotspots in the core genome of Staphylococcus aureus. Nat Commun, 5 pp. 3956. | Show Abstract | Read more

Horizontal gene transfer is an important driver of bacterial evolution, but genetic exchange in the core genome of clonal species, including the major pathogen Staphylococcus aureus, is incompletely understood. Here we reveal widespread homologous recombination in S. aureus at the species level, in contrast to its near-complete absence between closely related strains. We discover a patchwork of hotspots and coldspots at fine scales falling against a backdrop of broad-scale trends in rate variation. Over megabases, homoplasy rates fluctuate 1.9-fold, peaking towards the origin-of-replication. Over kilobases, we find core recombination hotspots of up to 2.5-fold enrichment situated near fault lines in the genome associated with mobile elements. The strongest hotspots include regions flanking conjugative transposon ICE6013, the staphylococcal cassette chromosome (SCC) and genomic island νSaα. Mobile element-driven core genome transfer represents an opportunity for adaptation and challenges our understanding of the recombination landscape in predominantly clonal pathogens, with important implications for genotype-phenotype mapping.

Walker TM, Lalor MK, Broda A, Saldana Ortega L, Morgan M, Parker L, Churchill S, Bennett K et al. 2014. Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007-12, with whole pathogen genome sequences: an observational study. Lancet Respir Med, 2 (4), pp. 285-292. | Show Abstract | Read more

BACKGROUND: Patients born outside the UK have contributed to a 20% rise in the UK's tuberculosis incidence since 2000, but their effect on domestic transmission is not known. Here we use whole-genome sequencing to investigate the epidemiology of tuberculosis transmission in an unselected population over 6 years. METHODS: We identified all residents with Oxfordshire postcodes with a Mycobacterium tuberculosis culture or a clinical diagnosis of tuberculosis between Jan 1, 2007, and Dec 31, 2012, using local databases and checking against the national Enhanced Tuberculosis Surveillance database. We used Illumina technology to sequence all available M tuberculosis cultures from identified cases. Sequences were clustered by genetic relatedness and compared retrospectively with contact investigations. The first patient diagnosed in each cluster was defined as the index case, with links to subsequent cases assigned first by use of any epidemiological linkage, then by genetic distance, and then by timing of diagnosis. FINDINGS: Although we identified 384 patients with a diagnosis of tuberculosis, country of birth was known for 380 and we sequenced isolates from 247 of 269 cases with culture-confirmed disease. 39 cases were genomically linked within 13 clusters, implying 26 local transmission events. Only 11 of 26 possible transmissions had been previously identified through contact tracing. Of seven genomically confirmed household clusters, five contained additional genomic links to epidemiologically unidentified non-household members. 255 (67%) patients were born in a country with high tuberculosis incidence, conferring a local incidence of 109 cases per 100,000 population per year in Oxfordshire, compared with 3·5 cases per 100,000 per year for those born in low-incidence countries. However, patients born in the low-incidence countries, predominantly UK, were more likely to have pulmonary disease (adjusted odds ratio 1·8 [95% CI 1·2-2·9]; p=0·009), social risk factors (4·4 [2·0-9·4]; p<0·0001), and be part of a local transmission cluster (4·8 [1·6-14·8]; p=0·006). INTERPRETATION: Although inward migration has contributed to the overall tuberculosis incidence, our findings suggest that most patients born in high-incidence countries reactivate latent infection acquired abroad and are not involved in local onward transmission. Systematic screening of new entrants could further improve tuberculosis control, but it is important that health care remains accessible to all individuals, especially high-risk groups, if tuberculosis control is not to be jeopardised. FUNDING: UK Clinical Research Collaboration (Wellcome Trust, Medical Research Council, National Institute for Health Research [NIHR]), and NIHR Oxford Biomedical Research Centre.

Votintseva AA, Fung R, Miller RR, Knox K, Godwin H, Wyllie DH, Bowden R, Crook DW, Walker AS. 2014. Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire. BMC Microbiol, 14 (1), pp. 63. | Show Abstract | Read more

BACKGROUND: Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as "spa-types", based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as "non-typeable". RESULTS: We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated "non-typeable" using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies. CONCLUSIONS: This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.

Miller RM, Price JR, Batty EM, Didelot X, Wyllie D, Golubchik T, Crook DW, Paul J et al. 2014. Healthcare-associated outbreak of meticillin-resistant Staphylococcus aureus bacteraemia: role of a cryptic variant of an epidemic clone. J Hosp Infect, 86 (2), pp. 83-89. | Show Abstract | Read more

BACKGROUND: New strains of meticillin-resistant Staphylococcus aureus (MRSA) may be associated with changes in rates of disease or clinical presentation. Conventional typing techniques may not detect new clonal variants that underlie changes in epidemiology or clinical phenotype. AIM: To investigate the role of clonal variants of MRSA in an outbreak of MRSA bacteraemia at a hospital in England. METHODS: Bacteraemia isolates of the major UK lineages (EMRSA-15 and -16) from before and after the outbreak were analysed by whole-genome sequencing in the context of epidemiological and clinical data. For comparison, EMRSA-15 and -16 isolates from another hospital in England were sequenced. A clonal variant of EMRSA-16 was identified at the outbreak hospital and a molecular signature test designed to distinguish variant isolates among further EMRSA-16 strains. FINDINGS: By whole-genome sequencing, EMRSA-16 isolates during the outbreak showed strikingly low genetic diversity (P < 1 × 10(-6), Monte Carlo test), compared with EMRSA-15 and EMRSA-16 isolates from before the outbreak or the comparator hospital, demonstrating the emergence of a clonal variant. The variant was indistinguishable from the ancestral strain by conventional typing. This clonal variant accounted for 64/72 (89%) of EMRSA-16 bacteraemia isolates at the outbreak hospital from 2006. CONCLUSIONS: Evolutionary changes in epidemic MRSA strains not detected by conventional typing may be associated with changes in disease epidemiology. Rapid and affordable technologies for whole-genome sequencing are becoming available with the potential to identify and track the emergence of variants of highly clonal organisms.

Dingle KE, Elliott B, Robinson E, Griffiths D, Eyre DW, Stoesser N, Vaughan A, Golubchik T et al. 2014. Evolutionary history of the Clostridium difficile pathogenicity locus. Genome Biol Evol, 6 (1), pp. 36-52. | Show Abstract | Read more

The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19 kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and nontoxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ∼30 years ago within a clade 1 genotype. The genetic organization of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related but nonconjugative and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting but clinically important genetic elements were thus characterized: the PaLoc, mobilized rarely via homologous recombination, and Tn6218, mobilized frequently through transposition.

Lamble S, Batty E, Attar M, Buck D, Bowden R, Lunter G, Crook D, El-Fahmawi B, Piazza P. 2013. Improved workflows for high throughput library preparation using the transposome-based Nextera system. BMC Biotechnol, 13 (1), pp. 104. | Show Abstract | Read more

BACKGROUND: The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs. RESULTS: We show that an initial bead-based normalisation step can remove the need for quantification and improves sample purity. A 75% cost reduction was achieved with a low-volume modified protocol which was tested over genomes with different GC content to demonstrate its robustness. Finally we developed a custom set of index tags and primers which increase the number of samples that can simultaneously be sequenced on a single lane of an Illumina instrument. CONCLUSIONS: We addressed the bottlenecks of Nextera library construction to produce a modified protocol which harnesses the full power of the Nextera kit and allows the reproducible construction of libraries on a high-throughput scale reducing the associated cost of the kit.

Wong TH, Dearlove BL, Hedge J, Giess AP, Piazza P, Trebes A, Paul J, Smit E et al. 2013. Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England. Virol J, 10 (1), pp. 335. | Show Abstract | Read more

BACKGROUND: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. METHOD: Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. RESULTS: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. CONCLUSION: Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.

Eyre DW, Babakhani F, Griffiths D, Seddon J, Del Ojo Elias C, Gorbach SL, Peto TE, Crook DW, Walker AS. 2014. Whole-genome sequencing demonstrates that fidaxomicin is superior to vancomycin for preventing reinfection and relapse of infection with Clostridium difficile. J Infect Dis, 209 (9), pp. 1446-1451. | Show Abstract | Read more

Whole-genome sequencing was used to determine whether the reductions in recurrence of Clostridium difficile infection observed with fidaxomicin in pivotal phase 3 trials occurred by preventing relapse of the same infection, by preventing reinfection with a new strain, or by preventing both outcomes. Paired isolates of C. difficile were available from 93 of 199 participants with recurrences (28 were treated with fidaxomicin, and 65 were treated with vancomycin). Given C. difficile evolutionary rates, paired samples ≤2 single-nucleotide variants (SNVs) apart were considered relapses, paired samples >10 SNVs apart were considered reinfection, and those 3-10 SNVs apart (or without whole-genome sequences) were considered indeterminate in a competing risks survival analysis. Fidaxomicin reduced the risk of both relapse (competing risks hazard ratio [HR], 0.40 [95% confidence interval {CI}, .25-.66]; P = .0003) and reinfection (competing risks HR, 0.33 [95% CI, 0.11-1.01]; P = .05).

Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, O'Connor L, Oakley SJ et al. 2013. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis, 13 (11), pp. 936-945. | Show Abstract | Read more

BACKGROUND: Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. METHODS: In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12,420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. FINDINGS: Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12-2·31). Multistage algorithms performed better than did standalone assays. INTERPRETATION: We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.

Eyre DW, Fawley WN, Best EL, Griffiths D, Stoesser NE, Crook DW, Peto TE, Walker AS, Wilcox MH. 2013. Comparison of multilocus variable-number tandem-repeat analysis and whole-genome sequencing for investigation of Clostridium difficile transmission. J Clin Microbiol, 51 (12), pp. 4141-4149. | Show Abstract | Read more

No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ≥2 SNVs but ≤10 STRDs, and 69 (5%) had ≤2 SNVs but ≥10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

Eyre DW, Cule ML, Wilson DJ, Griffiths D, Vaughan A, O'Connor L, Ip CL, Golubchik T et al. 2013. Diverse sources of C. difficile infection identified on whole-genome sequencing. N Engl J Med, 369 (13), pp. 1195-1205. | Show Abstract | Read more

BACKGROUND: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions. METHODS: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location. RESULTS: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile. CONCLUSIONS: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).

Juhas M, Dimopoulou I, Robinson E, Elamin A, Harding R, Hood D, Crook D. 2013. Identification of another module involved in the horizontal transfer of the Haemophilus genomic island ICEHin1056 Plasmid, 70 (2), pp. 277-283. | Show Abstract | Read more

A significant part of horizontal gene transfer is facilitated by genomic islands. Haemophilus influenzae genomic island ICE. Hin1056 is an archetype of a genomic island that accounts for pandemic spread of antibiotics resistance. ICE. Hin1056 has modular structure and harbors modules involved in type IV secretion and integration. Previous studies have shown that ICE. Hin1056 encodes a functional type IV secretion system; however, other modules have not been characterized yet. Here we show that the module on the 5' extremity of ICE. Hin1056 consists of 15 genes that are well conserved in a number of related genomic islands. Furthermore by disrupting six genes of the investigated module of ICE. Hin1056 by site-specific mutagenesis we demonstrate that in addition to type IV secretion system module, the investigated module is also important for the successful conjugal transfer of ICE. Hin1056 from donor to recipient cells. © 2013 Elsevier Inc.

Mullane KM, Cornely OA, Crook DW, Golan Y, Louie TJ, Miller MA, Josephson MA, Gorbach SL. 2013. Renal impairment and clinical outcomes of Clostridium difficile infection in two randomized trials. Am J Nephrol, 38 (1), pp. 1-11. | Show Abstract | Read more

BACKGROUND/AIMS: Patients with chronic kidney disease (CKD) have increased risk for Clostridium difficile infection (CDI) and for subsequent mortality. We determined the effect of CKD on response to treatment for CDI. METHODS: This is a post hoc analysis of two randomized controlled phase 3 trials that enrolled patients with CDI. Patients received either fidaxomicin 200 mg b.i.d. or vancomycin 125 mg q.i.d. for 10 days. Univariate and multivariate analyses compared end points by treatment received and CKD stage. RESULTS: At baseline, 27, 21, and 9% of the patients had stage 2 (60-89 ml/min/1.73 m(2)), stage 3 (30-59), and stage 4 or higher (<30) CKD. Cure rates were similar for normal (91%) and stage 2 CKD (92%), but declined to 80% for stage 3 and to 75% for stage 4 CKD (p < 0.001 for trend). Time to resolution of diarrhea (TTROD) increased with stage 3 and stage 4 CKD. CDI recurrence rates 4 weeks after treatment were 16, 20, 27, and 24% for normal, stage 2, stage 3, and stage 4 or higher CKD, respectively. Mortality increased with CKD stage. In multivariate analyses, stage 3 or higher CKD correlated with lower odds of cure, greater chance of recurrence, and lower odds of sustained response 28 days after treatment. Initial cure rates were similar in the vancomycin or fidaxomicin groups; however, the rate of recurrence was higher following vancomycin treatment independent of renal function. The presence of immunosuppression did not alter this effect. CONCLUSION: Progressive CKD is associated with increased TTROD, lower cure rates, and higher recurrence rates with treatment of CDI.

Walker AS, Eyre DW, Crook DW, Wilcox MH, Peto TE. 2013. Regarding "Clostridium difficile ribotype does not predict severe infection". Clin Infect Dis, 56 (12), pp. 1845-1846. | Read more

Juhas M, Dimopoulou I, Robinson E, Elamin A, Harding R, Hood D, Crook D. 2013. Identification of another module involved in the horizontal transfer of the Haemophilus genomic island ICEHin1056. Plasmid, 70 (2), pp. 277-283. | Show Abstract | Read more

A significant part of horizontal gene transfer is facilitated by genomic islands. Haemophilus influenzae genomic island ICEHin1056 is an archetype of a genomic island that accounts for pandemic spread of antibiotics resistance. ICEHin1056 has modular structure and harbors modules involved in type IV secretion and integration. Previous studies have shown that ICEHin1056 encodes a functional type IV secretion system; however, other modules have not been characterized yet. Here we show that the module on the 5' extremity of ICEHin1056 consists of 15 genes that are well conserved in a number of related genomic islands. Furthermore by disrupting six genes of the investigated module of ICEHin1056 by site-specific mutagenesis we demonstrate that in addition to type IV secretion system module, the investigated module is also important for the successful conjugal transfer of ICEHin1056 from donor to recipient cells.

Batty EM, Wong TH, Trebes A, Argoud K, Attar M, Buck D, Ip CL, Golubchik T et al. 2013. A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples. PLoS One, 8 (6), pp. e66129. | Show Abstract | Read more

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.

Stoesser N, Batty EM, Eyre DW, Morgan M, Wyllie DH, Del Ojo Elias C, Johnson JR, Walker AS, Peto TE, Crook DW. 2013. Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data. J Antimicrob Chemother, 68 (10), pp. 2234-2244. | Show Abstract | Read more

OBJECTIVES: Whole-genome sequencing potentially represents a single, rapid and cost-effective approach to defining resistance mechanisms and predicting phenotype, and strain type, for both clinical and epidemiological purposes. This retrospective study aimed to determine the efficacy of whole genome-based antimicrobial resistance prediction in clinical isolates of Escherichia coli and Klebsiella pneumoniae. METHODS: Seventy-four E. coli and 69 K. pneumoniae bacteraemia isolates from Oxfordshire, UK, were sequenced (Illumina HiSeq 2000). Resistance phenotypes were predicted from genomic sequences using BLASTn-based comparisons of de novo-assembled contigs with a study database of >100 known resistance-associated loci, including plasmid-associated and chromosomal genes. Predictions were made for seven commonly used antimicrobials: amoxicillin, co-amoxiclav, ceftriaxone, ceftazidime, ciprofloxacin, gentamicin and meropenem. Comparisons were made with phenotypic results obtained in duplicate by broth dilution (BD Phoenix). Discrepancies, either between duplicate BD Phoenix results or between genotype and phenotype, were resolved with gradient diffusion analyses. RESULTS: A wide variety of antimicrobial resistance genes were identified, including blaCTX-M, blaLEN, blaOKP, blaOXA, blaSHV, blaTEM, aac(3')-Ia, aac-(3')-IId, aac-(3')-IIe, aac(6')-Ib-cr, aadA1a, aadA4, aadA5, aadA16, aph(6')-Id, aph(3')-Ia, qnrB and qnrS, as well as resistance-associated mutations in chromosomal gyrA and parC genes. The sensitivity of genome-based resistance prediction across all antibiotics for both species was 0.96 (95% CI: 0.94-0.98) and the specificity was 0.97 (95% CI: 0.95-0.98). Very major and major error rates were 1.2% and 2.1%, respectively. CONCLUSIONS: Our method was as sensitive and specific as routinely deployed phenotypic methods. Validation against larger datasets and formal assessments of cost and turnaround time in a routine laboratory setting are warranted.

Walker AS, Eyre DW, Crook DW, Peto TE, Wilcox MH. 2013. Reply to Walk et al. Clin Infect Dis, 57 (4), pp. 626-627. | Read more

Eyre DW, Cule ML, Griffiths D, Crook DW, Peto TE, Walker AS, Wilson DJ. 2013. Detection of mixed infection from bacterial whole genome sequence data allows assessment of its role in Clostridium difficile transmission. PLoS Comput Biol, 9 (5), pp. e1003059. | Show Abstract | Read more

Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections-infection with ≥2 unrelated strains of the same species where only one is sequenced-potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between the pairs of cases under investigation. These results demonstrate that mixed infections can be detected without additional sequencing effort, and this will be important in assessing the extent of cryptic transmission in our hospitals.

Walker AS, Eyre DW, Wyllie DH, Dingle KE, Griffiths D, Shine B, Oakley S, O'Connor L et al. 2013. Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection. Clin Infect Dis, 56 (11), pp. 1589-1600. | Show Abstract | Read more

BACKGROUND: Despite substantial interest in biomarkers, their impact on clinical outcomes and variation with bacterial strain has rarely been explored using integrated databases. METHODS: From September 2006 to May 2011, strains isolated from Clostridium difficile toxin enzyme immunoassay (EIA)-positive fecal samples from Oxfordshire, United Kingdom (approximately 600,000 people) underwent multilocus sequence typing. Fourteen-day mortality and levels of 15 baseline biomarkers were compared between consecutive C. difficile infections (CDIs) from different clades/sequence types (STs) and EIA-negative controls using Cox and normal regression adjusted for demographic/clinical factors. RESULTS: Fourteen-day mortality was 13% in 2222 adults with 2745 EIA-positive samples (median, 78 years) vs 5% in 20,722 adults with 27,550 EIA-negative samples (median, 74 years) (absolute attributable mortality, 7.7%; 95% CI, 6.4%-9.0%). Mortality was highest in clade 5 CDIs (25% [16 of 63]; polymerase chain reaction (PCR) ribotype 078/ST 11), then clade 2 (20% [111 of 560]; 99% PCR ribotype 027/ST 1) versus clade 1 (12% [137 of 1168]; adjusted P < .0001). Within clade 1, 14-day mortality was only 4% (3 of 84) in ST 44 (PCR ribotype 015) (adjusted P = .05 vs other clade 1). Mean baseline neutrophil counts also varied significantly by genotype: 12.4, 11.6, and 9.5 × 10(9) neutrophils/L for clades 5, 2 and 1, respectively, vs 7.0 × 10(9) neutrophils/L in EIA-negative controls (P < .0001) and 7.9 × 10(9) neutrophils/L in ST 44 (P = .08). There were strong associations between C. difficile-type-specific effects on mortality and neutrophil/white cell counts (rho = 0.48), C-reactive-protein (rho = 0.43), eosinophil counts (rho = -0.45), and serum albumin (rho = -0.47). Biomarkers predicted 30%-40% of clade-specific mortality differences. CONCLUSIONS: C. difficile genotype predicts mortality, and excess mortality correlates with genotype-specific changes in biomarkers, strongly implicating inflammatory pathways as a major influence on poor outcome after CDI. PCR ribotype 078/ST 11 (clade 5) leads to severe CDI; thus ongoing surveillance remains essential.

Stoesser NE, Martin J, Mawer D, Eyre DW, Walker AS, Peto TE, Crook DW, Wilcox MH. 2013. Risk factors for Clostridium difficile acquisition in infants: importance of study design. Clin Infect Dis, 56 (11), pp. 1680-1681. | Read more

Golubchik T, Batty EM, Miller RR, Farr H, Young BC, Larner-Svensson H, Fung R, Godwin H et al. 2013. Within-host evolution of Staphylococcus aureus during asymptomatic carriage. PLoS One, 8 (5), pp. e61319. | Show Abstract | Read more

BACKGROUND: Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. PRINCIPAL FINDINGS: We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). SIGNIFICANCE: This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Eyre DW, Walker AS, Freeman J, Baines SD, Fawley WN, Chilton CH, Griffiths D, Vaughan A, Crook DW, Peto TE, Wilcox MH. 2013. Short-term genome stability of serial Clostridium difficile ribotype 027 isolates in an experimental gut model and recurrent human disease. PLoS One, 8 (5), pp. e63540. | Show Abstract | Read more

BACKGROUND: Clostridium difficile whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals. METHODS: Serial isolates from two scenarios were whole genome sequenced. Firstly, 62 isolates from 29 timepoints from three in vitro gut models, inoculated with a NAP1/027 strain. Secondly, 122 isolates from 44 patients (2-8 samples/patient) with mostly recurrent/on-going symptomatic NAP-1/027 C. difficile infection. Reference-based mapping was used to identify single nucleotide variants (SNVs). RESULTS: Across three gut model inductions, two with antibiotic treatment, total 137 days, only two new SNVs became established. Pre-existing minority SNVs became dominant in two models. Several SNVs were detected, only present in the minority of colonies at one/two timepoints. The median (inter-quartile range) [range] time between patients' first and last samples was 60 (29.5-118.5) [0-561] days. Within-patient C. difficile evolution was 0.45 SNVs/called genome/year (95%CI 0.00-1.28) and within-host diversity was 0.28 SNVs/called genome (0.05-0.53). 26/28 gut model and patient SNVs were non-synonymous, affecting a range of gene targets. CONCLUSIONS: The consistency of whole genome sequencing data from gut model C. difficile isolates, and the high stability of genomic sequences in isolates from patients, supports the use of whole genome sequencing in detailed transmission investigations.

Ammerlaan HS, Harbarth S, Buiting AG, Crook DW, Fitzpatrick F, Hanberger H, Herwaldt LA, van Keulen PH et al. 2013. Secular trends in nosocomial bloodstream infections: antibiotic-resistant bacteria increase the total burden of infection. Clin Infect Dis, 56 (6), pp. 798-805. | Show Abstract | Read more

BACKGROUND: It is unknown whether rising incidence rates of nosocomial bloodstream infections (BSIs) caused by antibiotic-resistant bacteria (ARB) replace antibiotic-susceptible bacteria (ASB), leaving the total BSI rate unaffected. METHODS: We investigated temporal trends in annual incidence densities (events per 100 000 patient-days) of nosocomial BSIs caused by methicillin-resistant Staphylococcus aureus (MRSA), ARB other than MRSA, and ASB in 7 ARB-endemic and 7 ARB-nonendemic hospitals between 1998 and 2007. RESULTS: 33 130 nosocomial BSIs (14% caused by ARB) yielded 36 679 microorganisms. From 1998 to 2007, the MRSA incidence density increased from 0.2 to 0.7 (annual increase, 22%) in ARB-nonendemic hospitals, and from 3.1 to 11.7 (annual increase, 10%) in ARB-endemic hospitals (P = .2), increasing the incidence density difference between ARB-endemic and ARB-nonendemic hospitals from 2.9 to 11.0. The non-MRSA ARB incidence density increased from 2.8 to 4.1 (annual increase, 5%) in ARB-nonendemic hospitals, and from 1.5 to 17.4 (annual increase, 22%) in ARB-endemic hospitals (P < .001), changing the incidence density difference from -1.3 to 13.3. Trends in ASB incidence densities were similar in both groups (P = .7). With annual increases of 3.8% and 5.4% of all nosocomial BSIs in ARB-nonendemic and ARB-endemic hospitals, respectively (P < .001), the overall incidence density difference of 3.8 increased to 24.4. CONCLUSIONS: Increased nosocomial BSI rates due to ARB occur in addition to infections caused by ASB, increasing the total burden of disease. Hospitals with high ARB infection rates in 2005 had an excess burden of BSI of 20.6 per 100 000 patient-days in a 10-year period, mainly caused by infections with ARB.

Louie TJ, Miller MA, Crook DW, Lentnek A, Bernard L, High KP, Shue YK, Gorbach SL. 2013. Effect of age on treatment outcomes in Clostridium difficile infection. J Am Geriatr Soc, 61 (2), pp. 222-230. | Show Abstract | Read more

OBJECTIVES: To determine the effect of advancing age on the clinical outcomes of Clostridium difficile (CDI) treatment. DESIGN: Regression modeling of results from two double-blind randomized multicenter studies on the treatment of primary and first recurrent cases of CDI to examine for effects of age and study drug on outcomes of cure (resolution of diarrhea), recurrence within 4 weeks of completing successful therapy, and cure without recurrence. SETTING: Participants were randomized into studies in the United States, Canada, and Europe. PARTICIPANTS: Nine hundred ninety-nine individuals with toxin-positive CDI were randomized to receive vancomycin (125 mg 4 times daily) or fidaxomicin (200 mg twice daily) for 10 days. MEASUREMENTS: The effect of advancing age in those aged 18 to 40 years and in 10-year increments thereafter was examined. RESULTS: The model predicts a 17% lower clinical cure, 17% greater recurrence, and 13% lower sustained clinical response by advancing decade than in those younger than 40 (P < .01 each). Clinical cure was similar in the fidaxomicin and vancomycin treatment groups, although fidaxomicin was associated with a more than 50% lower relative risk for recurrence than vancomycin (P < .001). Multivariate regression modeling showed that risk factors accounting for poorer outcomes with advancing age include infection with the BI strain type, inpatient status, renal insufficiency, leukocytosis, hypoalbuminemia, and concomitant medication exposure. CONCLUSION: Measurable and progressive deterioration in CDI treatment outcomes occurred with advancing age in those aged 40 and older, highlighting the need for prevention and treatment strategies. Fidaxomicin treatment was associated with a 60% lower risk of recurrence than vancomycin after adjusting for age, concomitant antibiotics, and C. difficile strain.

Price J, Gordon NC, Crook D, Llewelyn M, Paul J. 2013. The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections. Clin Microbiol Infect, 19 (9), pp. 784-789. | Show Abstract | Read more

Staphylococcus aureus remains a leading cause of hospital-acquired and community-associated infection worldwide. The burden of disease is exacerbated by the emergence of virulent strains with reduced susceptibility to commonly used antibiotics and their dissemination in healthcare settings and in the community. Whole genome sequencing (WGS) has the potential to revolutionize our understanding and management of S. aureus infection. As a research tool, WGS has provided insights into the origins of antibiotic-resistant strains, the genetic basis of virulence, the emergence and spread of lineages, and the population structure of S. aureus. As a frontline tool, WGS offers the prospect of a method that could be used to predict resistance, assess virulence, and type isolates at the highest possible resolution. The results generated could be used to guide clinical management and infection control practice. Studies using bench-top sequencing machines have already demonstrated the feasibility of such approaches. Infection control management is compromised by our incomplete understanding of transmission, which in turn reflects the suboptimal resolution offered by conventional typing methods. As the costs of sequencing begin to approach those of conventional methods, high-resolution typing with WGS could realistically be implemented for hospital infection control, as well as for local and national surveillance practice. Translation into routine practice will require the development of a knowledge base, reliable automated bioinformatic tools, the capacity to store, exchange and interrogate large volumes of genomic data, and an acceptance of WGS by clinicians, infection control specialists, and laboratory staff.

Eyre DW, Griffiths D, Vaughan A, Golubchik T, Acharya M, O'Connor L, Crook DW, Walker AS, Peto TE. 2013. Asymptomatic Clostridium difficile colonisation and onward transmission. PLoS One, 8 (11), pp. e78445. | Show Abstract | Read more

INTRODUCTION: Combined genotyping/whole genome sequencing and epidemiological data suggest that in endemic settings only a minority of Clostridium difficile infection, CDI, is acquired from other cases. Asymptomatic patients are a potential source for many unexplained cases. METHODS: We prospectively screened a cohort of medical inpatients in a UK teaching hospital for asymptomatic C. difficile carriage using stool culture. Electronic and questionnaire data were used to determine risk factors for asymptomatic carriage by logistic regression. Carriage isolates were compared with all hospital/community CDI cases from the same geographic region, from 12 months before the study to 3 months after, using whole genome sequencing and hospital admission data, assessing particularly for evidence of onward transmission from asymptomatic cases. RESULTS: Of 227 participants recruited, 132 provided ≥1 stool samples for testing. 18 participants were culture-positive for C. difficile, 14/132(11%) on their first sample. Independent risk factors for asymptomatic carriage were patient reported loose/frequent stool (but not meeting CDI criteria of ≥3 unformed stools in 24 hours), previous overnight hospital stay within 6 months, and steroid/immunosuppressant medication in the last 6 months (all p≤0.02). Surprisingly antibiotic exposure in the last 6 months was independently associated with decreased risk of carriage (p = 0.005). The same risk factors were identified excluding participants reporting frequent/loose stool. 13/18(72%) asymptomatically colonised patients carried toxigenic strains from common disease-causing lineages found in cases. Several plausible transmission events to asymptomatic carriers were identified, but in this relatively small study no clear evidence of onward transmission from an asymptomatic case was seen. CONCLUSIONS: Transmission events from any one asymptomatic carrier are likely to be relatively rare, but as asymptomatic carriage is common, it may still be an important source of CDI, which could be quantified in larger studies. Risk factors established for asymptomatic carriage may help identify patients for inclusion in such studies.

Didelot X, Eyre DW, Cule M, Ip CL, Ansari MA, Griffiths D, Vaughan A, O'Connor L et al. 2012. Microevolutionary analysis of Clostridium difficile genomes to investigate transmission. Genome Biol, 13 (12), pp. R118. | Show Abstract | Read more

BACKGROUND: The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing. RESULTS: Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions. CONCLUSIONS: Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.

Dingle KE, Didelot X, Ansari MA, Eyre DW, Vaughan A, Griffiths D, Ip CL, Batty EM et al. 2013. Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing. J Infect Dis, 207 (4), pp. 675-686. | Show Abstract | Read more

BACKGROUND: Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30%. The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster. Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens. METHODS: Whole-genome sequences were determined for 57 C. difficile isolates representative of the population structure and different clinical phenotypes. Phylogenetic analyses were performed on their genomic region (>63 kb) spanning the cwp cluster. RESULTS: Genetic diversity across the cwp cluster peaked within slpA, cwp66 (adhesin), and secA2 (secretory translocase). These genes formed a 10-kb cassette, of which 12 divergent variants were found. Homologous recombination involving this cassette caused it to associate randomly with genotype. One cassette contained a novel insertion (length, approximately 24 kb) that resembled S-layer glycosylation gene clusters. CONCLUSIONS: Genetic exchange of S-layer cassettes parallels polysaccharide capsular switching in other species. Both cause major antigenic shifts, while the remainder of the genome is unchanged. C. difficile genotype is therefore not predictive of antigenic type. S-layer switching and immune escape could help explain temporal and geographic variation in C. difficile epidemiology and may inform genotyping and vaccination strategies.

Price JR, Didelot X, Crook DW, Llewelyn MJ, Paul J. 2013. Whole genome sequencing in the prevention and control of Staphylococcus aureus infection. J Hosp Infect, 83 (1), pp. 14-21. | Show Abstract | Read more

BACKGROUND: Staphylococcus aureus remains a leading cause of hospital-acquired infection but weaknesses inherent in currently available typing methods impede effective infection prevention and control. The high resolution offered by whole genome sequencing has the potential to revolutionise our understanding and management of S. aureus infection. AIM: To outline the practicalities of whole genome sequencing and discuss how it might shape future infection control practice. METHODS: We review conventional typing methods and compare these with the potential offered by whole genome sequencing. FINDINGS: In contrast with conventional methods, whole genome sequencing discriminates down to single nucleotide differences and allows accurate characterisation of transmission events and outbreaks and additionally provides information about the genetic basis of phenotypic characteristics, including antibiotic susceptibility and virulence. However, translating its potential into routine practice will depend on affordability, acceptable turnaround times and on creating a reliable standardised bioinformatic infrastructure. CONCLUSION: Whole genome sequencing has the potential to provide a universal test that facilitates outbreak investigation, enables the detection of emerging strains and predicts their clinical importance.

Walker TM, Ip CL, Harrell RH, Evans JT, Kapatai G, Dedicoat MJ, Eyre DW, Wilson DJ et al. 2013. Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study. Lancet Infect Dis, 13 (2), pp. 137-146. | Show Abstract | Read more

BACKGROUND: Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. METHODS: In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit-variable-number tandem-repeat data. FINDINGS: We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis. The estimated rate of change in DNA sequences was 0.5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0.3-0.7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0.0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters. INTERPRETATION: Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts. FUNDING: Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.

Didelot X, Bowden R, Wilson DJ, Peto TE, Crook DW. 2012. Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet, 13 (9), pp. 601-612. | Show Abstract | Read more

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.

Schlackow I, Walker AS, Dingle K, Griffiths D, Oakley S, Finney J, Vaughan A, Gill MJ, Crook DW, Peto TE, Wyllie DH. 2012. Surveillance of infection severity: a registry study of laboratory diagnosed Clostridium difficile. PLoS Med, 9 (7), pp. e1001279. | Show Abstract | Read more

BACKGROUND: Changing clinical impact, as virulent clones replace less virulent ones, is a feature of many pathogenic bacterial species and can be difficult to detect. Consequently, innovative techniques monitoring infection severity are of potential clinical value. METHODS AND FINDINGS: We studied 5,551 toxin-positive and 20,098 persistently toxin-negative patients tested for Clostridium difficile infection between February 1998 and July 2009 in a group of hospitals based in Oxford, UK, and investigated 28-day mortality and biomarkers of inflammation (blood neutrophil count, urea, and creatinine concentrations) collected at diagnosis using iterative sequential regression (ISR), a novel joinpoint-based regression technique suitable for serial monitoring of continuous or dichotomous outcomes. Among C. difficile toxin-positive patients in the Oxford hospitals, mean neutrophil counts on diagnosis increased from 2003, peaked in 2006-2007, and then declined; 28-day mortality increased from early 2006, peaked in late 2006-2007, and then declined. Molecular typing confirmed these changes were likely due to the ingress of the globally distributed severe C. difficile strain, ST1. We assessed the generalizability of ISR-based severity monitoring in three ways. First, we assessed and found strong (p<0.0001) associations between isolation of the ST1 severe strain and higher neutrophil counts at diagnosis in two unrelated large multi-centre studies, suggesting the technique described might be useful elsewhere. Second, we assessed and found similar trends in a second group of hospitals in Birmingham, UK, from which 5,399 cases were analysed. Third, we used simulation to assess the performance of this surveillance system given the ingress of future severe strains under a variety of assumptions. ISR-based severity monitoring allowed the detection of the severity change years earlier than mortality monitoring. CONCLUSIONS: Automated electronic systems providing early warning of the changing severity of infectious conditions can be established using routinely collected laboratory hospital data. In the settings studied here these systems have higher performance than those monitoring mortality, at least in C. difficile infection. Such systems could have wider applicability for monitoring infections presenting in hospital.

Darton T, Jones C, Waddington C, Dougan G, Sztein M, Levine M, Angus B, Farrar J et al. 2012. Demonstration of primary and asymptomatic DNAaemia in participants challenged with Salmonella Typhi (Quailes strain) during the development of a human model of typhoid infection INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E215-E215. | Read more

Crook D, Cornely O, Esposito R, Poirier A, Somero M, Weiss K, Tillotson G. 2012. Clostridium difficile in 7 European countries and North America: Fidaxomicin vs vancomycin therapy INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E242-E242. | Read more

Jones C, Waddington C, Darton T, Bowman J, Farrar J, Dougan G, Levine M, Lockhart S et al. 2012. Quantification of antibody secreting cell responses in a human challenge model of Salmonella Typhi infection INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E224-E224. | Read more

Waddington C, Darton T, Jones C, Haworth K, Peters A, Kerridge S, Crook D, Lockhart S et al. 2012. Variations in attack rate in a single-blind, dose escalation challenge study of Salmonella Typhi in healthy adult volunteers INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E244-E244. | Read more

Schlackow I, Stoesser N, Walker AS, Crook DW, Peto TE, Wyllie DH, Infections in Oxfordshire Research Database Team. 2012. Increasing incidence of Escherichia coli bacteraemia is driven by an increase in antibiotic-resistant isolates: electronic database study in Oxfordshire 1999-2011. J Antimicrob Chemother, 67 (6), pp. 1514-1524. | Show Abstract | Read more

OBJECTIVES: To investigate trends in Escherichia coli resistance, bacteraemia rates and post-bacteraemia outcomes over time. METHODS: Trends in E. coli bacteraemia incidence were monitored from January 1999 to June 2011 using an infection surveillance database including microbiological, clinical risk factor, infection severity and outcome data in Oxfordshire, UK, with imported temperature/rainfall data. RESULTS: A total of 2240 E. coli (from 2080 patients) were studied, of which 1728 (77%) were susceptible to co-amoxiclav, cefotaxime, ciprofloxacin and gentamicin. E. coli bacteraemia incidence increased from 3.4/10,000 bedstays in 1999 to 5.7/10,000 bedstays in 2011. The increase was fastest around 2006, and was essentially confined to organisms resistant to ciprofloxacin, co-amoxiclav, cefotaxime and/or aminoglycosides. Resistant E. coli isolation rates increased similarly in those with and without recent hospital contact. The sharp increase also occurred in urinary isolates, with similar timing. In addition to these long-term trends, increases in ambient temperature, but not rainfall, were associated with increased E. coli bacteraemia rates. It is unclear whether resistant E. coli bacteraemia rates are currently still increasing [incidence rate ratio = 1.07 per annum (95% CI = 0.99-1.16), P = 0.07], whereas current susceptible E. coli bacteraemia rates are not changing significantly [incidence rate ratio = 1.01 (95% CI = 0.99-1.02)]. However, neither mortality nor biomarkers associated with mortality (blood creatinine, urea/albumin concentrations, neutrophil counts) changed during the study. CONCLUSIONS: E. coli bacteraemia rates have risen due to rising rates of resistant organisms; little change occurred in susceptible E. coli. Although the severity of resistant infections, and their outcome, appear similar to susceptible E. coli in the setting studied, the increasing burden of highly resistant organisms is alarming and merits on-going surveillance.

Golubchik T, Brueggemann AB, Street T, Gertz RE, Spencer CC, Ho T, Giannoulatou E, Link-Gelles R et al. 2012. Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event. Nat Genet, 44 (3), pp. 352-355. | Show Abstract | Read more

Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age(1). The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States(2,3) provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination(4), showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases.

Walker AS, Eyre DW, Wyllie DH, Dingle KE, Harding RM, O'Connor L, Griffiths D, Vaughan A et al. 2012. Characterisation of Clostridium difficile hospital ward-based transmission using extensive epidemiological data and molecular typing. PLoS Med, 9 (2), pp. e1001172. | Show Abstract | Read more

BACKGROUND: Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals, hindering the identification of sources and routes of transmission based on shared time and space alone. This may compromise rational control despite costly prevention strategies. This study aimed to investigate ward-based transmission of C. difficile, by subdividing outbreaks into distinct lineages defined by multi-locus sequence typing (MLST). METHODS AND FINDINGS: All C. difficile toxin enzyme-immunoassay-positive and culture-positive samples over 2.5 y from a geographically defined population of ~600,000 persons underwent MLST. Sequence types (STs) were combined with admission and ward movement data from an integrated comprehensive healthcare system incorporating three hospitals (1,700 beds) providing all acute care for the defined geographical population. Networks of cases and potential transmission events were constructed for each ST. Potential infection sources for each case and transmission timescales were defined by prior ward-based contact with other cases sharing the same ST. From 1 September 2007 to 31 March 2010, there were means of 102 tests and 9.4 CDIs per 10,000 overnight stays in inpatients, and 238 tests and 15.7 CDIs per month in outpatients/primary care. In total, 1,276 C. difficile isolates of 69 STs were studied. From MLST, no more than 25% of cases could be linked to a potential ward-based inpatient source, ranging from 37% in renal/transplant, 29% in haematology/oncology, and 28% in acute/elderly medicine to 6% in specialist surgery. Most of the putative transmissions identified occurred shortly (≤ 1 wk) after the onset of symptoms (141/218, 65%), with few >8 wk (21/218, 10%). Most incubation periods were ≤ 4 wk (132/218, 61%), with few >12 wk (28/218, 13%). Allowing for persistent ward contamination following ward discharge of a CDI case did not increase the proportion of linked cases after allowing for random meeting of matched controls. CONCLUSIONS: In an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic enzyme-immunoassay-positive patients cannot account for most new CDI cases.

Cited:

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Didelot X, Bowden R, Wilson DJ, Peto TEA, Crook DW. 2012. Transforming clinical microbiology with bacterial genome sequencing Nature Reviews Genetics, 13 (9), pp. 601-612. | Show Abstract | Read more

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow. © 2012 Macmillan Publishers Limited. All rights reserved.

Cornely OA, Miller MA, Louie TJ, Crook DW, Gorbach SL. 2012. Treatment of first recurrence of Clostridium difficile infection: fidaxomicin versus vancomycin. Clin Infect Dis, 55 Suppl 2 (suppl 2), pp. S154-S161. | Show Abstract | Read more

Recurrence of Clostridium difficile infection (CDI) occurs in approximately 25% of successfully treated patients. Two phase 3 randomized, double-blind trials were conducted at 154 sites in the United States, Canada, and Europe to compare fidaxomicin vs vancomycin in treating CDI. Patients with CDI received fidaxomicin 200 mg twice daily or vancomycin 125 mg 4 times daily for 10 days. The primary end point was clinical cure of CDI at end of treatment, and a secondary end point was recurrence during the 28 days following clinical cure. In all, 1164 subjects were enrolled, of which a subgroup of 128 in the per-protocol population had another recent episode of CDI prior to the CDI diagnosis at study enrollment. In the analysis of this subgroup, initial response to therapy was similar for both drugs (>90% cure). However, recurrence within 28 days occurred in 35.5% of patients treated with vancomycin and 19.7% of patients treated with fidaxomicin (-15.8% difference; 95% confidence interval, -30.4% to -0.3%; P = .045). Early recurrence (within 14 days) was reported in 27% of patients treated with vancomycin and 8% of patients treated with fidaxomicin (P = .003). In patients with a first recurrence of CDI, fidaxomicin was similar to vancomycin in achieving a clinical response at end of therapy but superior in preventing a second recurrence within 28 days.

Eyre DW, Walker AS, Wyllie D, Dingle KE, Griffiths D, Finney J, O'Connor L, Vaughan A et al. 2012. Predictors of first recurrence of Clostridium difficile infection: implications for initial management. Clin Infect Dis, 55 Suppl 2 (suppl 2), pp. S77-S87. | Show Abstract | Read more

Symptomatic recurrence of Clostridium difficile infection (CDI) occurs in approximately 20% of patients and is challenging to treat. Identifying those at high risk could allow targeted initial management and improve outcomes. Adult toxin enzyme immunoassay-positive CDI cases in a population of approximately 600,000 persons from September 2006 through December 2010 were combined with epidemiological/clinical data. The cumulative incidence of recurrence ≥ 14 days after the diagnosis and/or onset of first-ever CDI was estimated, treating death without recurrence as a competing risk, and predictors were identified from cause-specific proportional hazards regression models. A total of 1678 adults alive 14 days after their first CDI were included; median age was 77 years, and 1191 (78%) were inpatients. Of these, 363 (22%) experienced a recurrence ≥ 14 days after their first CDI, and 594 (35%) died without recurrence through March 2011. Recurrence risk was independently and significantly higher among patients admitted as emergencies, with previous gastrointestinal ward admission(s), last discharged 4-12 weeks before first diagnosis, and with CDI diagnosed at admission. Recurrence risk also increased with increasing age, previous total hours admitted, and C-reactive protein level at first CDI (all P < .05). The 4-month recurrence risk increased by approximately 5% (absolute) for every 1-point increase in a risk score based on these factors. Risk factors, including increasing age, initial disease severity, and hospital exposure, predict CDI recurrence and identify patients likely to benefit from enhanced initial CDI treatment.

Sears P, Crook DW, Louie TJ, Miller MA, Weiss K. 2012. Fidaxomicin attains high fecal concentrations with minimal plasma concentrations following oral administration in patients with Clostridium difficile infection. Clin Infect Dis, 55 Suppl 2 (suppl 2), pp. S116-S120. | Show Abstract | Read more

Fidaxomicin has recently been approved for the treatment of Clostridium difficile infection (CDI). As part of phase III studies, plasma and fecal samples were analyzed for concentrations of fidaxomicin and its metabolite, OP-1118. Plasma samples were collected before and after dose receipt on the first and last days of therapy, and fecal samples were collected on the last day of therapy. Samples were analyzed for fidaxomicin and OP-1118 (metabolite), using validated liquid chromatography/tandem mass spectrometric methods. Plasma concentrations were low for both fidaxomicin (mean [± standard deviation {SD}], 22.8 ± 26.7 ng/mL and 28.5 ± 33.4 ng/mL on the first and last days of therapy, respectively) and OP-1118 (mean [± SD], 44.5 ± 50.4 ng/mL and 85.6 ± 131 ng/mL, respectively). In contrast, fecal levels were >1000 µg/g for fidaxomicin and >800 µg/g for OP-1118. Fidaxomicin mean fecal levels were >5000 times the minimum inhibitory concentration for C. difficile of 0.25 µg/mL.

Crook DW, Walker AS, Kean Y, Weiss K, Cornely OA, Miller MA, Esposito R, Louie TJ et al. 2012. Fidaxomicin versus vancomycin for Clostridium difficile infection: meta-analysis of pivotal randomized controlled trials. Clin Infect Dis, 55 Suppl 2 (suppl 2), pp. S93-103. | Show Abstract | Read more

Two recently completed phase 3 trials (003 and 004) showed fidaxomicin to be noninferior to vancomycin for curing Clostridium difficile infection (CDI) and superior for reducing CDI recurrences. In both studies, adults with active CDI were randomized to receive blinded fidaxomicin 200 mg twice daily or vancomycin 125 mg 4 times a day for 10 days. Post hoc exploratory intent-to-treat (ITT) time-to-event analyses were undertaken on the combined study 003 and 004 data, using fixed-effects meta-analysis and Cox regression models. ITT analysis of the combined 003/004 data for 1164 patients showed that fidaxomicin reduced persistent diarrhea, recurrence, or death by 40% (95% confidence interval [CI], 26%-51%; P < .0001) compared with vancomycin through day 40. A 37% (95% CI, 2%-60%; P = .037) reduction in persistent diarrhea or death was evident through day 12 (heterogeneity P = .50 vs 13-40 days), driven by 7 (1.2%) fidaxomicin versus 17 (2.9%) vancomycin deaths at <12 days. Low albumin level, low eosinophil count, and CDI treatment preenrollment were risk factors for persistent diarrhea or death at 12 days, and CDI in the previous 3 months was a risk factor for recurrence (all P < .01). Fidaxomicin has the potential to substantially improve outcomes from CDI.

Juhas M, Stark M, von Mering C, Lumjiaktase P, Crook DW, Valvano MA, Eberl L. 2012. High confidence prediction of essential genes in Burkholderia cenocepacia. PLoS One, 7 (6), pp. e40064. | Show Abstract | Read more

BACKGROUND: Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia. CONCLUSIONS/SIGNIFICANCE: We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life.

Young BC, Golubchik T, Batty EM, Fung R, Larner-Svensson H, Votintseva AA, Miller RR, Godwin H et al. 2012. Evolutionary dynamics of Staphylococcus aureus during progression from carriage to disease. Proc Natl Acad Sci U S A, 109 (12), pp. 4550-4555. | Show Abstract | Read more

Whole-genome sequencing offers new insights into the evolution of bacterial pathogens and the etiology of bacterial disease. Staphylococcus aureus is a major cause of bacteria-associated mortality and invasive disease and is carried asymptomatically by 27% of adults. Eighty percent of bacteremias match the carried strain. However, the role of evolutionary change in the pathogen during the progression from carriage to disease is incompletely understood. Here we use high-throughput genome sequencing to discover the genetic changes that accompany the transition from nasal carriage to fatal bloodstream infection in an individual colonized with methicillin-sensitive S. aureus. We found a single, cohesive population exhibiting a repertoire of 30 single-nucleotide polymorphisms and four insertion/deletion variants. Mutations accumulated at a steady rate over a 13-mo period, except for a cluster of mutations preceding the transition to disease. Although bloodstream bacteria differed by just eight mutations from the original nasally carried bacteria, half of those mutations caused truncation of proteins, including a premature stop codon in an AraC-family transcriptional regulator that has been implicated in pathogenicity. Comparison with evolution in two asymptomatic carriers supported the conclusion that clusters of protein-truncating mutations are highly unusual. Our results demonstrate that bacterial diversity in vivo is limited but nonetheless detectable by whole-genome sequencing, enabling the study of evolutionary dynamics within the host. Regulatory or structural changes that occur during carriage may be functionally important for pathogenesis; therefore identifying those changes is a crucial step in understanding the biological causes of invasive bacterial disease.

Eyre DW, Golubchik T, Gordon NC, Bowden R, Piazza P, Batty EM, Ip CL, Wilson DJ et al. 2012. A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance. BMJ Open, 2 (3), pp. e001124-e001124. | Show Abstract | Read more

OBJECTIVES: To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management. DESIGN: The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. SETTING: Isolates were obtained from potential outbreaks associated with three UK hospitals. PARTICIPANTS: Isolates were sequenced from a cluster of eight MRSA carriers and an associated bacteraemia case in an intensive care unit, another MRSA cluster of six cases and two clusters of C difficile. Additionally, all C difficile isolates from cases over 6 weeks in a single hospital were rapidly sequenced and compared with local strain sequences obtained in the preceding 3 years. MAIN OUTCOME MEASURE: Whole-genome genetic relatedness of the isolates within each epidemiological cluster. RESULTS: Twenty-six MRSA and 15 C difficile isolates were successfully sequenced and analysed within 5 days of culture. Both MRSA clusters were identified as outbreaks, with most sequences in each cluster indistinguishable and all within three single nucleotide variants (SNVs). Epidemiologically unrelated isolates of the same spa-type were genetically distinct (≥21 SNVs). In both C difficile clusters, closely epidemiologically linked cases (in one case sharing the same strain type) were shown to be genetically distinct (≥144 SNVs). A reconstruction applying rapid sequencing in C difficile surveillance provided early outbreak detection and identified previously undetected probable community transmission. CONCLUSIONS: This benchtop sequencing technology is widely generalisable to human bacterial pathogens. The findings provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection and therefore improve hospital infection control and patient outcomes in routine clinical practice.

Saha SK, Al Emran HM, Hossain B, Darmstadt GL, Saha S, Islam M, Chowdhury AI, Foster D et al. 2012. Streptococcus pneumoniae serotype-2 childhood meningitis in Bangladesh: a newly recognized pneumococcal infection threat. PLoS One, 7 (3), pp. e32134. | Show Abstract | Read more

BACKGROUND: Streptococcus pneumoniae is a leading cause of meningitis in countries where pneumococcal conjugate vaccines (PCV) targeting commonly occurring serotypes are not routinely used. However, effectiveness of PCV would be jeopardized by emergence of invasive pneumococcal diseases (IPD) caused by serotypes which are not included in PCV. Systematic hospital based surveillance in Bangladesh was established and progressively improved to determine the pathogens causing childhood sepsis and meningitis. This also provided the foundation for determining the spectrum of serotypes causing IPD. This article reports an unprecedented upsurge of serotype 2, an uncommon pneumococcal serotype, without any known intervention. METHODS AND FINDINGS: Cases with suspected IPD had blood or cerebrospinal fluid (CSF) collected from the beginning of 2001 till 2009. Pneumococcal serotypes were determined by capsular swelling of isolates or PCR of culture-negative CSF specimens. Multicenter national surveillance, expanded from 2004, identified 45,437 patients with suspected bacteremia who were blood cultured and 10,618 suspected meningitis cases who had a lumber puncture. Pneumococcus accounted for 230 culture positive cases of meningitis in children <5 years. Serotype-2 was the leading cause of pneumococcal meningitis, accounting for 20.4% (45/221; 95% CI 15%-26%) of cases. Ninety eight percent (45/46) of these serotype-2 strains were isolated from meningitis cases, yielding the highest serotype-specific odds ratio for meningitis (29.6; 95% CI 3.4-256.3). The serotype-2 strains had three closely related pulsed field gel electrophoresis types. CONCLUSIONS: S. pneumoniae serotype-2 was found to possess an unusually high potential for causing meningitis and was the leading serotype-specific cause of childhood meningitis in Bangladesh over the past decade. Persisting disease occurrence or progressive spread would represent a major potential infection threat since serotype-2 is not included in PCVs currently licensed or under development.

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Golubchik T, Brueggemann AB, Street T, Gertz RE, Spencer CCA, Ho T, Giannoulatou E, Link-Gelles R et al. 2012. Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event Nature Genetics, 44 (3), pp. 352-355. | Show Abstract | Read more

Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age. The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination, showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases. © 2012 Nature America, Inc. All rights reserved.

Cornely OA, Crook DW, Esposito R, Poirier A, Somero MS, Weiss K, Sears P, Gorbach S, OPT-80-004 Clinical Study Group. 2012. Fidaxomicin versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis, 12 (4), pp. 281-289. | Show Abstract | Read more

BACKGROUND: Infection with Clostridium difficile is the primary infective cause of antibiotic-associated diarrhoea. We aimed to compare efficacy and safety of fidaxomicin and vancomycin to treat patients with C difficile infection in Europe, Canada, and the USA. METHODS: In this multicentre, double-blind, randomised, non-inferiority trial, we enrolled patients from 45 sites in Europe and 41 sites in the USA and Canada between April 19, 2007, and Dec 11, 2009. Eligible patients were aged 16 years or older with acute, toxin-positive C difficile infection. Patients were randomly allocated (1:1) to receive oral fidaxomicin (200 mg every 12 h) or oral vancomycin (125 mg every 6 h) for 10 days. The primary endpoint was clinical cure, defined as resolution of diarrhoea and no further need for treatment. An interactive voice-response system and computer-generated randomisation schedule gave a randomisation number and medication kit number for each patient. Participants and investigators were masked to treatment allocation. Non-inferiority was prespecified with a margin of 10%. Modified intention-to-treat and per-protocol populations were analysed. This study is registered with ClinicalTrials.gov, number NCT00468728. FINDINGS: Of 535 patients enrolled, 270 were assigned fidaxomicin and 265 vancomycin. After 26 patients were excluded, 509 were included in the modified intention-to-treat (mITT) population. 198 (91·7%) of 216 patients in the per-protocol population given fidaxomicin achieved clinical cure, compared with 213 (90·6%) of 235 given vancomycin, meeting the criterion for non-inferiority (one-sided 97·5% CI -4·3%). Non-inferiority was also shown for clinical cure in the mITT population, with 221 (87·7%) of 252 patients given fidaxomicin and 223 (86·8%) of 257 given vancomycin cured (one-sided 97·5% CI -4·9%). In most subgroup analyses of the primary endpoint in the mITT population, outcomes in the two treatment groups did not differ significantly; although patients receiving concomitant antibiotics for other infections had a higher cure rate with fidaxomicin (46 [90·2%] of 51) than with vancomycin (33 [73·3%] of 45; p=0·031). Occurrence of treatment-emergent adverse events did not differ between groups. 20 (7·6%) of 264 patients given at least one dose of fidaxomicin and 17 (6·5%) of 260 given vancomycin died. INTERPRETATION: Fidaxomicin could be an alternative treatment for infection with C difficile, with similar efficacy and safety to vancomycin. FUNDING: Optimer Pharmaceuticals.

Eyre DW, Walker AS, Griffiths D, Wilcox MH, Wyllie DH, Dingle KE, Crook DW, Peto TE. 2012. Clostridium difficile mixed infection and reinfection. J Clin Microbiol, 50 (1), pp. 142-144. | Show Abstract | Read more

Isolates from consecutive Clostridium difficile infection (CDI) fecal samples underwent multilocus sequence typing. Potential reinfections with different genotypes were identified in 88/560 (16%) sample pairs taken 1 to 1,414 days (median, 24; interquartile range [IQR], 1 to 52 days) apart; odds of reinfection increased by 58% for every doubling of time between samples. Of 109 sample pairs taken on the same day, 3 (3%) had different genotypes. Considering samples 0 to 7 days apart as the same CDI, 7% of cases had mixed infections with >1 genotype.

Stoesser N, Crook DW, Moore CE, Phetsouvanh R, Chansamouth V, Newton PN, Jones N. 2012. Characteristics of CTX-M ESBL-producing Escherichia coli isolates from the Lao People's Democratic Republic, 2004-09. J Antimicrob Chemother, 67 (1), pp. 240-242. | Read more

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Ibarz-Pavon AB, MacLennan J, Andrews NJ, Gray SJ, Urwin R, Clarke SC, Walker AM, Evans MR et al. 2011. Changes in Serogroup and Genotype Prevalence Among Carried Meningococci in the United Kingdom During Vaccine Implementation JOURNAL OF INFECTIOUS DISEASES, 204 (7), pp. 1046-1053. | Show Abstract | Read more

Background. Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population. Methods. Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed. Results. A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear. Conclusions. Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction. © The Author 2011.

Ibarz-Pavón AB, Maclennan J, Andrews NJ, Gray SJ, Urwin R, Clarke SC, Walker AM, Evans MR et al. 2011. Changes in serogroup and genotype prevalence among carried meningococci in the United Kingdom during vaccine implementation. J Infect Dis, 204 (7), pp. 1046-1053. | Show Abstract | Read more

BACKGROUND: Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population. METHODS: Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed. RESULTS: A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear. CONCLUSIONS: Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction.

Wyllie D, Paul J, Crook D. 2011. Waves of trouble: MRSA strain dynamics and assessment of the impact of infection control. J Antimicrob Chemother, 66 (12), pp. 2685-2688. | Show Abstract | Read more

There has been a sustained decline in bloodstream infections due to methicillin-resistant Staphylococcus aureus (MRSA) throughout the UK. The UK MRSA epidemic, which began in the 1990s, has been dominated by two epidemic MRSA (EMRSA) clones {EMRSA-15, of clonal complex (CC) 22 [sequence type (ST) 22], and EMRSA-16, of CC30 (ST36)}. It appears that both these clones followed a wave trajectory (initial expansion, relative stasis, then decline). Three recent studies have shown that ST36 has declined faster than ST22, a change that appears to have begun before the recent intensification of intensive control measures in the UK. The biological basis of infectious disease waves, including those of MRSA, is discussed, as are the implications of such waves for the assessment of the impact of infection control measures.

Stoesser N, Crook DW, Fung R, Griffiths D, Harding RM, Kachrimanidou M, Keshav S, Peto TE, Vaughan A, Walker AS, Dingle KE. 2011. Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection. J Clin Microbiol, 49 (11), pp. 3994-3996. | Show Abstract | Read more

Molecular analysis of Clostridium difficile (28 isolates) from children (n = 128) in Oxfordshire, United Kingdom, identified eight toxigenic genotypes. Six of these were isolated from 27% of concurrent adult C. difficile-associated infections studied (n = 83). No children carried hypervirulent PCR ribotype 027. Children could participate in the transmission of some adult disease-causing genotypes.

Wyllie DH, Walker AS, Miller R, Moore C, Williamson SR, Schlackow I, Finney JM, O'Connor L, Peto TE, Crook DW. 2011. Decline of meticillin-resistant Staphylococcus aureus in Oxfordshire hospitals is strain-specific and preceded infection-control intensification. BMJ Open, 1 (1), pp. e000160. | Show Abstract | Read more

Background In the past, strains of Staphylococcus aureus have evolved, expanded, made a marked clinical impact and then disappeared over several years. Faced with rising meticillin-resistant S aureus (MRSA) rates, UK government-supported infection control interventions were rolled out in Oxford Radcliffe Hospitals NHS Trust from 2006 onwards. Methods Using an electronic Database, the authors identified isolation of MRS among 611 434 hospital inpatients admitted to acute hospitals in Oxford, UK, 1 April 1998 to 30 June 2010. Isolation rates were modelled using segmented negative binomial regression for three groups of isolates: from blood cultures, from samples suggesting invasion (eg, cerebrospinal fluid, joint fluid, pus samples) and from surface swabs (eg, from wounds). Findings MRSA isolation rates rose rapidly from 1998 to the end of 2003 (annual increase from blood cultures 23%, 95% CI 16% to 30%), and then declined. The decline accelerated from mid-2006 onwards (annual decrease post-2006 38% from blood cultures, 95% CI 29% to 45%, p=0.003 vs previous decline). Rates of meticillin-sensitive S aureus changed little by comparison, with no evidence for declines 2006 onward (p=0.40); by 2010, sensitive S aureus was far more common than MRSA (blood cultures: 2.9 vs 0.25; invasive samples 14.7 vs 2.0 per 10 000 bedstays). Interestingly, trends in isolation of erythromycin-sensitive and resistant MRSA differed. Erythromycin-sensitive strains rose significantly faster (eg, from blood cultures p=0.002), and declined significantly more slowly (p=0.002), than erythromycin-resistant strains (global p<0.0001). Bacterial typing suggests this reflects differential spread of two major UK MRSA strains (ST22/36), ST36 having declined markedly 2006-2010, with ST22 becoming the dominant MRSA strain. Conclusions MRSA isolation rates were falling before recent intensification of infection-control measures. This, together with strain-specific changes in MRSA isolation, strongly suggests that incompletely understood biological factors are responsible for the much recent variation in MRSA isolation. A major, mainly meticillin-sensitive, S aureus burden remains.

Dingle KE, Griffiths D, Didelot X, Evans J, Vaughan A, Kachrimanidou M, Stoesser N, Jolley KA et al. 2011. Clinical Clostridium difficile: clonality and pathogenicity locus diversity. PLoS One, 6 (5), pp. e19993. | Show Abstract | Read more

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings. The major virulence determinants are large clostridial toxins, toxin A (tcdA) and toxin B (tcdB), encoded within the pathogenicity locus (PaLoc). Isolates vary in pathogenicity from hypervirulent PCR-ribotypes 027 and 078 with high mortality, to benign non-toxigenic strains carried asymptomatically. The relative pathogenicity of most toxigenic genotypes is still unclear, but may be influenced by PaLoc genetic variant. This is the largest study of C. difficile molecular epidemiology performed to date, in which a representative collection of recent isolates (n = 1290) from patients with CDI in Oxfordshire, UK, was genotyped by multilocus sequence typing. The population structure was described using NeighborNet and ClonalFrame. Sequence variation within toxin B (tcdB) and its negative regulator (tcdC), was mapped onto the population structure. The 69 Sequence Types (ST) showed evidence for homologous recombination with an effect on genetic diversification four times lower than mutation. Five previously recognised genetic groups or clades persisted, designated 1 to 5, each having a strikingly congruent association with tcdB and tcdC variants. Hypervirulent ST-11 (078) was the only member of clade 5, which was divergent from the other four clades within the MLST loci. However, it was closely related to the other clades within the tcdB and tcdC loci. ST-11 (078) may represent a divergent formerly non-toxigenic strain that acquired the PaLoc (at least) by genetic recombination. This study focused on human clinical isolates collected from a single geographic location, to achieve a uniquely high density of sampling. It sets a baseline of MLST data for future comparative studies investigating genotype virulence potential (using clinical severity data for these isolates), possible reservoirs of human CDI, and the evolutionary origins of hypervirulent strains.

Chapman SJ, Khor CC, Vannberg FO, Rautanen A, Walley A, Segal S, Moore CE, Davies RJ et al. 2010. Common NFKBIL2 polymorphisms and susceptibility to pneumococcal disease: a genetic association study. Crit Care, 14 (6), pp. R227. | Show Abstract | Read more

INTRODUCTION: Streptococcus pneumoniae remains a major global health problem and a leading cause of death in children worldwide. The factors that influence development of pneumococcal sepsis remain poorly understood, although increasing evidence points towards a role for genetic variation in the host's immune response. Recent insights from the study of animal models, rare human primary immunodeficiency states, and population-based genetic epidemiology have focused attention on the role of the proinflammatory transcription factor NF-κB in pneumococcal disease pathogenesis. The possible role of genetic variation in the atypical NF-κB inhibitor IκB-R, encoded by NFKBIL2, in susceptibility to invasive pneumococcal disease has not, to our knowledge, previously been reported upon. METHODS: An association study was performed examining the frequencies of nine common NFKBIL2 polymorphisms in two invasive pneumococcal disease case-control groups: European individuals from hospitals in Oxfordshire, UK (275 patients and 733 controls), and African individuals from Kilifi District Hospital, Kenya (687 patients with bacteraemia, of which 173 patients had pneumococcal disease, together with 550 controls). RESULTS: Five polymorphisms significantly associated with invasive pneumococcal disease susceptibility in the European study, of which two polymorphisms also associated with disease in African individuals. Heterozygosity at these loci was associated with protection from invasive pneumococcal disease (rs760477, Mantel-Haenszel 2 × 2 χ(2) = 11.797, P = 0.0006, odds ratio = 0.67, 95% confidence interval = 0.53 to 0.84; rs4925858, Mantel-Haenszel 2 × 2 χ(2) = 9.104, P = 0.003, odds ratio = 0.70, 95% confidence interval = 0.55 to 0.88). Linkage disequilibrium was more extensive in European individuals than in Kenyans. CONCLUSIONS: Common NFKBIL2 polymorphisms are associated with susceptibility to invasive pneumococcal disease in European and African populations. These findings further highlight the importance of control of NF-κB in host defence against pneumococcal disease.

Foster D, Walker AS, Paul J, Griffiths D, Knox K, Peto TE, Crook DW, Oxford Invasive Pneumococcal Surveillance Group. 2011. Reduction in invasive pneumococcal disease following implementation of the conjugate vaccine in the Oxfordshire region, England. J Med Microbiol, 60 (Pt 1), pp. 91-97. | Show Abstract | Read more

Pneumococcal conjugate vaccine to seven capsular types has been highly effective in the US since its introduction in 2000. The same vaccine was adopted by the UK in 2006. Ongoing surveillance since 1995 of invasive pneumococcal disease (IPD) in Oxfordshire, UK, allowed assessment of the impact of vaccine intervention. The vaccine significantly reduced IPD among the target group, children under 2 years of age; incidence rate ratio (IRR)=0.62 (95 % CI 0.43-0.90) (P=0.008) comparing the 3 years pre- and post-implementation with a residual incidence of 22.4/100 000 children. The reduction was even greater when comparing 11 years pre- with the 3 years post-implementation of vaccine; IRR=0.53 (0.39-0.70) (P<0.0001). There was a marked direct effect of the vaccine evidenced by substantial reductions in the seven serotypes contained in the vaccine. There was also a clear reduction in IPD for those serotypes contained in the vaccine among those older than 2 years when comparing both the 3 and 11 year pre-PCV7 time periods, with IRR=0.57 (0.47-0.69) (P<0.0001) and IRR=0.50 (0.43-0.58) (P<0.0001), respectively, indicating a strong herd effect. There was a significant, though moderate, rise in the serotypes not contained in the vaccine, with clear evidence for replacement in some serotypes.

Robinson E, Juhas M, Hood D, Crook D. 2010. Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae. J Microbiol Methods, 83 (3), pp. 330-334. | Show Abstract | Read more

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp.

Moore CE, Sengduangphachanh A, Thaojaikong T, Sirisouk J, Foster D, Phetsouvanh R, McGee L, Crook DW, Newton PN, Peacock SJ. 2010. Enhanced determination of Streptococcus pneumoniae serotypes associated with invasive disease in Laos by using a real-time polymerase chain reaction serotyping assay with cerebrospinal fluid. Am J Trop Med Hyg, 83 (3), pp. 451-457. | Show Abstract | Read more

A prospective hospital-based study was undertaken to define the incidence of invasive pneumococcal disease (IPD) and circulating serotypes in Laos. Of 10,799 patients with hemocultures and 353 patients with cerebrospinal fluid samples, 0.21% and 5.4%, respectively, were positive for Streptococcus pneumoniae, giving a total of 35 IPD patients. We developed a real-time polymerase chain reaction to detect serotypes represented in the 13-valent pneumococcal vaccine. A blinded evaluation comparing serotype as defined by the Quellung reaction versus the polymerase chain reaction demonstrated 100% concordance. The most frequent serotype (n = 33 patients) was 1 (n = 6), followed by serotypes 5, 6A/B/C, 14, and 23F. Serotypes represented in the 7-valent polysaccharide-protein conjugate vaccine (PCV-7) infected 39% of patients, with 73% coverage for the PCV-10 and PCV-13 vaccines. Although the sample size is small, these data suggest that the PCV-7 vaccine may have relatively low efficacy in Laos. Further studies are urgently needed to guide pneumococcal vaccine policy in Laos.

Miller R, Walker AS, Knox K, Wyllie D, Paul J, Haworth E, Mant D, Peto T, Crook DW. 2010. 'Feral' and 'wild'-type methicillin-resistant Staphylococcus aureus in the United Kingdom. Epidemiol Infect, 138 (5), pp. 655-665. | Show Abstract | Read more

Circulation of methicillin-resistant Staphylococcus aureus (MRSA) outside hospitals could alter the impact of hospital-based control strategies. We investigated two groups of cases (each matched to controls with MRSA): 61 'community cases' not in acute hospital in the year before MRSA isolation; and 21 cases with ciprofloxacin-sensitive (CipS) MRSA. Multi-locus sequence typing, spa-typing and Panton-Valentine leukocidin gene testing were performed and demographics obtained. Additional questionnaires were completed by community case GPs. Community cases comprised 6% of Oxfordshire MRSA. Three community cases had received no regular healthcare or antibiotics: one was infected with CipS. Ninety-one percent of community cases had healthcare-associated sequence type (ST)22/36; CipS MRSA cases had heterogeneous STs but many had recent healthcare exposure. A substantial minority of UK MRSA transmission may occur outside hospitals. Hospital strains are becoming 'feral' or persisting in long-term carriers in the community with regular healthcare contacts; those with recent healthcare exposure may nevertheless acquire non-hospital epidemic MRSA strains in the community.

Darmstadt GL, Choi Y, Arifeen SE, Bari S, Rahman SM, Mannan I, Seraji HR, Winch PJ et al. 2010. Evaluation of a cluster-randomized controlled trial of a package of community-based maternal and newborn interventions in Mirzapur, Bangladesh. PLoS One, 5 (3), pp. e9696. | Show Abstract | Read more

BACKGROUND: To evaluate a delivery strategy for newborn interventions in rural Bangladesh. METHODS: A cluster-randomized controlled trial was conducted in Mirzapur, Bangladesh. Twelve unions were randomized to intervention or comparison arm. All women of reproductive age were eligible to participate. In the intervention arm, community health workers identified pregnant women; made two antenatal home visits to promote birth and newborn care preparedness; made four postnatal home visits to negotiate preventive care practices and to assess newborns for illness; and referred sick neonates to a hospital and facilitated compliance. Primary outcome measures were antenatal and immediate newborn care behaviours, knowledge of danger signs, care seeking for neonatal complications, and neonatal mortality. FINDINGS: A total of 4616 and 5241 live births were recorded from 9987 and 11153 participants in the intervention and comparison arm, respectively. High coverage of antenatal (91% visited twice) and postnatal (69% visited on days 0 or 1) home visitations was achieved. Indicators of care practices and knowledge of maternal and neonatal danger signs improved. Adjusted mortality hazard ratio in the intervention arm, compared to the comparison arm, was 1.02 (95% CI: 0.80-1.30) at baseline and 0.87 (95% CI: 0.68-1.12) at endline. Primary causes of death were birth asphyxia (49%) and prematurity (26%). No adverse events associated with interventions were reported. CONCLUSION: Lack of evidence for mortality impact despite high program coverage and quality assurance of implementation, and improvements in targeted newborn care practices suggests the intervention did not adequately address risk factors for mortality. The level and cause-structure of neonatal mortality in the local population must be considered in developing interventions. Programs must ensure skilled care during childbirth, including management of birth asphyxia and prematurity, and curative postnatal care during the first two days of life, in addition to essential newborn care and infection prevention and management. TRIAL REGISTRATION: Clinicaltrials.gov NCT00198627.

Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW, Fung R, Golubchik T, Harding RM et al. 2010. Multilocus sequence typing of Clostridium difficile. J Clin Microbiol, 48 (3), pp. 770-778. | Show Abstract | Read more

A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.

Robinson E, Juhas M, Hood D, Crook D. 2010. Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae Journal of Microbiological Methods, 83 (3), pp. 330-334. | Show Abstract | Read more

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp. © 2010 Elsevier B.V.

Chapman SJ, Vannberg FO, Khor CC, Rautanen A, Maskell NA, Davies CW, Moore CE, Day NP, Crook DW, Davies RJ, Hill AV. 2010. Mannose-binding lectin genotypes: lack of association with susceptibility to thoracic empyema. BMC Med Genet, 11 (1), pp. 5. | Show Abstract | Read more

BACKGROUND: The role of the innate immune protein mannose-binding lectin (MBL) in host defence against severe respiratory infection remains controversial. Thoracic empyema is a suppurative lung infection that arises as a major complication of pneumonia and is associated with a significant mortality. Although the pathogenesis of thoracic empyema is poorly understood, genetic susceptibility loci for this condition have recently been identified. The possible role of MBL genotypic deficiency in susceptibility to thoracic empyema has not previously been reported. METHODS: To investigate this further we compared the frequencies of the six functional MBL polymorphisms in 170 European individuals with thoracic empyema and 225 healthy control individuals. RESULTS: No overall association was observed between MBL genotypic deficiency and susceptibility to thoracic empyema (2 x 2 Chi square = 0.02, P = 0.87). Furthermore, no association was seen between MBL deficiency and susceptibility to the Gram-positive or pneumococcal empyema subgroups. MBL genotypic deficiency did not associate with progression to death or requirement for surgery. CONCLUSIONS: Our results suggest that MBL genotypic deficiency does not associate with susceptibility to thoracic empyema in humans.

Stoesser N, Griffiths D, Fung R, Vaughan A, Crook DW, Dingle K. 2009. RE-VISITING CLOSTRIDIUM DIFFICILE IN CHILDREN: RESERVOIR, VICTIMS, BOTH OR NONE? JOURNAL OF INFECTION, 59 (6), pp. S429-S430.

Chapman SJ, Khor CC, Vannberg FO, Rautanen A, Segal S, Moore CE, Davies RJ, Day NP et al. 2010. NFKBIZ polymorphisms and susceptibility to pneumococcal disease in European and African populations. Genes Immun, 11 (4), pp. 319-325. | Show Abstract | Read more

The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) has a central role in host defence against pneumococcal disease. Both rare mutations and common polymorphisms in the NFKBIA gene encoding the NF-kappaB inhibitor, IkappaB-alpha, associate with susceptibility to bacterial disease, but the possible role of polymorphisms within the related IkappaB-zeta gene NFKBIZ in the development of invasive pneumococcal disease (IPD) has not been reported previously. To investigate this further, we examined the frequencies of 22 single-nucleotide polymorphisms spanning NFKBIZ in two case-control studies, comprising UK Caucasian (n=1008) and Kenyan (n=723) individuals. Nine polymorphisms within a single UK linkage disequilibrium (LD) block and all four polymorphisms within the equivalent, shorter Kenyan LD block displayed either a significant association with IPD or a trend towards association. For each polymorphism, heterozygosity was associated with protection from IPD when compared with the combined homozygous states (for example, for rs600718, Mantel-Haenszel 2 x 2 chi(2)=7.576, P=0.006, odds ratio (OR)=0.67, 95% confidence interval (95% CI) for OR: 0.51-0.88; for rs616597, Mantel-Haenszel 2 x 2 chi(2)=8.715, P=0.003, OR=0.65, 95% CI: 0.49-0.86). We conclude that multiple NFKBIZ polymorphisms associate with susceptibility to IPD in humans. The study of multiple populations may aid in fine mapping of associations within extensive regions of strong LD ('transethnic mapping').

Darmstadt GL, Saha SK, Choi Y, El Arifeen S, Ahmed NU, Bari S, Rahman SM, Mannan I et al. 2009. Population-based incidence and etiology of community-acquired neonatal bacteremia in Mirzapur, Bangladesh: an observational study. J Infect Dis, 200 (6), pp. 906-915. | Show Abstract | Read more

BACKGROUND: To devise treatment strategies for neonatal infections, the population-level incidence and antibiotic susceptibility of pathogens must be defined. METHODS: Surveillance for suspected neonatal sepsis was conducted in Mirzapur, Bangladesh, from February 2004 through November 2006. Community health workers assessed neonates on postnatal days 0, 2, 5, and 8 and referred sick neonates to a hospital, where blood was collected for culture from neonates with suspected sepsis. We estimated the incidence and pattern of community-acquired neonatal bacteremia and determined the antibiotic susceptibility profile of pathogens. RESULTS: The incidence rate of community-acquired neonatal bacteremia was 3.0 per 1000 person-neonatal periods. Among the 30 pathogens identified, the most common was Staphylococcus aureus (n = 10); half of all isolates were gram positive. Nine were resistant to ampicillin and gentamicin or to ceftiaxone, and 13 were resistant to cotrimoxazole. CONCLUSION: S. aureus was the most common pathogen to cause community-acquired neonatal bacteremia. Nearly 40% of infections were identified on days 0-3, emphasizing the need to address maternal and environmental sources of infection. The combination of parenteral procaine benzyl penicillin and an aminoglycoside is recommended for the first-line treatment of serious community-acquired neonatal infections in rural Bangladesh, which has a moderate level of neonatal mortality. Additional population-based data are needed to further guide national and global strategies.

Juhas M, van der Meer JR, Gaillard M, Harding RM, Hood DW, Crook DW. 2009. Genomic islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol Rev, 33 (2), pp. 376-393. | Show Abstract | Read more

Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

Juhas M, Crook DW, Hood DW. 2008. Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence. Cell Microbiol, 10 (12), pp. 2377-2386. | Show Abstract | Read more

Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains.

Pandit H, Vlychou M, Whitwell D, Crook D, Luqmani R, Ostlere S, Murray DW, Athanasou NA. 2008. Necrotic granulomatous pseudotumours in bilateral resurfacing hip arthoplasties: evidence for a type IV immune response. Virchows Arch, 453 (5), pp. 529-534. | Show Abstract | Read more

Clinical, radiological and histological findings were analysed in four patients who developed bilateral pseudotumours following metal-on-metal (MoM) resurfacing arthroplasties of both hips. Using a panel of monoclonal antibodies directed against HLA-DR, macrophages (CD14, CD68), dendritic cells (DC-SIGN, S100, CD11c), B cells (CD20), and T cells (CD3, CD4, CD8), the nature of the heavy inflammatory response seen in these cases was examined. Bilateral masses developed in periprosthetic soft tissues following the second MoM arthroplasty; these were characterised histologically by extensive coagulative necrosis, a heavy macrophage infiltrate and the presence of granulomas containing macrophages and giant cells; there was also a diffuse lymphocyte and variable plasma cell and eosinophil polymorph infiltrate. Immunohistochemistry showed strong expression of HLA-DR, CD14 and CD68 in both granulomatous and necrotic areas; lymphocytes were predominantly CD3+/CD4+ T cells. The clinical, morphological and immunophenotypic features of these necrotic granulomatous pseudotumours, which in all cases develop following a second resurfacing hip arthroplasty, is suggestive of a type IV immune response, possibly to MoM metal alloy components.

Saha SK, Darmstadt GL, Baqui AH, Hossain B, Islam M, Foster D, Al-Emran H, Naheed A et al. 2008. Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design. PLoS One, 3 (10), pp. e3576. | Show Abstract | Read more

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Oh SY, Griffiths D, John T, Lee YC, Yu LM, McCarthy N, Heath PT, Crook D, Ramsay M, Moxon ER, Pollard AJ. 2008. School-aged children: a reservoir for continued circulation of Haemophilus influenzae type b in the United Kingdom. J Infect Dis, 197 (9), pp. 1275-1281. | Show Abstract | Read more

BACKGROUND: A resurgence of Haemophilus influenzae type b (Hib) disease occurred in the United Kingdom between 1999 and 2003 and was partially attributed to lower immunogenicity of combination vaccines. The reservoir for Hib that led to transmission in this period is unknown. METHODS: We estimated the point prevalence of Hib carriage in school-aged children and adults, using oropharyngeal swabbing and selective media. We characterized the Hib isolates by multilocus sequence typing (MLST) and measured Hib antibody concentrations in adults by enzyme-linked immunosorbent assay. RESULTS: Point prevalence for Hib carriage in 855 children aged 6-16 years was 4.2% (95% confidence interval [CI], 2.5%-5.9%). Five clonal groups of Hib were identified by MLST, 86% from the lineage of sequence type 6. No Hib was isolated in 385 adults (upper limit of 95% CI, 0.95%). The geometric mean concentration of serum antibody to polyribosylribitol phosphate was 0.47 microg/mL (95% CI, 0.37-0.59 mirog/mL) in adults. CONCLUSIONS: Hib carriage is common in school-aged children, who are a significant reservoir for ongoing transmission of Hib to susceptible individuals in the United Kingdom. Surveillance of transmission and immunity across all ages of the population is essential to monitor the evolution of Hib epidemiology.

Foster D, Knox K, Walker AS, Griffiths DT, Moore H, Haworth E, Peto T, Brueggemann AB, Crook DW, Oxford Invasive Pneumococcal Surveillance Group. 2008. Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England. J Med Microbiol, 57 (Pt 4), pp. 480-487. | Show Abstract | Read more

A 10-year invasive pneumococcal disease (IPD) enhanced surveillance project in the Oxfordshire region of the UK between 1996 and 2005 identified a total of 2691 Streptococcus pneumoniae isolates from all ages that provided a comprehensive description of pneumococcal epidemiology. All isolates were serotyped and those from children under 5 years of age were genotyped and a matched case-control study using adults hospitalized between 1995 and 2000 was performed to estimate the effectiveness of the pneumococcal polysaccharide vaccine in the local population. Fifty-one serotypes were isolated, with different age distributions. The overall incidence of IPD was 9.2 cases per 100 000 population per annum [95 % confidence interval (CI), 8.6-9.9] and that of meningitis was 0.7 per 100 000 population per annum (95 % CI 0.5-0.9). After adjusting for age, serotype 1 was found to be less likely to be associated with meningitis versus other IPD, compared with the most common serotype 14, whereas serotype 12F was more likely to cause meningitis than other IPD. There were significant temporal changes in IPD incidence of four serotypes, with decreases in serotypes 1, 12F and 14 and increases in serotype 8. A possible novel variant (from serotype 6A to 6B) was found using multilocus sequence typing analysis. From the matched case-control study of adults, the pneumococcal polysaccharide vaccine effectiveness was estimated to be 43 % (2-68 %), which did not change significantly after adjustment for pre-existing co-morbidities. The data provide a baseline against which the impact of the pneumococcal conjugate vaccine introduced in the UK in 2006 could be measured.

Miller R, Esmail H, Peto T, Walker S, Crook D, Wyllie D. 2008. Is MRSA admission bacteraemia community-acquired? A case control study. J Infect, 56 (3), pp. 163-170. | Show Abstract | Read more

OBJECTIVES: To compare characteristics of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible S. aureus (MSSA) bacteraemia detected on admission to a UK hospital and to determine whether these organisms are community-acquired. METHODS: Consecutive cases of MRSA bacteraemia admitted to general medicine between 2003 and 2006 were identified and compared to MSSA age-matched and unmatched controls (35, 35 and 34 patients, respectively). Demographics, MRSA risk factors, previous health-care contact and clinical presentation were compared using patient notes. Multi-locus sequence typing was performed. RESULTS: 34/35 strains of admission MRSA bacteraemia were the health-care associated Sequence Types (ST)-22 (77%) or ST-36 (21%), whereas 20 different MSSA strains were identified. No MRSA cases fitted the CDC definition of community-acquired MRSA. Compatible with health-care associated acquisition, after matching for age MRSA cases had significantly higher levels of previous hospital exposure than MSSA controls, and more co-morbidities. Notably, 63% of MRSA cases were admitted from their own home, as opposed to secondary care facilities. Clinical presentation of MRSA and MSSA bacteraemias was similar. CONCLUSIONS: MRSA strains associated with health-care were responsible for almost all cases of MRSA bacteraemia on admission to hospital during the period studied. Despite this the majority of cases with MRSA admission bacteraemia were admitted from their own homes. Further research is needed into the determinants of MRSA bacteraemia among patients outside hospital.

Maiden MC, Ibarz-Pavón AB, Urwin R, Gray SJ, Andrews NJ, Clarke SC, Walker AM, Evans MR et al. 2008. Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity. J Infect Dis, 197 (5), pp. 737-743. | Show Abstract | Read more

BACKGROUND: In 1999, meningococcal serogroup C conjugate (MCC) vaccines were introduced in the United Kingdom for those under 19 years of age. The impact of this intervention on asymptomatic carriage of meningococci was investigated to establish whether serogroup replacement or protection by herd immunity occurred. METHODS: Multicenter surveys of carriage were conducted during vaccine introduction and on 2 successive years, resulting in a total of 48,309 samples, from which 8599 meningococci were isolated and characterized by genotyping and phenotyping. RESULTS: A reduction in serogroup C carriage (rate ratio, 0.19) was observed that lasted at least 2 years with no evidence of serogroup replacement. Vaccine efficacy against carriage was 75%, and vaccination had a disproportionate impact on the carriage of sequence type (ST)-11 complex serogroup C meningococci that (rate ratio, 0.06); these meningococci also exhibited high rates of capsule expression. CONCLUSIONS: The impact of vaccination with MCC vaccine on the prevalence of carriage of group C meningococci was consistent with herd immunity. The high impact on the carriage of ST-11 complex serogroup C could be attributed to high levels of capsule expression. High vaccine efficacy against disease in young children, who were not protected long-term by the schedule initially used, is attributed to the high vaccine efficacy against carriage in older age groups.

Warwick RM, Magee JG, Leeming JP, Graham JC, Hannan MM, Chadwick M, Crook DW, Yearsley CP, Rayner A, Parker R. 2008. Mycobacteria and allograft heart valve banking: an international survey. J Hosp Infect, 68 (3), pp. 255-261. | Show Abstract | Read more

Since the 1970s many tissue banks have been testing allograft heart valves (HVs) for Mycobacterium tuberculosis (MTB). Donor selection for low risk of tuberculosis (TB) was introduced in the 1980s and appears to have reduced the risk of TB transmission. Regulatory guidance does not specify testing for TB, but does exclude donors with a recent history of TB. This survey of HV international bank practices revealed variations in donor selection, testing and processing of valves. Participant banks (from Europe and the USA) reported that over a period of 15 years, HV tissues from 38,413 donors were banked and 32,289 donors were tested for TB, none being positive. HV-associated tissue from 27,840 donors was stained and underwent microscopy; none of these were positive for acid-fast bacilli (AFB). Non-tuberculosis mycobacteria (NTBM) were detected by culture on 24 HVs. It is recommended that HV banks employ donor selection to exclude donors at risk of TB, to culture material for mycobacteria, and to investigate potential sources when clusters of NTBM are found to facilitate corrective and preventative actions.

Walker S, Peto TE, O'Connor L, Crook DW, Wyllie D. 2008. Are there better methods of monitoring MRSA control than bacteraemia surveillance? An observational database study. PLoS One, 3 (6), pp. e2378. | Show Abstract | Read more

BACKGROUND: Despite a substantial burden of non-bacteraemic methicillin resistant Staphylococcus aureus (MRSA) disease, most MRSA surveillance schemes are based on bacteraemias. Using bacteraemia as an outcome, trends at hospital level are difficult to discern, due to random variation. We investigated rates of nosocomial bacteraemic and non-bacteraemic MRSA infection as surveillance outcomes. METHODS AND FINDINGS: We used microbiology and patient administration system data from an Oxford hospital to estimate monthly rates of first nosocomial MRSA bacteraemia, and nosocomial MRSA isolation from blood/respiratory/sterile site specimens ("sterile sites") or all clinical samples (screens excluded) in all patients admitted from the community for at least 2 days between April 1998 and June 2006. During this period there were 441 nosocomial MRSA bacteraemias, 1464 MRSA isolations from sterile sites, and 3450 isolations from clinical specimens (8% blood, 15% sterile site, 10% respiratory, 59% surface swabs, 8% urine) in over 2.6 million patient-days. The ratio of bacteraemias to sterile site and all clinical isolations was similar over this period (around 3 and 8-fold lower respectively), during which rates of nosocomial MRSA bacteraemia increased by 27% per year to July 2003 before decreasing by 18% per year thereafter (heterogeneity p<0.001). Trends in sterile site and all clinical isolations were similar. Notably, a change in rate of all clinical MRSA isolations in December 2002 could first be detected with conventional statistical significance by August 2003 (p = 0.03). In contrast, when monitoring MRSA bacteraemia, identification of probable changes in trend took longer, first achieving p<0.05 in July 2004. CONCLUSIONS: MRSA isolation from all sites of suspected infection, including bacteraemic and non-bacteraemic isolation, is a potential new surveillance method for MRSA control. It occurs about 8 times more frequently than bacteraemia, allowing robust statistical determination of changing rates over substantially shorter times or smaller areas than using bacteraemia as an outcome.

Walker AS, Spiegelhalter D, Crook DW, Wyllie D, Morris J, Peto TE. 2008. Fairness of financial penalties to improve control of Clostridium difficile. BMJ, 337 (nov20 2), pp. a2097. | Read more

Saha SK, Darmstadt GL, Baqui AH, Islam N, Qazi S, Islam M, El Arifeen S, Santosham M, Black RE, Crook DW. 2008. Direct detection of the multidrug resistance genome of Haemophilus influenzae in cerebrospinal fluid of children: implications for treatment of meningitis. Pediatr Infect Dis J, 27 (1), pp. 49-53. | Show Abstract | Read more

BACKGROUND: Multidrug resistance (MDR), specifically to ampicillin and chloramphenicol, has complicated the treatment of Haemophilus influenzae type b (Hib) meningitis. This is worsened by use of prior antibiotics, which limits identification of the causative agent by culture and increases reliance on antigen detection. OBJECTIVE: We aimed to develop a PCR assay for detecting the family of Haemophilus integrating and conjugative elements (ICEs) represented by ICEHin1056 among antibiotic resistant Hib, and then apply this directly to CSF to diagnose Hib meningitis and predict organism susceptibility, irrespective of culture results. STUDY DESIGN: Primers specific for orf 51 of ICEHin1056 were designed and multiplexed with Bex primers, specific for H. influenzae, and tested on culture positive and negative cases. RESULTS: Of 73 Hib isolates, orf 51 PCR amplicons, predicting the presence of ICEs, were found in all 33 MDR isolates while only in 1 of 33 sensitive strains. The remaining 7 ampicillin susceptible, chloramphenicol and tetracycline resistant strains did not produce a PCR product to orf 51. PCR amplification from CSF specimens of these culture positive cases produced identical results with 100% and 97% positive and negative predictive values, respectively. Multiplex PCR to detect Bex and orf 51 identified another 16 MDR Hib cases among 81 culture-negative CSF samples. CONCLUSIONS: Direct PCR for orf 51 in CSF identified resistance pattern of 51% more Hib strains than culture alone (110 versus 73). The ability to detect MDR, in culture negative Hib meningitis cases has significant implications for better directing antibiotic treatment of meningitis cases and thus for preventing disability and death.

Wyllie DH, Walker AS, Peto TE, Crook DW. 2007. Hospital exposure in a UK population, and its association with bacteraemia. J Hosp Infect, 67 (4), pp. 301-307. | Show Abstract | Read more

Despite the importance of healthcare-associated infection, few studies have quantified the association between severe infection and hospital exposure in UK populations. Our aim was to estimate the proportion of the population with recent hospital admission, together with rates of infection in hospital-exposed and hospital-naïve populations. We studied bacteraemia as a marker of severe infection in a population of 550,000, served by two hospitals, between 1 April 2000 and 31 March 2005. Hospital-exposed persons accounted for 8.3% of the population, defined as having been resident in a hospital in the last year. The hospital-exposed population accounted for 55% of all admissions, and 42% of emergency admissions to medical, paediatric or surgery departments. After adjustment for age, the hospital-exposed group had much higher rates of admission bacteraemia. Age-standardised incidence rate ratios relative to hospital-naïve patients were 43 [95% confidence interval (CI): 22-85] for meticillin-resistant Staphylococcus aureus (MRSA), 20 (15-27) for S. aureus other than MRSA, 7.3 (5.2-10) for Streptococcus pneumoniae, and 14 (11-18) for E. coli. MRSA was common among hospital-exposed admissions, including emergencies in hospital-exposed men, rates of admission MRSA bacteraemia (31 per 100,000 per annum) and S. pneumoniae bacteraemia (33 per 100,000 per annum) were similar. This quantitative analysis confirms that prior hospital admission is a major risk factor for bacteraemia on hospital admission; it is unclear whether acquisition of pathogens in hospital, co-morbidity or other factors explain this.

Bisharat N, Cohen DI, Maiden MC, Crook DW, Peto T, Harding RM. 2007. The evolution of genetic structure in the marine pathogen, Vibrio vulnificus. Infect Genet Evol, 7 (6), pp. 685-693. | Show Abstract | Read more

Multi-locus sequence types (MLST) from a global collection of Vibrio vulnificus isolates were analysed for the contribution of recombination to the evolution of two divergent clusters of strains and a human-pathogenic hybrid genotype, which caused a disease outbreak in Israel. Recombination contributes more substantially than mutation to generating strain diversity. For allelic diversity within loci, the ratio of recombination to mutation events is approximately 2:1. The role of recombination relative to mutation in the generation of new MLST variants of V. vulnificus within the clusters is comparable to that of other highly recombining bacteria such as Neisseria meningitidis. However, across the divide between the two major clusters of V. vulnificus strains, there is substantial linkage disequilibrium, lower estimates for recombination rates and shorter estimates of recombination tract length. We account for these differences between V. vulnificus and N. meningitidis by attributing them to the presence of the unusual genetic structure within V. vulnificus. The reason for the presence of distinct and divergent genomes remains unresolved. Two possible explanations put forward for future study are first, ecologically based population structure within V. vulnificus and second, a recombination donor from a phenotypically differentiated species.

Brueggemann AB, Pai R, Crook DW, Beall B. 2007. Vaccine escape recombinants emerge after pneumococcal vaccination in the United States. PLoS Pathog, 3 (11), pp. e168. | Show Abstract | Read more

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.

Chung A, Perera R, Brueggemann AB, Elamin AE, Harnden A, Mayon-White R, Smith S, Crook DW, Mant D. 2007. Effect of antibiotic prescribing on antibiotic resistance in individual children in primary care: prospective cohort study. BMJ, 335 (7617), pp. 429. | Show Abstract | Read more

OBJECTIVE: To assess the effect of community prescribing of an antibiotic for acute respiratory infection on the prevalence of antibiotic resistant bacteria in an individual child. STUDY DESIGN: Observational cohort study with follow-up at two and 12 weeks. SETTING: General practices in Oxfordshire. PARTICIPANTS: 119 children with acute respiratory tract infection, of whom 71 received a beta lactam antibiotic. MAIN OUTCOME MEASURES: Antibiotic resistance was assessed by the geometric mean minimum inhibitory concentration (MIC) for ampicillin and presence of the ICEHin1056 resistance element in up to four isolates of Haemophilus species recovered from throat swabs at recruitment, two weeks, and 12 weeks. RESULTS: Prescribing amoxicillin to a child in general practice more than triples the mean minimum inhibitory concentration for ampicillin (9.2 microg/ml v 2.7 microg/ml, P=0.005) and doubles the risk of isolation of Haemophilus isolates possessing homologues of ICEHin1056 (67% v 36%; relative risk 1.9, 95% confidence interval 1.2 to 2.9) two weeks later. Although this increase is transient (by 12 weeks ampicillin resistance had fallen close to baseline), it is in the context of recovery of the element from 35% of children with Haemophilus isolates at recruitment and from 83% (76% to 89%) at some point in the study. CONCLUSION: The short term effect of amoxicillin prescribed in primary care is transitory in the individual child but sufficient to sustain a high level of antibiotic resistance in the population.

Chapman SJ, Khor CC, Vannberg FO, Frodsham A, Walley A, Maskell NA, Davies CW, Segal S et al. 2007. IkappaB genetic polymorphisms and invasive pneumococcal disease. Am J Respir Crit Care Med, 176 (2), pp. 181-187. | Show Abstract | Read more

RATIONALE: Increasing evidence supports a key role for the transcription factor nuclear factor (NF)-kappaB in the host response to pneumococcal infection. Control of NF-kappaB activity is achieved through interactions with the IkappaB family of inhibitors, encoded by the genes NFKBIA, NFKBIB, and NFKBIE. Rare NFKBIA mutations cause immunodeficiency with severe bacterial infection, raising the possibility that common IkappaB gene polymorphisms confer susceptibility to common bacterial disease. OBJECTIVES: To determine whether polymorphisms in NFKBIA, NFKBIB, and NFKBIE associate with susceptibility to invasive pneumococcal disease (IPD) and thoracic empyema. METHODS: We studied the frequencies of 62 single-nucleotide polymorphisms (SNPs) across NFKBIA, NFKBIB, and NFKBIE in individuals with IPD and control subjects (n=1,060). Significantly associated SNPs were then studied in a group of individuals with thoracic empyema and a second control group (n=632). MEASUREMENTS AND MAIN RESULTS: Two SNPs in the NFKBIA promoter region were associated with protection from IPD in both the initial study group and the pneumococcal empyema subgroup. Significant protection from IPD was observed for carriage of mutant alleles at these two loci on combining the groups (SNP rs3138053: Mantel-Haenszel 2x2 chi2=13.030, p=0.0003; odds ratio [OR], 0.60; 95% confidence interval [CI], 0.45-0.79; rs2233406: Mantel-Haenszel 2x2 chi2=18.927, p=0.00001; OR, 0.55; 95% CI, 0.42-0.72). An NFKBIE SNP associated with susceptibility to IPD but not pneumococcal empyema. None of the NFKBIB SNPs associated with IPD susceptibility. CONCLUSIONS: NFKBIA polymorphisms associate with susceptibility to IPD. Genetic variation in an inhibitor of NF-kappaB therefore not only causes a very rare immunodeficiency state but may also influence the development of common infectious disease.

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Harnden A, Perera R, Brueggemann AB, Mayon-White R, Crook DW, Thomson A, Mant D. 2007. Respiratory infections for which general practitioners consider prescribing an antibiotic: a prospective study ARCHIVES OF DISEASE IN CHILDHOOD, 92 (7), pp. 594-597. | Show Abstract | Read more

Objective: To determine the viral aetiology of respiratory infections in children presenting to primary care with "more than a simple cold". Design: Observational study in 18 Oxfordshire general practices over four winters (2000-01 to 2003-04). Patients: 425 children aged 6 months to 12 years with cough and fever for whom general practitioners considered prescribing an antibiotic. Methods: Nasopharyngeal aspirate obtained from 408 (96%) children was subjected to PCR for respiratory viruses. Parents completed an illness diary for the duration of illness. Results: A viral cause of infection was detected in most (77%) children. Clinical symptoms correctly identified the infecting virus in 45% of cases. The duration of illness was short and the time course was very similar for all infecting viruses. One third of children were prescribed an antibiotic (34%), but this made no difference to the rate of parent-assessed recovery (Kruskal-Wallis, p = 0.67). About one in five children with influenza who did not receive an antibiotic had persistent fever on day 7 compared to no children receiving antibiotics (p = 0.02); this difference remained after adjustment for severity and other factors and was not seen with other viruses. Conclusions: Most children receiving antibiotics for respiratory symptoms in general practice have an identifiable viral illness. In routine clinical practice, neither the specific infecting virus nor the use of antibiotics has a significant effect on the time course of illness. Antibiotics may reduce the duration of fever in children with influenza which could reflect an increased risk of secondary bacterial infection for such children.

Harnden A, Perera R, Brueggemann AB, Mayon-White R, Crook DW, Thomson A, Mant D. 2007. Respiratory infections for which general practitioners consider prescribing an antibiotic: a prospective study. Arch Dis Child, 92 (7), pp. 594-597. | Show Abstract | Read more

OBJECTIVE: To determine the viral aetiology of respiratory infections in children presenting to primary care with "more than a simple cold". DESIGN: Observational study in 18 Oxfordshire general practices over four winters (2000-01 to 2003-04). PATIENTS: 425 children aged 6 months to 12 years with cough and fever for whom general practitioners considered prescribing an antibiotic. METHODS: Nasopharyngeal aspirate obtained from 408 (96%) children was subjected to PCR for respiratory viruses. Parents completed an illness diary for the duration of illness. RESULTS: A viral cause of infection was detected in most (77%) children. Clinical symptoms correctly identified the infecting virus in 45% of cases. The duration of illness was short and the time course was very similar for all infecting viruses. One third of children were prescribed an antibiotic (34%), but this made no difference to the rate of parent-assessed recovery (Kruskal-Wallis, p = 0.67). About one in five children with influenza who did not receive an antibiotic had persistent fever on day 7 compared to no children receiving antibiotics (p = 0.02); this difference remained after adjustment for severity and other factors and was not seen with other viruses. CONCLUSIONS: Most children receiving antibiotics for respiratory symptoms in general practice have an identifiable viral illness. In routine clinical practice, neither the specific infecting virus nor the use of antibiotics has a significant effect on the time course of illness. Antibiotics may reduce the duration of fever in children with influenza which could reflect an increased risk of secondary bacterial infection for such children.

Dimopoulou ID, Kartali SI, Harding RM, Peto TE, Crook DW. 2007. Diversity of antibiotic resistance integrative and conjugative elements among haemophili. J Med Microbiol, 56 (Pt 6), pp. 838-846. | Show Abstract | Read more

The objective of this study was to investigate the sequence diversity in a single country of a family of integrative and conjugative elements (ICEs) that are vectors of antibiotic resistance in Haemophilus influenzae and Haemophilus parainfluenzae, and test the hypothesis that they emerged from a single lineage. Sixty subjects aged 9 months - 13 years were recruited and oropharyngeal samples cultured. Up to 10 morphologically distinct Pasteurellaceae spp. were purified, and then the species were determined and differentiated by partial sequence analysis of 16S rDNA and mdh, respectively. ICEs were detected by PCR directed at five genes distributed evenly across the ICE. These amplicons were sequenced and aligned by the neighbour-joining algorithm. A total of 339 distinguishable isolates were cultured. ICEs with all 5 genes present were found in 9 of 110 (8 %) H. influenzae and 21 of 211 (10 %) H. parainfluenzae, respectively. ICEs were not detected among the other Pasteurellaceae. A total of 20 of 60 (33 %) children carried at least 1 oropharyngeal isolate with an ICE possessing all 5 genes. One of the five genes, integrase, however, consisted of two lineages, one of which was highly associated with H. influenzae. The topology of neighbour-joining trees of the remaining four ICE genes was compared and showed a lack of congruence; though, the genes form a common pool among H. influenzae and H. parainfluenzae. This family of antibiotic resistance ICEs was prevalent among the children studied, was genetically diverse, formed a large gene pool, transferred between H. influenzae and H. parainfluenzae, lacked population structure and possessed features suggestive of panmixia, all indicating it has not recently emerged from a single source.

Chapman SJ, Vannberg FO, Khor CC, Segal S, Moore CE, Knox K, Day NP, Davies RJ, Crook DW, Hill AV. 2007. Functional polymorphisms in the FCN2 gene are not associated with invasive pneumococcal disease. Mol Immunol, 44 (12), pp. 3267-3270. | Show Abstract | Read more

L-ficolin is a pattern-recognition molecule which binds lipoteichoic acid and Gram-positive bacteria and activates the lectin pathway of complement. Five common functional polymorphisms have recently been identified in the FCN2 gene which encodes L-ficolin: three promoter polymorphisms (at positions -986, -602 and -4) which affect serum L-ficolin concentration, and two non-synonymous polymorphisms (Thr236Met and Ala258Ser) which influence carbohydrate binding. We studied the frequencies of these polymorphisms in individuals with invasive pneumococcal disease (IPD) and a control group. Although the five FCN2 polymorphisms were each present in the UK Caucasian population studied, no significant associations were observed between the FCN2 polymorphisms and susceptibility to IPD. This is in contrast to mannose-binding lectin deficiency, which we have previously shown to be associated with increased susceptibility to IPD. Although we are unable to exclude small effects of FCN2 genetic variation on susceptibility to IPD, the result suggests that L-ficolin may not be critical for host defence against pneumococcal infection.

Khor CC, Chapman SJ, Vannberg FO, Dunne A, Murphy C, Ling EY, Frodsham AJ, Walley AJ et al. 2007. A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis. Nat Genet, 39 (4), pp. 523-528. | Show Abstract | Read more

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.

Juhas M, Crook DW, Dimopoulou ID, Lunter G, Harding RM, Ferguson DJP, Hood DW. 2007. Functional analysis of the novel Type IV secretion system involved in propagation of genomic islands. PLASMID, 57 (2), pp. 221-221.

Juhas M, Crook DW, Dimopoulou ID, Lunter G, Harding RM, Ferguson DJ, Hood DW. 2007. Novel type IV secretion system involved in propagation of genomic islands. J Bacteriol, 189 (3), pp. 761-771. | Show Abstract | Read more

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.

Juhas M, Power PM, Harding RM, Ferguson DJ, Dimopoulou ID, Elamin AR, Mohd-Zain Z, Hood DW et al. 2007. Sequence and functional analyses of Haemophilus spp. genomic islands. Genome Biol, 8 (11), pp. R237. | Show Abstract | Read more

BACKGROUND: A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily. RESULTS: These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts. CONCLUSION: Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.

Cited:

82

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Brueggemann AB, Pai R, Crook DW, Beall B. 2007. Vaccine escape recombinants emerge after pneumococcal vaccination in the United States PLoS Pathogens, 3 (11), pp. 1628-1636. | Show Abstract | Read more

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.

Saha SK, Darmstadt GL, Baqui AH, Crook DW, Islam MN, Islam M, Hossain M, El Arifeen S, Santosham M, Black RE. 2006. Molecular basis of resistance displayed by highly ciprofloxacin-resistant Salmonella enterica serovar Typhi in Bangladesh. J Clin Microbiol, 44 (10), pp. 3811-3813. | Show Abstract | Read more

Highly ciprofloxacin-resistant (MIC, 512 microg/ml) strains of Salmonella enterica serovar Typhi were isolated from the blood of typhoid patients in Dhaka, Bangladesh. The strains were indistinguishable by their antibiograms, biotypes, and variable-number tandem repeat types and had matching point mutations at positions 83 and 87 of the gyrA gene. The isolation of these strains in an area of high endemicity indicates the need for continuous surveillance of antibiotic resistance of S. enterica serovar Typhi and for the rationalized use of ciprofloxacin.

Sleeman KL, Griffiths D, Shackley F, Diggle L, Gupta S, Maiden MC, Moxon ER, Crook DW, Peto TE. 2006. Capsular serotype-specific attack rates and duration of carriage of Streptococcus pneumoniae in a population of children. J Infect Dis, 194 (5), pp. 682-688. | Show Abstract | Read more

BACKGROUND: The relative invasiveness rates (attack rates) of Streptococcus pneumoniae of different capsular serotypes in children are not known. Estimates of capsular serotype invasiveness (designated "invasive odds ratios") that are based on cross-sectional prevalence carriage data have been published, but these estimates could be biased by variation in the duration of carriage. METHODS: The relative attack rates of invasive pneumococci were measured using national UK surveillance data on invasive pneumococcal disease (IPD) incidence and data on incidence of pneumococcal acquisition from longitudinal studies of nasopharyngeal pneumococcal carriage. RESULTS: We found significant differences in capsular serotype-specific attack rates. For example, capsular serotypes 4, 14, 7F, 9V, and 18C were associated with rates of >20 IPD cases/100,000 acquisitions, whereas capsular serotypes 23F, 6A, 19F, 16F, 6B, and 15B/C were associated with <10 IPD cases/100,000 acquisitions. There was an inverse relationship between duration of carriage and attack rate by capsular serotype (P<.0001). Attack rates were significantly correlated with invasive odds ratios (P<.0001). CONCLUSIONS: The capsular serotype is a major determinant of both pneumococcal duration of carriage and attack rate. Published invasive odds ratios are a reliable and practical method of determining capsular serotype invasiveness and will be valuable for investigating and characterizing emerging capsular serotypes in the context of conjugate vaccination.

Wyllie DH, Crook DW, Peto TE. 2006. Mortality after Staphylococcus aureus bacteraemia in two hospitals in Oxfordshire, 1997-2003: cohort study. BMJ, 333 (7562), pp. 281. | Show Abstract | Read more

OBJECTIVE: To determine the incidence of methicillin resistant and methicillin sensitive Staphylococcus aureus (MRSA and MSSA) bacteraemia in inpatients and associated mortality within 30 days after diagnosis. DESIGN: Anonymised record linkage study of data from hospital information systems and microbiology databases. SETTING: Teaching hospital and district general hospital in Oxfordshire. PARTICIPANTS: Inpatients aged 18 or over admitted to a teaching hospital between 1 April 1997 and 31 March 2004 and to a district general hospital between 1 April 1999 and 31 March 2004. The main part of the study comprised 216 644 inpatients; patients admitted to haematology, nephrology, or oncology services were not included because most were managed as outpatients. OUTCOME MEASURES: Nosocomial MSSA and MRSA bacteraemia; death in hospital within 30 days after bacteraemia. RESULTS: Rates of S aureus bacteraemia rose between 1997 and 2003, and MRSA was responsible for this increase. Overall mortality 30 days after bacteraemia was 29%. The crude odds ratio for death after MRSA bacteraemia compared with MSSA bacteraemia was 1.49 (95% confidence interval 0.99 to 2.26). CONCLUSION: The spread of MRSA has greatly increased the overall number of cases of S aureus bacteraemia and has contributed to short term mortality after S aureus bacteraemia.

Chapman SJ, Khor CC, Vannberg FO, Maskell NA, Davies CW, Hedley EL, Segal S, Moore CE et al. 2006. PTPN22 and invasive bacterial disease. Nat Genet, 38 (5), pp. 499-500. | Read more

Jones N, Oliver K, Jones Y, Haines A, Crook D. 2006. Carriage of group B streptococcus in pregnant women from Oxford, UK. J Clin Pathol, 59 (4), pp. 363-366. | Show Abstract | Read more

OBJECTIVE: To investigate asymptomatic vagino-rectal carriage of group B streptococcus (GBS) in pregnant women. METHODS: Women in the final trimester of pregnancy were recruited. A single vagino-rectal swab was taken, with consent, for culture of GBS. Two microbiological methods for isolation of GBS from vagino-rectal swabs were compared. The distribution of capsular serotypes of the GBS identified was determined. Epidemiological data for a subset (n = 167) of the pregnant women participating were examined. RESULTS: 21.3% were colonised vagino-rectally with GBS. Risk factors for neonatal GBS disease (maternal fever, prolonged rupture of membranes, and preterm delivery) were present in 34 of 167 women (20.4%), and the presence of these factors correlated poorly with GBS carriage. Capsular serotypes III (26.4%), IA (25.8%), V (18.9%), and IB (15.7%) were prevalent in the GBS isolates. Selective broth culture of vagino-rectal swabs was superior to selective plate culture, but the combination of both methods was associated with increased detection of GBS (7.5%). An algorithm for the identification of GBS from vagino-rectal swabs was developed. CONCLUSIONS: GBS carriage is prevalent in pregnant women in Oxfordshire, UK. The poor correlation between risk factors and GBS carriage requires further investigation in larger groups, given that the identification of these surrogate markers is recommended to guide administration of intrapartum antibiotic prophylaxis by the Royal College of Obstetricians of the UK. A selective broth culture detected more GBS carriers than a selective plate culture.

Jones N, Oliver KA, Barry J, Harding RM, Bisharat N, Spratt BG, Peto T, Crook DW, Oxford Group B Streptococcus Consortium. 2006. Enhanced invasiveness of bovine-derived neonatal sequence type 17 group B streptococcus is independent of capsular serotype. Clin Infect Dis, 42 (7), pp. 915-924. | Show Abstract | Read more

BACKGROUND: A defined geographical area (Oxford, United Kingdom) was investigated for the role of group B Streptococcus (GBS) as a human pathogen. METHODS: GBS carriage in pregnant women and invasive disease in neonates and adults >60 years of age was studied over a 3-year period. Multilocus sequence typing and capsular serotyping were used to study 369 isolates of GBS from carriage in pregnant women (n=190) and invasive disease in neonates (n=109) and adults >60 years of age (n=70). RESULTS: A total of 20.3% of pregnant women carried GBS. Invasive GBS disease occurred at a rate of 0.9 cases per 1000 live births and 11 cases per 100,000 population >60 years of age per annum. Four sequence types (STs) (ST-17, ST-19, ST-23, and ST-1) that were identified with use of multilocus sequence typing accounted for >50% of carried and invasive strains. A single sequence type (ST-17), previously shown to be phylogenetically of bovine origin, was significantly associated with increased invasiveness in neonates (P=.00002), and this was independent of capsular serotype III. In contrast, among adults >60 years of age, no STs exhibited increased invasiveness, compared with STs carried in pregnant women. CONCLUSIONS: Enhanced invasiveness associated with ST-17 is specific to neonates and is independent of capsular serotype.

Crook DW. 2006. Capsular type and the pneumococcal human host-parasite relationship. Clin Infect Dis, 42 (4), pp. 460-462. | Read more

Rose PW, Ziebland S, Harnden A, Mayon-White R, Mant D, Oxford Childhood Infection Study group (OXCIS). 2006. Why do general practitioners prescribe antibiotics for acute infective conjunctivitis in children? Qualitative interviews with GPs and a questionnaire survey of parents and teachers. Fam Pract, 23 (2), pp. 226-232. | Show Abstract | Read more

BACKGROUND: Acute infective conjunctivitis in children is a common presentation in primary care. Treatment is usually with antibiotics and prescribing may be affected by non-clinical factors. AIMS: To investigate the non-clinical determinants of the management of acute infective conjunctivitis in children. DESIGN: Qualitative interviews with GPs and a questionnaire survey of parents of children with acute infective conjunctivitis and teachers. SETTING: GPs in Sheffield and Berkshire and parents of children with acute infective conjunctivitis and schools in Oxfordshire. METHODS: Semi-structured telephone interviews of 39 GPs. Questionnaire survey of 326 parents of children enrolled into a trial of acute infective conjunctivitis treatment. Questionnaire survey of 223 nurseries and primary schools in Oxfordshire. RESULTS: All three groups agreed that acute infective conjunctivitis was a mild condition. Parents were certain about the benefits of antibiotic treatment and sought early consultations with their GP in a desire to get their child back to school. GPs sometimes collude with a parent's request to prescribe to enable school attendance. Despite this 54.2% (95%CI 48.5-59.8%) children missed a mean of 1.85 days from school and 28.6% of parents (95%CI 23.5-33.7%) missed a mean of 1.5 days off work. CONCLUSION: Social factors, including the need for children to attend day care or school and parents to go to work, contribute to the decision to prescribe antibiotics for children with acute infective conjunctivitis. Understanding these issues and changing school policies in line with national guidance may reduce pressure on GPs to prescribe for this condition.

Wyllie DH, Peto TE, Crook D. 2005. MRSA bacteraemia in patients on arrival in hospital: a cohort study in Oxfordshire 1997-2003. BMJ, 331 (7523), pp. 992. | Show Abstract | Read more

OBJECTIVE: To describe the incidence and determinants of methicillin resistant and methicillin sensitive Staphylococcus aureus (MRSA and MSSA) bacteraemia in patients presenting to acute hospitals. DESIGN: Anonymised record linkage study with information from hospital information systems and microbiology databases. SETTING: One teaching hospital and one district general hospital in Oxfordshire. PARTICIPANTS: All patients admitted to a teaching hospital 1 April 1997 to 31 March 2003 and to a district general hospital 1 April 1999 to 31 March 2003. MAIN OUTCOME MEASURES: Detection of MRSA and MSSA from blood cultures taken during the first two days of admission to hospital. RESULTS: In the teaching hospital, there were 479 patients with MSSA and 116 with MRSA bacteraemia admitted from the community. Among this group, which comprised 24% of all hospital MRSA cases, 31% (36 cases) of patients had been admitted to renal, oncology, or haematology services for intensive day case therapy. The 69% remaining were most commonly patients admitted as medical or surgical emergencies. At least 91% had been in hospital previously; the median time since discharge was 46 days. About half of cases were in patients in whom MRSA had not been isolated before. Similar epidemiology was observed in the district general hospital. CONCLUSION: Diagnostic algorithms and policies on use of antibiotics need to reflect the fact that a quarter of hospital MRSA cases occur in patients who have previously been in hospital and are subsequently readmitted.

Erwin AL, Nelson KL, Mhlanga-Mutangadura T, Bonthuis PJ, Geelhood JL, Morlin G, Unrath WC, Campos J et al. 2005. Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae. Infect Immun, 73 (9), pp. 5853-5863. | Show Abstract | Read more

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Rose PW, Harnden A, Brueggemann AB, Perera R, Sheikh A, Crook D, Mant D. 2005. Chloramphenicol treatment for acute infective conjunctivitis in children in primary care: a randomised double-blind placebo-controlled trial. Lancet, 366 (9479), pp. 37-43. | Show Abstract | Read more

BACKGROUND: One in eight schoolchildren have an episode of acute infective conjunctivitis every year. Standard clinical practice is to prescribe a topical antibiotic, although the evidence to support this practice is scarce. We undertook a randomised double-blind trial to compare the effectiveness of chloramphenicol eye drops with placebo in children with infective conjunctivitis in primary care. METHODS: Our study included 326 children aged 6 months to 12 years with a clinical diagnosis of conjunctivitis who were recruited from 12 general medical practices in the UK. We assigned 163 children to receive chloramphenicol eye drops and 163 to receive placebo eye drops. Eye swabs were taken for bacterial and viral analysis. The primary outcome was clinical cure at day 7, which was assessed from diaries completed by parents. All children were followed up for 6 weeks to identify relapse. Survival statistics were used for comparison, and analysis was by intention to treat. FINDINGS: Nine children were lost to follow-up (one in chloramphenicol group; eight in placebo group). Clinical cure by day 7 occurred in 128 (83%) of 155 children with placebo compared with 140 (86%) of 162 with chloramphenicol (risk difference 3.8%, 95% CI -4.1% to 11.8%). Seven (4%) children with chloramphenicol and five (3%) with placebo had further conjunctivitis episodes within 6 weeks (1.2%, -2.9% to 5.3%). Adverse events were rare and evenly distributed between each group. INTERPRETATION: Most children presenting with acute infective conjunctivitis in primary care will get better by themselves and do not need treatment with an antibiotic.

Bisharat N, Jones N, Marchaim D, Block C, Harding RM, Yagupsky P, Peto T, Crook DW. 2005. Population structure of group B streptococcus from a low-incidence region for invasive neonatal disease. Microbiology, 151 (Pt 6), pp. 1875-1881. | Show Abstract | Read more

The population structure of group B streptococcus (GBS) from a low-incidence region for invasive neonatal disease (Israel) was investigated using multilocus genotype data. The strain collection consisted of isolates from maternal carriage (n=104) and invasive neonatal disease (n=50), resolving into 46 sequence types. The most prevalent sequence types were ST-1 (17.5 %), ST-19 (10.4 %), ST-17 (9.7 %), ST-22 (8.4 %) and ST-23 (6.5 %). Serotype III was the most common, accounting for 29.2 % of the isolates. None of the serotypes was significantly associated with invasive neonatal disease. burst analysis resolved the 46 sequence types into seven lineages (clonal complexes), from which only lineage ST-17, expressing serotype III only, was significantly associated with invasive neonatal disease. Lineage ST-22 expressed mainly serotype II, and was significantly associated with carriage. The distribution of the various sequence types and lineages, and the association of lineage ST-17 with invasive disease, are consistent with the results of analyses from a global GBS isolate collection. These findings could imply that the global variation in disease incidence is independent of the circulating GBS populations, and may be more affected by other risk factors for invasive GBS disease, or by different prevention strategies.

Bennett JS, Griffiths DT, McCarthy ND, Sleeman KL, Jolley KA, Crook DW, Maiden MC. 2005. Genetic diversity and carriage dynamics of Neisseria lactamica in infants. Infect Immun, 73 (4), pp. 2424-2432. | Show Abstract | Read more

Neisseria lactamica, a harmless human commensal found predominantly in the upper respiratory tracts of infants, is closely related to Neisseria meningitidis, a pathogen of global significance. Colonization with N. lactamica may be responsible for the increase in immunity to meningococcal disease that occurs during childhood, when rates of meningococcal carriage are low. This observation has led to the suggestion that N. lactamica whole cells or components are potential constituents of novel meningococcal vaccines. However, the dynamics of carriage and population diversity of N. lactamica in children are poorly understood, presenting difficulties for the choice of representative isolates for use in vaccine development. This problem was addressed by the multilocus sequence typing of N. lactamica isolates from two longitudinal studies of bacterial carriage in infants. The studies comprised 100 and 216 subjects, with N. lactamica carriage monitored from age 4 weeks until age 96 weeks and from age 2 weeks until age 24 weeks, respectively. The maximum observed carriage rate was 44% at 56 weeks of age, with isolates obtained on multiple visits for the majority (54 of 75, 72%) of carriers. The N. lactamica isolates were genetically diverse, with 69 distinct genotypes recovered from the 75 infants. Carriage was generally long-lived, with an average rate of loss of under 1% per week during the 28 weeks following acquisition. Only 11 of the 75 infants carried more than one genotypically unique isolate during the course of the study. Some participants shared identical isolates with siblings, but none shared identical isolates with their parents. These findings have implications for the design of vaccines based on this organism.

Sleeman KL, Daniels L, Gardiner M, Griffiths D, Deeks JJ, Dagan R, Gupta S, Moxon ER, Peto TE, Crook DW. 2005. Acquisition of Streptococcus pneumoniae and nonspecific morbidity in infants and their families: a cohort study. Pediatr Infect Dis J, 24 (2), pp. 121-127. | Show Abstract | Read more

BACKGROUND: Most children are believed to acquire Streptococcus pneumoniae asymptomatically, with only a few developing overt S. pneumoniae disease. This study investigates the relationship between acquisition of S. pneumoniae and mild nonspecific infection leading to general practitioner (GP) consultation. METHODS: A prospective birth cohort study of 213 infants assessed at home 9 times during 24 weeks by nasopharyngeal swab and parental interview was conducted. RESULTS: All positive S. pneumoniae swabs (including acquisition and carriage) were significantly associated with GP consultations for infection by the study infant compared with infants with negative swabs [odds ratio (OR), 1.6; 95% confidence interval (CI) 1.1-2.2; P = 0.005]. There was a stronger association with S. pneumoniae acquisition alone (OR 2.1; 95% CI 1.3-3.4; P = 0.001) than with carriage only (OR 1.4; 95% CI 0.9-2.0; P = 0.1). Multivariate analysis confirmed that S. pneumoniae acquisition by the study subject was independently associated with GP consultations: adjusted hazard ratio, 1.8 (95% CI 1.1-2.9); P = 0.02. A similar and independent association was found between S. pneumoniae acquisition by the study subject, and GP consultations for infection by the family (adjusted hazard ratio, 1.8; 95% CI 1.1-2.8; P = 0.01). CONCLUSION: Acquisition of S. pneumoniae by the study infant was significantly associated with GP consultations for infection by the infant or family.

Bisharat N, Cohen DI, Harding RM, Falush D, Crook DW, Peto T, Maiden MC. 2005. Hybrid Vibrio vulnificus. Emerg Infect Dis, 11 (1), pp. 30-35. | Show Abstract | Read more

The recent emergence of the human-pathogenic Vibrio vulnificus in Israel was investigated by using multilocus genotype data and modern molecular evolutionary analysis tools. We show that this pathogen is a hybrid organism that evolved by the hybridization of the genomes from 2 distinct and independent populations. These findings provide clear evidence of how hybridization between 2 existing and nonpathogenic forms has apparently led to the emergence of an epidemic infectious disease caused by this pathogenic variant. This novel observation shows yet another way in which epidemic organisms arise.

Mohd-Zain Z, Turner SL, Cerdeño-Tárraga AM, Lilley AK, Inzana TJ, Duncan AJ, Harding RM, Hood DW, Peto TE, Crook DW. 2004. Transferable antibiotic resistance elements in Haemophilus influenzae share a common evolutionary origin with a diverse family of syntenic genomic islands. J Bacteriol, 186 (23), pp. 8114-8122. | Show Abstract | Read more

Transferable antibiotic resistance in Haemophilus influenzae was first detected in the early 1970s. After this, resistance spread rapidly worldwide and was shown to be transferred by a large 40- to 60-kb conjugative element. Bioinformatics analysis of the complete sequence of a typical H. influenzae conjugative resistance element, ICEHin1056, revealed the shared evolutionary origin of this element. ICEHin1056 has homology to 20 contiguous sequences in the National Center for Biotechnology Information database. Systematic comparison of these homologous sequences resulted in identification of a conserved syntenic genomic island consisting of up to 33 core genes in 16 beta- and gamma-Proteobacteria. These diverse genomic islands shared a common evolutionary origin, insert into tRNA genes, and have diverged widely, with G+C contents ranging from 40 to 70% and amino acid homologies as low as 20 to 25% for shared core genes. These core genes are likely to account for the conjugative transfer of the genomic islands and may even encode autonomous replication. Accessory gene clusters were nestled among the core genes and encode the following diverse major attributes: antibiotic, metal, and antiseptic resistance; degradation of chemicals; type IV secretion systems; two-component signaling systems; Vi antigen capsule synthesis; toxin production; and a wide range of metabolic functions. These related genomic islands include the following well-characterized structures: SPI-7, found in Salmonella enterica serovar Typhi; PAP1 or pKLC102, found in Pseudomonas aeruginosa; and the clc element, found in Pseudomonas sp. strain B13. This is the first report of a diverse family of related syntenic genomic islands with a deep evolutionary origin, and our findings challenge the view that genomic islands consist only of independently evolving modules.

Brueggemann AB, Peto TE, Crook DW, Butler JC, Kristinsson KG, Spratt BG. 2004. Temporal and geographic stability of the serogroup-specific invasive disease potential of Streptococcus pneumoniae in children. J Infect Dis, 190 (7), pp. 1203-1211. | Show Abstract | Read more

A meta-analysis study design was used to analyze 7 data sets of invasive and carriage pneumococcal isolates recovered from children, to determine whether invasive disease potential differs for each serotype and, if so, whether it has changed over time or differs geographically. Serotype- and serogroup-specific odds ratios (ORs) were calculated for each study and as a pooled estimate, with use of serotype 14 as the reference group. ORs varied widely: the serotypes with the highest ORs (1, 5, and 7) were 60-fold more invasive than those with the lowest ORs (3, 6A, and 15). There was a significant inverse correlation between invasive disease and carriage prevalence for the serotypes that we considered, which implies that the most invasive serotypes and serogroups were the least commonly carried and that the most frequently carried were the least likely to cause invasive disease. There was no evidence of any temporal change or major geographical differences in serotype- or serogroup-specific invasive disease potential.

Bisharat N, Crook DW, Leigh J, Harding RM, Ward PN, Coffey TJ, Maiden MC, Peto T, Jones N. 2004. Hyperinvasive neonatal group B streptococcus has arisen from a bovine ancestor. J Clin Microbiol, 42 (5), pp. 2161-2167. | Show Abstract | Read more

The genetic relatedness and evolutionary relationships between group B streptococcus (GBS) isolates from humans and those from bovines were investigated by phylogenetic analysis of multilocus sequence typing data. The collection of isolates consisted of 111 GBS isolates from cows with mastitis and a diverse global collection of GBS isolates from patients with invasive disease (n = 83) and carriers (n = 69). Cluster analysis showed that the majority of the bovine isolates (93%) grouped into one phylogenetic cluster. The human isolates showed greater diversity and clustered separately from the bovine population. However, the homogeneous human sequence type 17 (ST-17) complex, known to be significantly associated with invasive neonatal disease, was the only human lineage found to be clustered within the bovine population and was distinct from all the other human lineages. Split decomposition analysis revealed that the human isolate ST-17 complex, the major hyperinvasive neonatal clone, has recently arisen from a bovine lineage.

Dingle KE, Crook D, Jeffery K. 2004. Stable and noncompetitive RNA internal control for routine clinical diagnostic reverse transcription-PCR. J Clin Microbiol, 42 (3), pp. 1003-1011. | Show Abstract | Read more

Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G+C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.

Peacock SJ, Justice A, Griffiths D, de Silva GD, Kantzanou MN, Crook D, Sleeman K, Day NP. 2003. Determinants of acquisition and carriage of Staphylococcus aureus in infancy. J Clin Microbiol, 41 (12), pp. 5718-5725. | Show Abstract | Read more

Nasal carriage of Staphylococcus aureus is a major risk factor for invasive S. aureus disease. The aim of this study was to define factors associated with carriage. We conducted a prospective, longitudinal community-based study of infants and their mothers for a period of 6 months following delivery. The epidemiology of carriage was examined for 100 infant-mother pairs. Infant carriage varied significantly with age, falling from 40 to 50% during the first 8 weeks to 21% by 6 months. Determinants of infant S. aureus carriage included maternal carriage, breastfeeding, and number of siblings. Bacterial typing of S. aureus was performed by pulsed-field gel electrophoresis and multilocus sequence typing. The majority of individuals carried a single strain of S. aureus over time, and the mother was the usual source for colonizing isolates in infants. The effect of other components of the normal nasal flora on the development of S. aureus carriage was examined in 157 consecutive infants. Negative associations (putative bacterial interference) between S. aureus and other species occurred early in infancy but were not sustained. An increasing antistaphylococcal effect observed over time was not attributable to bacterial interference. S. aureus carriage in infants is likely to be determined by a combination of host, environmental, and bacterial factors, but bacterial interference does not appear to be an ultimate determinant of carrier status.

Grant CC, Harnden AR, Jewell G, Knox K, Peto TE, Crook DW. 2003. Invasive pneumococcal disease in Oxford, 1985-2001: a retrospective case series. Arch Dis Child, 88 (8), pp. 712-714. | Show Abstract | Read more

AIMS: To describe a series of children with invasive pneumococcal disease (IPD). METHODS: A review of patient records for children aged 0-18 years admitted to the John Radcliffe Hospital with IPD from 1985 to 2001. Social deprivation was measured by the Jarman index. The proportion of children with congenital abnormalities was compared with national data. RESULTS: We identified 140 children with IPD; complete data were available for 136 children. The median age at diagnosis was 1.5 years. The social deprivation score of households of children with IPD was higher than that of the average Oxfordshire household (-2.5 v -7.3, p < 0.001). Forty four per cent of cases had at least one preceding health problem. The children with preceding health problems were significantly older than those with no preceding problems (median age 2.67 years, interquartile range 1.21 to 6.20 versus 1.11 years, interquartile range 0.51 to 2.21; p < 0.001). There was an increased risk of IPD for children with central nervous system malformations (OR = 99, 95% CI 31 to 236), congenital heart disease (OR = 62, 95% CI 24 to 131), and chromosomal abnormalities (OR = 32, 95% CI 6.6 to 96). CONCLUSIONS: There is an increased risk of IPD associated with increased social deprivation; and also with central nervous system malformations, congenital heart disease, and chromosomal abnormalities.

Jones N, Bohnsack JF, Takahashi S, Oliver KA, Chan MS, Kunst F, Glaser P, Rusniok C et al. 2003. Multilocus sequence typing system for group B streptococcus. J Clin Microbiol, 41 (6), pp. 2530-2536. | Show Abstract | Read more

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (n = 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.

Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG. 2003. Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-specific differences in invasive disease potential. J Infect Dis, 187 (9), pp. 1424-1432. | Show Abstract | Read more

By use of multilocus sequence typing, Streptococcus pneumoniae isolates causing invasive disease (n=150) were compared with those from nasopharyngeal carriage (n=351) among children in Oxford. The prevalence of individual clones (sequence types) and serotypes among isolates from invasive disease was related to their prevalence in carriage, and an odds ratio (OR) for invasive disease was calculated for the major clones and serotypes. All major carried clones and serotypes caused invasive disease, although their ability to do so varied greatly. Thus, 2 serotype 14 clones were approximately 10-fold overrepresented among disease isolates, compared with carriage isolates, whereas a serotype 3 clone was approximately 10-fold underrepresented. The lack of heterogeneity between the ORs of different clones of the same serotype, and analysis of isolates of the same genotype, but different serotype, suggested that capsular serotype may be more important than genotype in the ability of pneumococci to cause invasive disease.

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Harnden A, Brueggemann A, Shepperd S, White J, Hayward AC, Zambon M, Crook D, Mant D. 2003. Near patient testing for influenza in children in primary care: comparison with laboratory test BRITISH MEDICAL JOURNAL, 326 (7387), pp. 480-480. | Read more

Harnden A, Brueggemann A, Shepperd S, White J, Hayward AC, Zambon M, Crook D, Mant D. 2003. Near patient testing for influenza in children in primary care: comparison with laboratory test. BMJ, 326 (7387), pp. 480. | Read more

Dimopoulou ID, Kartali SI, Kartalis GN, Manolas KI, Simopoulos KE, Vargemezis BA, Theodoropoulou-Rodiou G, Bowler IC, Crook DW. 2003. Relationship between nosocomial Acinetobacter species occurring in two geographical areas (Greece and the UK). J Hosp Infect, 54 (3), pp. 207-211. | Show Abstract | Read more

Fifty-two isolates of Acinetobacter spp. obtained from three Greek and one UK hospital, were studied using partial 16 S ribosomal DNA sequence analysis, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) mediated fingerprinting and DNA macro-restriction analysis. The aim was twofold: first, to discern the major differences in the population of Acinetobacter spp. between the two countries. Second, to compare a simple PCR-based typing scheme with pulsed-field gel electrophoresis (PFGE). The multi-resistant Greek isolates were within DNA groups 2 and TU13, and clustered into three types both by REP-PCR and PFGE. By contrast, the more susceptible Oxford isolates were heterogeneous on 16 S RNA sequence analysis and distinguishable on typing. The need for studies that elucidate the phylogeny of Acinetobacter spp. inside and outside hospitals are important, as this will help clarify the relationship between organisms that are increasingly recognized as causes of severe infections.

Meats E, Brueggemann AB, Enright MC, Sleeman K, Griffiths DT, Crook DW, Spratt BG. 2003. Stability of serotypes during nasopharyngeal carriage of Streptococcus pneumoniae. J Clin Microbiol, 41 (1), pp. 386-392. | Show Abstract | Read more

Serotype changes among natural isolates of Streptococcus pneumoniae are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.

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Roy S, Hill AVS, Knox K, Griffiths D, Crook D. 2002. Association of common genetic variant with susceptibility to invasive pneumococcal disease BRITISH MEDICAL JOURNAL, 324 (7350), pp. 1369-1369. | Read more

Roy S, Hill AV, Knox K, Griffiths D, Crook D. 2002. Research pointers: Association of common genetic variant with susceptibility to invasive pneumococcal disease. BMJ, 324 (7350), pp. 1369.

Roy S, Knox K, Segal S, Griffiths D, Moore CE, Welsh KI, Smarason A, Day NP et al. 2002. MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet, 359 (9317), pp. 1569-1573. | Show Abstract | Read more

BACKGROUND: Streptococcus pneumoniae is a major cause of morbidity and mortality in developed and developing countries. No common genetic determinants of susceptibility have been defined. Mannose-binding lectin (MBL) is a key mediator of innate host immunity that activates the complement pathway and directly opsonises some infectious pathogens. Mutations in three codons in the MBL gene have been identified, and individuals homozygous for a mutant genotype have very little or no serum MBL. We did a case-control study in the UK to assess whether these mutant genotypes were associated with invasive pneumococcal disease. METHODS: The frequencies of genotypes defined by the three mutations in codons 52, 54, and 57, and a functional promoter polymorphism at -221, were compared in a two-stage study of 337 patients with invasive pneumococcal disease and 1032 controls. All individuals were recruited from an ethnically homogeneous white population in Oxfordshire, UK. Patients had S pneumoniae isolated from a normally sterile site. FINDINGS: In our initial set of participants, 28 (12%) of 229 patients and 18 (5%) of 353 controls were homozygotes for MBL codon variants (odds ratio 2.59 [95% CI 1.39-4.83], p=0.002). Neither heterozygosity for these codon variants nor the promoter polymorphism was associated with susceptibility. In a confirmatory study, 11 (10%) of 108 patients were MBL homozygotes compared with 36 (5%) of 679 controls (p=0.046). INTERPRETATION: Homozygotes for MBL codon variants, who represent about 5% of north Europeans and north Americans and larger proportions of populations in many developing countries, could be at substantially increased risk of invasive pneumococcal disease.

Dimopoulou ID, Russell JE, Mohd-Zain Z, Herbert R, Crook DW. 2002. Site-specific recombination with the chromosomal tRNA(Leu) gene by the large conjugative Haemophilus resistance plasmid. Antimicrob Agents Chemother, 46 (5), pp. 1602-1603. | Show Abstract | Read more

Characterization of the sequences involved in recombination of the Haemophilus plasmid p1056 with the Haemophilus influenzae chromosome produced evidence indicating site-specific recombination with chromosomal tRNA(Leu). attP sequences identical to those of p1056 were found in six plasmids of diverse origin, suggesting that a family of Haemophilus plasmids recombines with chromosomal tRNA(Leu).

Grant C, Harnden A, Jewell G, Knox K, Griffiths D, Fuller A, Shepperd S, Mant D, Peto T, Crook D. 2002. Epidemiology of paediatric invasive pneumococcal disease in Oxford PEDIATRIC RESEARCH, 51 (4), pp. 276A-276A.

Grant C, Harnden A, Jewell G, Knox K, Griffiths D, Fuller A, Shepperd S, Mant D, Peto T, Crook D. 2002. Risk factors for meningitis or pneumonia in a cohort of children with invasive pneumococcal disease PEDIATRIC RESEARCH, 51 (4), pp. 275A-276A.

Banatvala JE, Roberts C, Crook D, Peto T. 2002. Infectious disease training: challenges and opportunities. Lancet Infect Dis, 2 (1), pp. 9-10. | Read more

Batchelor BIF, Crook DWM, Jones T, Bowler ICJW. 2002. Impact of guidelines for the diagnosis of urinary tract infection on trimethoprim susceptibility of Escherichia coli JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, 49 (1), pp. 223A-2224.

Sleeman K, Knox K, George R, Miller E, Waight P, Griffiths D, Efstratiou A, Broughton K et al. 2001. Invasive pneumococcal disease in England and Wales: vaccination implications. J Infect Dis, 183 (2), pp. 239-246. | Show Abstract | Read more

Knowledge of the epidemiology of invasive pneumococcal disease (IPD) will aid in planning the use of pneumococcal vaccines. A United Kingdom (UK)-based surveillance in England and Wales (1995-1997) of 11,528 individuals with IPD and a local enhanced surveillance in the Oxford (UK) area (1995-1999) have been analyzed. IPD has a high attack rate in children, with 37.1-48.1 cases per 100,000 infants <1 year old per year, and in older persons, with 21.2-36.2 cases per 100,000 persons >65 years old per year, for England, Wales, and Oxford. The 7-valent conjugate vaccine includes serotypes causing < or =79% of IPD in children <5 years old, but only 66% in adults >65 years old. The data also indicate that IPD varies by serotype, age, and country, emphasizing that the epidemiology of IPD is heterogeneous and requires continued surveillance.

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Shackley F, Knox K, Morris JB, Crook D, Griffiths D, Mayon-White R, George R, Willocks L, Moxon ER, Gr OPS. 2000. Outcome of invasive pneumococcal disease: a UK based study ARCHIVES OF DISEASE IN CHILDHOOD, 83 (3), pp. 231-233. | Show Abstract | Read more

Methods - The records of 106 children aged less than 5 years with invasive disease caused by Streptococcus pneumoniae were reviewed. Results - The clinical manifestations were meningitis (37%), upper respiratory tract infection (24%), pneumonia (19%), and occult bacteraemia (18%). One child died and seven had persisting neurological impairment. Five serotypes caused 83% of disease and 92% of the serotypes are included in the seven valent conjugate vaccines which are undergoing trials. Conclusions - These data suggest that S pneumoniae infection is associated with a low case fatality rate but substantial morbidity in the UK.

Shackley F, Knox K, Morris JB, Crook D, Griffiths D, Mayon-White R, George R, Willocks L, Moxon E. 2000. Outcome of invasive pneumococcal disease: a UK based study. Oxford Pneumococcal Surveillance Group. Arch Dis Child, 83 (3), pp. 231-233. | Show Abstract

METHODS: The records of 106 children aged less than 5 years with invasive disease caused by Streptococcus pneumoniae were reviewed. RESULTS: The clinical manifestations were meningitis (37%), upper respiratory tract infection (24%), pneumonia (19%), and occult bacteraemia (18%). One child died and seven had persisting neurological impairment. Five serotypes caused 83% of disease and 92% of the serotypes are included in the seven valent conjugate vaccines which are undergoing trials. CONCLUSIONS: These data suggest that S pneumoniae infection is associated with a low case fatality rate but substantial morbidity in the UK.

Enright MC, Knox K, Griffiths D, Crook DW, Spratt BG. 2000. Molecular typing of bacteria directly from cerebrospinal fluid. Eur J Clin Microbiol Infect Dis, 19 (8), pp. 627-630. | Show Abstract | Read more

Using Streptococcus pneumoniae as an example, the ability of multilocus sequence typing (MLST) to characterise isolates directly from cerebrospinal fluid (CSF) was investigated. A nested multiplex polymerase chain reaction method that amplifies the seven housekeeping gene fragments used for pneumococcal MLST was applied to 30 CSF samples from suspected cases of bacterial meningitis. The fragments were amplified from all 14 samples from which Streptococcus pneumoniae was cultured, and, after direct sequencing, the allelic profiles obtained from ten of the samples corresponded to those of clones previously associated with invasive pneumococcal disease. MLST could also predict the penicillin susceptibility and serotype of the CSF isolates.

Leaves NI, Dimopoulou I, Hayes I, Kerridge S, Falla T, Secka O, Adegbola RA, Slack MP, Peto TE, Crook DW. 2000. Epidemiological studies of large resistance plasmids in Haemophilus. J Antimicrob Chemother, 45 (5), pp. 599-604. | Show Abstract | Read more

The distribution of large conjugative Haemophilus influenzae plasmids in the nasopharyngeal haemophili of a group of people and in a large collection of 541 H. influenzae type b (Hib) isolates was studied. A newly developed PCR-based assay was used to detect the plasmids. The target sequences were chosen from sequence analysis of part of p1056, a large multiresistance plasmid isolated from a clinical Hib isolate, 1056. Fifty-nine per cent of people were found to carry beta-lactamase-positive (beta-lac(+)), ampicillin-resistant (ampR) haemophili with detectable plasmid sequences. Of these, 83% were in Haemophilus parainfluenzae and 17% were in H. influenzae. In the collection of 541 Hib, antibiotic resistance [beta-lac(+)ampR, beta-lac(+)ampR plus tetracycline resistance (tetR) or tetR] was highly correlated with large plasmids. It was found that 2.3% of the isolates contained large cryptic plasmids (i.e. these isolates were susceptible to antibiotics). The distribution of plasmids between invasive and carried Hib did not differ significantly (25 of 245 and 23 of 276, respectively). Isolates with large plasmids occur at high frequency in the nasopharynx of the normal human population and consist of two populations in Hib, one associated with specific antibiotic resistance traits and the other cryptic. These plasmids do not appear to influence the invasiveness of Hib.

Peacock SJ, Howe PA, Day NP, Crook DW, Winearls CG, Berendt AR. 2000. Outcome following staphylococcal peritonitis. Perit Dial Int, 20 (2), pp. 215-219. | Show Abstract

OBJECTIVE: Staphylococcus spp predominate as the causative pathogen of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis.This study evaluated the difference in morbidity and mortality between peritonitis caused by S. aureus and coagulase-negative staphylococci (CoNS). DESIGN: Prospective observational study. SETTING: A single regional dialysis unit in a teaching hospital. PATIENTS: Thirty-seven patients had S. aureus peritonitis and 65 patients had CoNS peritonitis between July 1990 and November 1995. MAIN OUTCOME MEASURES: Using the first recorded episode of peritonitis, survival analysis was performed for time to (1) death, (2) removal of peritoneal dialysis catheter, and (3) change to hemodialysis. Abdominal complications were recorded for the first and subsequent episodes. RESULTS: No difference in time to death was demonstrated for the two groups (p = 0.79), although two deaths that occurred during therapy for peritonitis were attributable to S. aureus infection. In addition, 5 patients developed serious abdominal complications related to an episode of S. aureus peritonitis. Patients with S. aureus peritonitis had a shorter time to both peritoneal dialysis catheter removal (p = 0.004) and change to hemodialysis (p = 0.014). The change in mode of dialysis was independent of catheter loss. CONCLUSION: This study highlights the serious nature of S. aureus peritonitis and confirms the need for effective preventive measures against infection by this pathogen.

Müller-Graf CD, Whatmore AM, King SJ, Trzcinski K, Pickerill AP, Doherty N, Paul J, Griffiths D, Crook D, Dowson CG. 1999. Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease. Microbiology, 145 ( Pt 11) (11), pp. 3283-3293. | Show Abstract | Read more

The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.

Dumpis U, Crook D, Oksi J. 1999. Tick-borne encephalitis. Clin Infect Dis, 28 (4), pp. 882-890. | Show Abstract | Read more

Tick-borne encephalitis (TBE) is a zoonotic arbovirus infection endemic to Russia and Eastern and Central Europe. Despite being a common and serious life-threatening disease for which a mass vaccination program was implemented in Austria, there is only limited reference to this disease in the English-language literature. TBE is transmitted to humans usually by the bite of a tick (either Ixodes persulcatus or Ixodes ricinus); occasionally, cases occur following consumption of infected unpasteurized milk. Transmission is seasonal and occurs in spring and summer, particularly in rural areas favored by the vector. TBE is a serious cause of acute central nervous system disease, which may result in death or long-term neurological sequelae. Effective vaccines are available in a few countries. The risk for travelers of acquiring TBE is increasing with the recent rise in tourism to areas of endemicity during spring and summer.

Fife AJ, Crook DWM. 1999. Automation in clinical microbiology METHODS IN MICROBIOLOGY, VOL 28, 28 pp. 1-15.

Scott JA, Hall AJ, Hannington A, Edwards R, Mwarumba S, Lowe B, Griffiths D, Crook D, Marsh K. 1998. Serotype distribution and prevalence of resistance to benzylpenicillin in three representative populations of Streptococcus pneumoniae isolates from the coast of Kenya. Clin Infect Dis, 27 (6), pp. 1442-1450. | Show Abstract | Read more

As surveillance data from sub-Saharan Africa are few, three representative populations of Streptococcus pneumoniae isolates were examined in Kenya for serotype distribution and Etest minimum inhibitory concentrations (MICs) of benzylpenicillin: (1) 75 lung aspirate or blood culture isolates from 301 consecutive adult patients with pneumonia, (2) 112 invasive isolates from continuous pediatric inpatient surveillance over 4 years, and (3) 97 nasopharyngeal isolates from systematically selected sick children. The proportions with benzylpenicillin MICs of > or = 0.1 microgram/mL were 0.27, 0.29, and 0.47, respectively. Vaccine-related serotypes accounted for 96% of invasive isolates from children and 90% of those from human immunodeficiency virus (HIV)-seropositive adults. Serotype 1 accounted for 44% of pneumococci from HIV-seronegative patients but only 5% of those from HIV-seropositive patients (P = .0002). Of serotype 1 isolates, 98% were susceptible to benzylpenicillin, but serogroups 13, 14, 19, and 23 were strongly associated with an MIC of > or = 0.1 microgram/mL.

Atkins BL, Athanasou N, Deeks JJ, Crook DW, Simpson H, Peto TE, McLardy-Smith P, Berendt AR. 1998. Prospective evaluation of criteria for microbiological diagnosis of prosthetic-joint infection at revision arthroplasty. The OSIRIS Collaborative Study Group. J Clin Microbiol, 36 (10), pp. 2932-2939. | Show Abstract

A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic joint infection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to the management and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue were obtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histology as the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using different cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom 297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could not be interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all samples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples taken. The isolation of an indistinguishable microorganism from three or more independent specimens was highly predictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sensitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performance of the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosis of infection be three or more operative specimens that yield an indistinguishable organism, and that because of its low level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.

Peacock SJ, Eddleston M, Emptage A, King A, Crook DW. 1998. Positive intravenous line tip cultures as predictors of bacteraemia. J Hosp Infect, 40 (1), pp. 35-38. | Show Abstract | Read more

Intravenous line tip cultures provide valuable information when taken in conjunction with blood culture, but in practice are often performed in isolation. This retrospective study has evaluated: (1) the frequency of isolated line tip culture; and (2) whether the species of microorganism isolated from line tip culture, using the Maki semi-quantitative culture method, is predictive of bacteraemia. Of 2753 line tip culture episodes in 1659 patients between May 1993 and August 1995, 2230 were performed in isolation (81%). Evaluation of 792 positive line tip culture episodes in 654 patients where blood cultures were performed in the period from 48 h before, to 24 h after tip culture, identified 825 line tip isolates. Of these, 194 were associated with a blood culture positive for the same species. The rate of positive blood culture, according to species, ranged from 10-72%. The highest rate was seen for methicillin-susceptible Staphylococcus aureus where 70 of 97 line tip episodes (72%) were associated with positive blood culture. This compared with a rate of 17% for coagulase-negative staphylococci (P < 0.0001). Patients with line tip cultures positive for S. aureus should be considered to be at high risk of bacteraemia.

Herbert MA, Crook D, Moxon ER. 1998. Molecular Methods for Haemophilus influenzae. Methods Mol Med, 15 pp. 243-263. | Show Abstract | Read more

The speciesHaemophilus influenzae belongs to the genus Haemophilus and the family Pasteurellaceae. H influenzae are small, nonmotile, nonspore forming, Gram-negative, pleomorphic rods that range in shape from coccobacilli to long filaments. They require X and V factors (hemin and NAD, respectively) for aerobic growth, and may be facultatively anaerobic (1) Encapsulated H. influenzae are classified into six antigenically distinct serotypes (a-f), and have a clonal population structure with two major global subdivisions (I and II) (2,3). Nonencapsulated H. infuenzae (NCHi) appear to have a nonclonal population composition, but a broader analysis of NCHi in the future may reveal that the population is not so distinct from encapsulated H influenzae (4). Both encapsulated H influenzae and NCHi exhibit wide genetic diversity (5).

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Akhter H, Berrios X, Collins J, Crook D, Fotherby K, Garza-Flores J, Hagenfeldt K, Hannaford P et al. 1998. Cardiovascular disease and steroid hormone contraception - Introduction CARDIOVASCULAR DISEASE AND STEROID HORMONE CONTRACEPTION, 877 pp. 1-89.

Bowler IC, Storr JA, Davies GJ, Crook DW. 1998. Guidelines for the management of patients colonized or infected with vancomycin-resistant enterococci. J Hosp Infect, 39 (1), pp. 75-77. | Read more

Crook DW, Spratt BG. 1998. Multiple antibiotic resistance in Streptococcus pneumoniae. Br Med Bull, 54 (3), pp. 595-610. | Show Abstract | Read more

Streptococcus pneumoniae remains a major pathogen responsible for high morbidity and mortality in both the developed and developing world. During the last few years there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci, and the emergence of isolates with high-level resistance to extended-spectrum cephalosporins. In several countries, 50-80% of pneumococcal isolates, including the great majority of isolates of the serotypes associated with disease and carriage in children, are penicillin-resistant. Penicillin-resistant pneumococci are diverse, but in several countries successful highly penicillin-resistant clones (which in most cases are resistant also to tetracycline, chloramphenicol, and cotrimoxazole, and increasingly to erythromycin) have emerged, and some of these have spread globally. The effect of antibiotic resistance on the clinical outcome of otitis media, pneumonia and meningitis, and the potential of the new conjugate vaccines for controlling pneumococcal disease, are discussed.

Heath PT, Bowen-Morris J, Griffiths D, Griffiths H, Crook DW, Moxon ER. 1997. Antibody persistence and Haemophilus influenzae type b carriage after infant immunisation with PRP-T. Arch Dis Child, 77 (6), pp. 488-492. | Show Abstract | Read more

OBJECTIVES: To assess the persistence of serum Haemophilus influenzae type b antibodies and the prevalence of H influenzae type b carriage in a group of preschool age children previously vaccinated in infancy. DESIGN: Names were randomly selected from immunisation records. Families were visited on five occasions over a period of 12 months and throat swabs were taken from all family members present, with blood obtained from children at the first and last visits. RESULTS: One hundred and fifty three children at a median age of 3.6 years had a geometric mean titre (GMT) of 1.06 micrograms/ml (95% CI 0.80 to 1.38). Eight per cent had an undetectable antibody concentration, received a booster dose of plain PRP vaccine, and responded with concentrations > 2 micrograms/ml. GMT at 4.5 years of age was 0.89 microgram/ml (0.69 to 1.16). Twelve children who had been exposed to H influenzae had a GMT of 4.7 v 0.8 micrograms/ml for those without exposure. CONCLUSIONS: Accelerated immunisation against H influenzae without a second year booster results in persistence of satisfactory serum concentrations of antibody to 4.5 years of age. In those with undetectable antibody, immunological memory may still be present.

Barnes PD, Crook DW. 1997. Culture negative endocarditis. J Infect, 35 (3), pp. 209-213. | Read more

Peacock S, Howe P, Crook D, Berendt A, Winearls C. 1997. Outcome following peritoneal dialysis-related staphylococcal peritonitis KIDNEY INTERNATIONAL, 52 (1), pp. 274-274.

Dimopoulou ID, Jordens JZ, Legakis NJ, Crook DW. 1997. A molecular analysis of Greek and UK Haemophilus influenzae conjugative resistance plasmids. J Antimicrob Chemother, 39 (3), pp. 303-307. | Show Abstract | Read more

Antibiotic resistance in Haemophilus influenzae has been associated with the presence of large, chromosomally integrated, conjugative plasmids. The plasmids of 10 beta-lactamase-positive, ampicillin-resistant strains, two from the UK and eight from Greece, were investigated. Plasmids were detected and isolated after transfer to a rec-deficient recipient. Purified whole plasmid was used as probe. In addition a 12 kb PstI fragment containing the putative point of recircularization in one plasmid, p1056, was cloned and used as a probe. All plasmids shared a high degree of sequence homology suggesting that plasmids of diverse geographical origin are highly related. All plasmids also shared sequence homology with the 12 kb PstI fragment containing the point of recircularization, suggesting that the sequences involved in excision and recircularization are conserved.

Weller TM, Crook DW, Crow MR, Ibrahim W, Pennington TH, Selkon JB. 1997. Methicillin susceptibility testing of staphylococci by Etest and comparison with agar dilution and mecA detection. J Antimicrob Chemother, 39 (2), pp. 251-253. | Show Abstract | Read more

The susceptibility to methicillin of 44 Staphylococcus aureus and 120 coagulase-negative staphylococci (CNS) was determined by Etest, agar dilution and presence of the mecA gene. There was agreement between the results of all methods when testing S. aureus. However, discrepancies occurred with CNS when cultural methods were compared with presence of the mecA gene. mecA-positive isolates tested as resistant more often with agar dilution on Columbia agar plus 5% NaCl than by Etest.

Lessing MP, Crook DW, Bowler IC, Gribbin B. 1996. Native-valve endocarditis caused by Staphylococcus lugdunensis. QJM, 89 (11), pp. 855-858. | Show Abstract | Read more

Coagulase-negative staphylococci cause about 5% of native-valve endocarditis. Staphylococcus lugdunensis, a recently-described species of coagulase-negative staphylococci, has been reported to cause destructive native-valve endocarditis with a high mortality. We report four consecutive cases of definite Staphylococcus lugdunensis native-valve endocarditis by the Duke criteria over a 4-year period. All patients required urgent aortic valve replacement 1-5 days after admission, and recovered. An intriguing, aspect in the presentation of these patients was a history of vasectomy and inguinal skin breaks in the immediate period preceding the occurrence of endocarditis.

Kayley J, Berendt AR, Snelling MJ, Moore H, Hamilton HC, Peto TE, Crook DW, Conlon CP. 1996. Safe intravenous antibiotic therapy at home: experience of a UK based programme. J Antimicrob Chemother, 37 (5), pp. 1023-1029. | Show Abstract | Read more

Outpatient i.v. antibiotic therapy is well developed in the United States, largely because of pressures from third-party payers to reduce costs of medical care. We have developed an outpatient i.v. antibiotic programme in Oxford, that has evolved from a desire to provide high quality i.v. therapy to AIDS patients with cytomegalovirus retinitis. We describe the rationale of the service and report on our first two years' experience. We treated 67 consecutive patients (eight with HIV infection) at home with i.v. antibiotics. This resulted in a saving of 2275 hospital days for those patients without HIV infection. HIV positive patients received 69 months of home i.v. therapy. Minor intravascular catheter complications occurred in only five patients (7.5%). The only serious complications were three episodes of catheter-related sepsis (4.5%), all occurring in AIDS patients who had lines in for more than six months. We have shown that home i.v. antibiotic therapy can be delivered safely to patients with a wide variety of infectious problems using the existing network of community nurses in the National Health Service. Essential components to the programme include a multidisciplinary team working between the hospital and community and a written shared care protocol. Such a programme can result in reduced lengths of hospital stay and patient, community nurse and physician satisfaction.

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Kayley J, Berendt AR, Snelling MJM, Moore H, Hamilton HC, Peto TEA, Crook DWM, Conlon CP. 1996. Antimicrobial practice - Safe intravenous antibiotic therapy at home: Experience of a UK based programme Journal of Antimicrobial Chemotherapy, 37 (5), pp. 1023-1029. | Show Abstract

Outpatient iv antibiotic therapy is well developed in the United States, largely because of pressures from third-party payers to reduce costs of medical care. We have developed an outpatient iv antibiotic programme in Oxford, that has evolved from a desire to provide high quality iv therapy to AIDS patients with cytomegalovirus retinitis. We describe the rationale of the service and report on our first two years' experience. We treated 67 consecutive patients (eight with HIV infection) at home with iv antibiotics. This resulted in a saving of 2275 hospital days for those patients without HIV infection. HIV positive patients received 69 months of home iv therapy. Minor intravascular catheter complications occurred in only five patients (7.5%). The only serious complications were three episodes of catheter-related sepsis (4.5%), all occurring in AIDS patients who had lines in for more than six months. We have shown that home iv antibiotic therapy can be delivered safely to patients with a wide variety of infectious problems using the existing network of community nurses in the National Health Service. Essential components to the programme include a multidisciplinary team working between the hospital and community and a written shared care protocol. Such a programme can result in reduced lengths of hospital stay and patient, community nurse and physician satisfaction.

Batchelor BI, Hunt AR, Bowler IC, Crook DW. 1996. Laboratory detection of leucocyte esterase and nitrite as an alternative to urine microscopy. Eur J Clin Microbiol Infect Dis, 15 (8), pp. 663-664. | Read more

Sung RY, Ling JM, Fung SM, Oppenheimer SJ, Crook DW, Lau JT, Cheng AF. 1995. Carriage of Haemophilus influenzae and Streptococcus pneumoniae in healthy Chinese and Vietnamese children in Hong Kong. Acta Paediatr, 84 (11), pp. 1262-1267. | Show Abstract | Read more

Nasopharyngeal carriage of Haemophilus influenzae and Streptococcus pneumoniae was studied in 621 healthy Chinese children and 300 healthy Vietnamese children aged from 2 months to 5 years in Hong Kong. The carriage rate of H. influenzae type b in Vietnamese children was 1.3% (CI 0.04-2.63); it was zero in Chinese. The carriage rate of non-typable H. influenzae was 5.8% (CI 1.4-7.6%) in Chinese and 65.4% (CI 58.9-69.8%) in Vietnamese. The carriage rates of S. pneumoniae were 10.8% (CI 8.3-13.2%) and 55.7% (CI 50.1-61.3%) in Chinese and Vietnamese children, respectively. Univariate and multivariate logistic regression analyses were performed to search for factors associated with differences in carriage rates of both H. influenzae and S. pneumoniae between Chinese and Vetnamese children. Although older age, smaller living area and parental smoking were associated with higher carriage rates, these could not explain the remarkably low carriage rates of both bacteria in Chinese children.

Emerton ME, Crook DW, Cooke PH. 1995. Streptococcus bovis-infected total hip arthroplasty. J Arthroplasty, 10 (4), pp. 554-555. | Show Abstract | Read more

The case of a 51-year-old man who underwent a total hip arthroplasty following failed AO screw fixation of a subcapital femoral neck fracture is reported. Infection of the prosthesis with Streptococcus bovis type 1 followed a febrile illness. Further investigation revealed an occult premalignant polyp in the proximal colon. Colonic neoplasia and S. bovis bacteremia are associated with endocarditis; however, S. bovis is a rare pathogen infecting joint prostheses and should raise the possibility of a gastrointestinal lesion.

Leaves NI, Falla TJ, Crook DW. 1995. The elucidation of novel capsular genotypes of Haemophilus influenzae type b with the polymerase chain reaction. J Med Microbiol, 43 (2), pp. 120-124. | Show Abstract | Read more

Molecular characterization is an important pre-requisite for post-vaccine studies of Haemophilus influenzae type b (Hib). Three capsular genotyping patterns, b(S), b(G) and b(V), have been described in the major phylogenetic lineage of Hib. However, in a recent series of prospective studies, three new hybridisation patterns were observed among 425 strains of Hib. Four pairs of polymerase chain reaction (PCR) primers were used to identify the capsular gene (cap) structure of these Hib strains. This showed that the strains possessed simple DNA re-arrangements. In two instances a change in restriction enzyme recognition site was the most likely cause of the new hybridisation pattern. The third strain possessed a cap b locus consisting of intact tandem repeats of cap b in a b(S) background. It was reasoned that a similar cap b locus would not be readily recognised by hybridisation in a b(G) background, and b(G) strains were therefore characterized by the PCR method. This showed one of 35 b(G) strains to possess a cap locus with intact tandem repeat copies of cap b. The novel capsular genotypes described here are rare, but can be detected rapidly and accurately by a combination of PCR and capsular genotyping hybridisation patterns.

Peacock SJ, Bowler IC, Crook DW. 1995. Positive predictive value of blood cultures growing coagulase-negative staphylococci. Lancet, 346 (8968), pp. 191-192. | Read more

Tang CM, Howe P, Crook DW. 1995. The treatment of candidemia. N Engl J Med, 332 (16), pp. 1101.

Falla TJ, Crook DW, Anderson EC, Ward JI, Santosham M, Eskola J, Moxon ER. 1995. Characterization of capsular genes in Haemophilus influenzae isolates from H. influenzae type b vaccine recipients. J Infect Dis, 171 (4), pp. 1075-1076. | Read more

Athanasou NA, Pandey R, de Steiger R, Crook D, Smith PM. 1995. Diagnosis of infection by frozen section during revision arthroplasty. J Bone Joint Surg Br, 77 (1), pp. 28-33. | Show Abstract

We assessed the efficacy of intraoperative frozen-section histology in detecting infection in failed arthroplasties in 106 hips and knees. We found inflammatory changes consistent with infection (an average of one or more neutrophil polymorphs or plasma cells per high-power field in several samples) in 18 cases; there was a significant growth on bacterial culture in 20 cases. Compared with the bacterial cultures, the frozen sections provided two false-negative results and three false-positive results (sensitivity, 90%; specificity, 96%; and accuracy, 95%). The positive predictive value was 88%, the negative value, 98%. These results support the inclusion of intra-operative frozen-section histology in any protocol for revision arthroplasty for loose components.

Barbour ML, Mayon-White RT, Coles C, Crook DW, Moxon ER. 1995. The impact of conjugate vaccine on carriage of Haemophilus influenzae type b. J Infect Dis, 171 (1), pp. 93-98. | Show Abstract | Read more

Conjugate vaccines against Haemophilus influenzae type b (Hib) may modify Hib pharyngeal colonization. Hib colonization was compared in 371 infants and their families. In Oxfordshire, infants received PRP-T (polyribosylribitol phosphate conjugated to tetanus toxoid) and in Buckinghamshire they did not (controls). Infants were followed at 6, 9, and 12 months of age. Also, 6 unvaccinated Hib carriers were vaccinated and followed for 6 weeks. Hib acquisition was lower in vaccinees than controls (P < .01). During surveillance, 1.5% of vaccinees and 6.3% of controls carried Hib (P = .04). Among those with family Hib exposure, the carriage rates were 8.7% and 38.5% (P = .07), respectively. Hiv carriage rates were lower among vaccinees' unvaccinated siblings. Giving conjugate vaccine to a child carrying Hib did not rapidly terminate carriage. Thus, a primary means by which herd immunity to Hib is induced in a vaccinated population may be through reduction or delay in the initial acquisition of Hib.

Amyes SG, Baird DR, Crook DW, Gillespie SH, Howard AJ, Oppenhiem BA, Pedler SJ, Paull A, Tompkins DS, Lawrie SA. 1994. A multicentre study of the in-vitro activity of cefotaxime, cefuroxime, ceftazidime, ofloxacin and ciprofloxacin against blood and urinary pathogens. J Antimicrob Chemother, 34 (5), pp. 639-648. | Show Abstract | Read more

The in-vitro susceptibilities of aerobic bacteria isolated from 1804 blood and 4529 urine specimens collected at nine hospitals in the UK were examined. An agar dilution method was used to determine the MICs of each isolate to three cephalosporins, cefotaxime, cefuroxime and ceftazidime, and to two fluoroquinolones, ofloxacin and ciprofloxacin. Sensitivities were then calculated using British Society for Antimicrobial Chemotherapy recommended breakpoints. Of the cephalosporins tested cefotaxime was the most active against the Enterobacteriaceae. All the systemic staphylococcus isolates collected were sensitive to both cefotaxime and cefuroxime. As expected, ceftazidime was the only cephalosporin active against the Pseudomonas isolates. Both quinolones were highly active against the Enterobacteriaceae and Pseudomonas spp. They also demonstrated good Gram-positive activity, particularly against Staphylococcus aureus and Enterococcus spp.

Falla TJ, Crook DW, Brophy LN, Maskell D, Kroll JS, Moxon ER. 1994. PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol, 32 (10), pp. 2382-2386. | Show Abstract

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.

Weller TM, Smith PM, Crook DW. 1994. Corynebacterium jeikeium osteomyelitis following total hip joint replacement. J Infect, 29 (1), pp. 113-114. | Read more

Barbour ML, Mayon-White RT, Booy R, Moxon ER, Crook DW. 1993. Cardiac transplantation and immunisation against Haemophilus influenzae. Lancet, 342 (8883), pp. 1363-1364. | Read more

A'Court CH, Garrard CS, Crook D, Bowler I, Conlon C, Peto T, Anderson E. 1993. Microbiological lung surveillance in mechanically ventilated patients, using non-directed bronchial lavage and quantitative culture. Q J Med, 86 (10), pp. 635-648. | Show Abstract | Read more

We surveyed bronchial microflora by alternate-day, non-directed bronchial lavage (NBL) in 150 patients requiring mechanical ventilation on an intensive care unit. This simple technique uses a 20 ml non-bronchoscopic lung lavage, then quantitative bacterial culture. NBL bacteriological findings were identical to those obtained by same-day bronchoscopic broncho-alveolar lavage on 16/20 occasions. Using serial NBLs, the bronchial bacterial population was characterized during 65 episodes of pneumonia defined by clinical and retrospective criteria. Mean bacterial colony counts increased significantly during the 2 days preceding the clinical onset of pneumonia, from < or = 10(3) cfu/ml to > or = 10(5) cfu/ml (p < 0.05). In 51 patients showing a clinical response to antibiotic treatment, mean colony counts fell significantly after antibiotic initiation (p < 0.05). By contrast, in 14 patients who showed progressive clinical deterioration or relapse, there was no significant fall in NBL counts, and serial NBLs revealed antibiotic resistance or superinfection. The surveillance data altered clinical management in 42% of patients. Positive NBLs guided the choice of antibiotics, whilst negative NBLs encouraged the withholding of antibiotics, or detection of alternative pathology. We propose routine bacteriological lung surveillance of mechanically ventilated patients using this simple, inexpensive and safe technique.

Barbour ML, Booy R, Crook DW, Griffiths H, Chapel HM, Moxon ER, Mayon-White D. 1993. Haemophilus influenzae type b carriage and immunity four years after receiving the Haemophilus influenzae oligosaccharide-CRM197 (HbOC) conjugate vaccine. Pediatr Infect Dis J, 12 (6), pp. 478-484. | Show Abstract | Read more

Late in 1991, before the implementation of a national immunization program against Haemophilus influenzae type b (Hib) in the United Kingdom, we performed a 4-year follow-up of 120 children who in 1987 had been enrolled in an immunogenicity trial in which 60 of them (vaccinees) received an Hib conjugate vaccine (HbOC) at the same time as diphtheria-tetanus toxoid-pertussis vaccine at the ages of 3, 5 and 9 months. Sixty others (controls) received only diphtheria-tetanus toxoid-pertussis vaccine at the same ages and were not subsequently immunized against Hib. We investigated Hib pharyngeal colonization using the antiserum agar method and the concentrations of serum IgG antibody to the type b capsule by enzyme-linked immunosorbent assay. At 4 years of age the Hib colonization rates in vaccinees and controls were 8% (5 of 60) and 5% (3 of 60), respectively. The children colonized with Hib had greater serum anti-capsular IgG concentrations than did noncolonized children (P < 0.001), and colonized vaccinees tended to have higher concentrations than colonized controls (P = 0.053). Regardless of Hib colonization status vaccinees had greater antibody concentrations than controls (P < 0.001). Forty-nine percent of vaccinees had an antibody concentration > 1 microgram/ml. There was an inverse relationship between the Hib colony count on culture and the serum IgG concentration. These data indicate that the increase in serum antibody concentration after immunization with an Hib conjugate vaccine is sustained and that immunization primes for a booster response on exposure to Hib. There may be a relationship between previous Hib conjugate immunization and the density of Hib colonization in children.

Falla TJ, Dobson SR, Crook DW, Kraak WA, Nichols WW, Anderson EC, Jordens JZ, Slack MP, Mayon-White D, Moxon ER. 1993. Population-based study of non-typable Haemophilus influenzae invasive disease in children and neonates. Lancet, 341 (8849), pp. 851-854. | Show Abstract | Read more

The extent of non-capsulate, non-serotypable Haemophilus influenzae (NST) as a cause of serious invasive disease in children has not been fully defined. We describe the epidemiology of these childhood infections from cases identified during a continuing prospective survey of invasive H influenzae disease in the Oxford region, UK. 408 strains of H influenzae were isolated from cases of invasive disease. 383 (94%) were H influenzae type b (Hib), 24 (6%) were NST strains, and 1 was a type f strain. 3 of the NST strains were non-capsulate type b mutants (b-), but the remaining 21 strains were from the phylogenetically distinct and heterogeneous population of non-capsulate H influenzae (NC). 10 of the NC strains were isolated from neonates with sepsis; crude mortality rate was 40%, with an incidence of 4.6 cases per 100,000 livebirths. 11 NC strains were isolated from children after the neonatal period and under 10 years of age, 4 (36%) of which had severe, unrelated, predisposing conditions. The incidence of NC invasive diseases in these children was 0.5 per 100,000 per year. The attributable mortality for these infections was 10%. Infections due to these H influenzae strains are, after the implementation of Hib vaccines, likely to persist and represent a substantial proportion of the serious infections caused by this species.

Barbour ML, Crook DW, Mayon-White RT. 1993. An improved antiserum agar method for detecting carriage of Haemophilus influenzae type b. Eur J Clin Microbiol Infect Dis, 12 (3), pp. 215-217. | Show Abstract | Read more

Enriched Columbia medium was tested against Levinthal medium for the isolation of Haemophilus influenzae type b. In both media, Haemophilus influenzae type b recovery and antigen-antibody precipitation halos were equivalent. Haemophilus influenzae type b colony size and iridescence were superior on enriched Columbia medium. Enriched Columbia medium is inexpensive, simply prepared and easily standardised.

Falla TJ, Anderson EC, Chappell MM, Slack MP, Crook DW. 1993. Cross-reaction of spontaneous capsule-deficient Haemophilus influenzae type b mutants with type-specific antisera. Eur J Clin Microbiol Infect Dis, 12 (2), pp. 147-148. | Read more

Paul J, White SH, Nicholls KM, Crook DW. 1992. Prosthetic joint infection due to Candida parapsilosis in the UK: case report and literature review. Eur J Clin Microbiol Infect Dis, 11 (9), pp. 847-849. | Show Abstract | Read more

Candida infection of joint replacements is a rare but increasingly reported phenomenon. A case of Candida parapsilosis prosthetic knee joint infection occurring in the UK is described. Cure followed removal of the prosthesis and treatment, first with a combination of amphotericin and 5-fluorocytosine, then ketoconazole.

Dimopoulou ID, Kraak WA, Anderson EC, Nichols WW, Slack MP, Crook DW. 1992. Molecular epidemiology of unrelated clusters of multiresistant strains of Haemophilus influenzae. J Infect Dis, 165 (6), pp. 1069-1075. | Show Abstract | Read more

Three epidemiologically unrelated clusters of Haemophilus influenzae resistant to ampicillin, chloramphenicol, and tetracycline were studied. The biotypes and cell-envelope protein patterns were determined for 17 nonencapsulated strains, 6 from Dundee and 11 from Cheltenham, and for 6 type b encapsulated strains from Guildford. After mobilization by conjugation, large 32- to 36-MDa plasmids were purified from all the strains. The restriction fragment patterns of the plasmids were determined by ethidium bromide staining of digested purified plasmid or by Southern hybridization of digested total cellular DNA of the parent strains, probed with purified plasmid. Evidence is presented for a chromosomal location of the plasmids in the parent strains, the spread in nature of a plasmid between distinguishable strains of H. influenzae, the person-to-person spread of a strain within a cluster, and a high degree of sequence homology between distinguishable plasmids, implying their close relatedness.

Willemen D, Paul J, White SH, Crook DW. 1991. Closed suction drainage following knee arthroplasty. Effectiveness and risks. Clin Orthop Relat Res, (264), pp. 232-234. | Show Abstract

A prospective investigation was performed to determine when to remove a suction drain following total knee arthroplasty (TKA). Forty-one TKAs were randomly allocated to closed suction drainage for either 24 or 48 hours. The drain was removed and the tip was cut off and processed by a method giving quantitative cultures. In the 48-hour group, 85% of the total volume was drained during the first 24 hours. During the following 24-hour period, a mean volume of only 50 ml was drained. No organism was isolated from cultures of drain tips sampled at 24 hours. However, at 48 hours, 25% of the drain tips yielded light growths of coagulase-negative staphylococci (four drain tips) and Staphylococcus aureus (one drain tip). Clinical evaluations of wound healing were comparable in the two groups. Clearly, nothing is to be gained by continuing drainage beyond 24 hours. If drainage is maintained for longer periods, there is an increased risk of contamination by bacteria.

Garrard CS, Crook DWM. 1991. Nosocomial pneumonia in the critically III Current Anaesthesia & Critical Care, 2 (3), pp. 155-160. | Show Abstract | Read more

Despite the large body of published work emphasising the importance of nosocomial pneumonia its true significance with respect to mortality is difficult to assess. This partially stems from the limitations imposed by clinical diagnostic criteria and from the interpretation of multivariate analyses which in themselves can only confirm correlations and associations. Two key questions need to be resolved. First, what is the true incidence of nosocomial pneumonia? Microbiological data would suggest the incidence to be much lower than previously suspected and this view is supported by the relative rarity of severe lung infections manifested as lung cavitation, empyema or bacteraemia. Second, what is the true mortality attributable to nosocomial pneumonia? Clinical studies need to examine the mode of death and not just confirm the presence of pneumonia at the time of death. Only in cases where overwhelming lung infection leads to an inability to maintain respiratory or haemodynamic homeostasis can death be genuinely attributed to nosocomial pneumonia. Clinical experience suggests that the attributable mortality is low, an impression that is reinforced by the relatively small impact of selective decontamination of the digestive tract (SDD) upon survival in patients with nosocomial pneumonia. Until these questions are answered satisfactorily it is appropriate to take reasonable measures to prevent nosocomial pneumonia but to embark on antibiotic therapy only when there is supportive microbiological evidence or in the presence of clinically indisputable lung infection. © 1991 Longman Group UK Ltd.

Haley S, Paul J, Crook DW, White SH. 1990. Acinetobacter spp L-form infection of a cemented Charnley total hip replacement. J Clin Pathol, 43 (9), pp. 781. | Read more

Brightman CA, Crook DW, Kraak WA, Dimopoulou ID, Anderson EC, Nichols WW, Slack MP. 1990. Family outbreak of chloramphenicol-ampicillin resistant Haemophilus influenzae type b disease. Lancet, 335 (8685), pp. 351-352. | Read more

Clifford CP, Crook DW, Conlon CP, Fraise AP, Day DG, Peto TE. 1990. Impact of waterborne outbreak of cryptosporidiosis on AIDS and renal transplant patients. Lancet, 335 (8703), pp. 1455-1456. | Read more

Das S, Lindemann C, Young BC, Muller J, Österreich B, Ternette N, Winkler AC, Paprotka K et al. 2016. Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation. Proc Natl Acad Sci U S A, 113 (22), pp. E3101-E3110. | Show Abstract | Read more

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Seale AC, Koech AC, Sheppard AE, Barsosio HC, Langat J, Anyango E, Mwakio S, Mwarumba S et al. 2016. Maternal colonisation with Streptococcus agalactiae, and associated stillbirth and neonatal disease in coastal Kenya. Nat Microbiol, 1 (7), pp. 16067-16067. | Show Abstract | Read more

Streptococcus agalactiae (Group B Streptococcus, GBS) causes neonatal disease and stillbirth, but its burden in sub-Saharan Africa is uncertain. We assessed maternal recto-vaginal GBS colonisation (7967 women), stillbirth and neonatal disease. Whole genome sequencing was used to determine serotypes, sequence types (ST), and phylogeny. We found low maternal GBS colonisation prevalence (934/7967, 12%), but comparatively high incidence of GBS-associated stillbirth and early onset neonatal disease (EOD) in hospital (0.91(0.25-2.3)/1000 births; 0.76(0.25-1.77)/1000 live-births respectively). However, using a population denominator, EOD incidence was considerably reduced (0.13(0.07-0.21)/1000 live-births). Treated cases of EOD had very high case fatality (17/36, 47%), especially within 24 hours of birth, making under-ascertainment of community-born cases highly likely, both here and in similar facility-based studies. Maternal GBS colonisation was less common in women with low socio-economic status, HIV infection and undernutrition, but when GBS-colonised, they were more likely colonised by the most virulent clone, CC17. CC17 accounted for 267/915(29%) of maternal colonising (265/267(99%) serotype III, 2/267(0.7%) serotype IV), and 51/73(70%) of neonatal disease cases (all serotype III). Trivalent (Ia/II/III) and pentavalent (Ia/Ib/II/III/V) vaccines would cover 71/73(97%) and 72/73(99%) of disease-causing serotypes respectively. Serotype IV should be considered for inclusion, with evidence of capsular switching in CC17 strains.

Sheppard AE, Stoesser N, Wilson DJ, Sebra R, Kasarskis A, Anson LW, Giess A, Pankhurst LJ et al. 2016. Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC. Antimicrob Agents Chemother, 60 (6), pp. 3767-3778. | Show Abstract | Read more

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

Sheppard AE, Vaughan A, Jones N, Turner P, Turner C, Efstratiou A, Patel D, Modernising Medical Microbiology Informatics Group et al. 2016. Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data. J Clin Microbiol, 54 (5), pp. 1388-1390. | Show Abstract | Read more

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types.

Quan TP, Fawcett NJ, Wrightson JM, Finney J, Wyllie D, Jeffery K, Jones N, Shine B et al. 2016. Increasing burden of community-acquired pneumonia leading to hospitalisation, 1998-2014. Thorax, 71 (6), pp. 535-542. | Show Abstract | Read more

BACKGROUND: Community-acquired pneumonia (CAP) is a major cause of mortality and morbidity in many countries but few recent large-scale studies have examined trends in its incidence. METHODS: Incidence of CAP leading to hospitalisation in one UK region (Oxfordshire) was calculated over calendar time using routinely collected diagnostic codes, and modelled using piecewise-linear Poisson regression. Further models considered other related diagnoses, typical administrative outcomes, and blood and microbiology test results at admission to determine whether CAP trends could be explained by changes in case-mix, coding practices or admission procedures. RESULTS: CAP increased by 4.2%/year (95% CI 3.6 to 4.8) from 1998 to 2008, and subsequently much faster at 8.8%/year (95% CI 7.8 to 9.7) from 2009 to 2014. Pneumonia-related conditions also increased significantly over this period. Length of stay and 30-day mortality decreased slightly in later years, but the proportions with abnormal neutrophils, urea and C reactive protein (CRP) did not change (p>0.2). The proportion with severely abnormal CRP (>100 mg/L) decreased slightly in later years. Trends were similar in all age groups. Streptococcus pneumoniae was the most common causative organism found; however other organisms, particularly Enterobacteriaceae, increased in incidence over the study period (p<0.001). CONCLUSIONS: Hospitalisations for CAP have been increasing rapidly in Oxfordshire, particularly since 2008. There is little evidence that this is due only to changes in pneumonia coding, an ageing population or patients with substantially less severe disease being admitted more frequently. Healthcare planning to address potential further increases in admissions and consequent antibiotic prescribing should be a priority.

Pankhurst LJ, Del Ojo Elias C, Votintseva AA, Walker TM, Cole K, Davies J, Fermont JM, Gascoyne-Binzi DM et al. 2016. Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study. Lancet Respir Med, 4 (1), pp. 49-58. | Show Abstract | Read more

BACKGROUND: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. METHODS: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. FINDINGS: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. INTERPRETATION: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. FUNDING: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.

Miller R, Walker AS, Knox K, Wyllie D, Paul J, Haworth E, Mant D, Peto T, Crook DW. 2010. 'Feral' and 'wild'-type methicillin-resistant Staphylococcus aureus in the United Kingdom. Epidemiol Infect, 138 (5), pp. 655-665. | Show Abstract | Read more

Circulation of methicillin-resistant Staphylococcus aureus (MRSA) outside hospitals could alter the impact of hospital-based control strategies. We investigated two groups of cases (each matched to controls with MRSA): 61 'community cases' not in acute hospital in the year before MRSA isolation; and 21 cases with ciprofloxacin-sensitive (CipS) MRSA. Multi-locus sequence typing, spa-typing and Panton-Valentine leukocidin gene testing were performed and demographics obtained. Additional questionnaires were completed by community case GPs. Community cases comprised 6% of Oxfordshire MRSA. Three community cases had received no regular healthcare or antibiotics: one was infected with CipS. Ninety-one percent of community cases had healthcare-associated sequence type (ST)22/36; CipS MRSA cases had heterogeneous STs but many had recent healthcare exposure. A substantial minority of UK MRSA transmission may occur outside hospitals. Hospital strains are becoming 'feral' or persisting in long-term carriers in the community with regular healthcare contacts; those with recent healthcare exposure may nevertheless acquire non-hospital epidemic MRSA strains in the community.

Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW, Fung R, Golubchik T, Harding RM et al. 2010. Multilocus sequence typing of Clostridium difficile. J Clin Microbiol, 48 (3), pp. 770-778. | Show Abstract | Read more

A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.

Juhas M, Crook DW, Hood DW. 2008. Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence. Cell Microbiol, 10 (12), pp. 2377-2386. | Show Abstract | Read more

Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains.

Saha SK, Darmstadt GL, Baqui AH, Hossain B, Islam M, Foster D, Al-Emran H, Naheed A et al. 2008. Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design. PLoS One, 3 (10), pp. e3576. | Show Abstract | Read more

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Foster D, Knox K, Walker AS, Griffiths DT, Moore H, Haworth E, Peto T, Brueggemann AB, Crook DW, Oxford Invasive Pneumococcal Surveillance Group. 2008. Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England. J Med Microbiol, 57 (Pt 4), pp. 480-487. | Show Abstract | Read more

A 10-year invasive pneumococcal disease (IPD) enhanced surveillance project in the Oxfordshire region of the UK between 1996 and 2005 identified a total of 2691 Streptococcus pneumoniae isolates from all ages that provided a comprehensive description of pneumococcal epidemiology. All isolates were serotyped and those from children under 5 years of age were genotyped and a matched case-control study using adults hospitalized between 1995 and 2000 was performed to estimate the effectiveness of the pneumococcal polysaccharide vaccine in the local population. Fifty-one serotypes were isolated, with different age distributions. The overall incidence of IPD was 9.2 cases per 100 000 population per annum [95 % confidence interval (CI), 8.6-9.9] and that of meningitis was 0.7 per 100 000 population per annum (95 % CI 0.5-0.9). After adjusting for age, serotype 1 was found to be less likely to be associated with meningitis versus other IPD, compared with the most common serotype 14, whereas serotype 12F was more likely to cause meningitis than other IPD. There were significant temporal changes in IPD incidence of four serotypes, with decreases in serotypes 1, 12F and 14 and increases in serotype 8. A possible novel variant (from serotype 6A to 6B) was found using multilocus sequence typing analysis. From the matched case-control study of adults, the pneumococcal polysaccharide vaccine effectiveness was estimated to be 43 % (2-68 %), which did not change significantly after adjustment for pre-existing co-morbidities. The data provide a baseline against which the impact of the pneumococcal conjugate vaccine introduced in the UK in 2006 could be measured.

Miller R, Esmail H, Peto T, Walker S, Crook D, Wyllie D. 2008. Is MRSA admission bacteraemia community-acquired? A case control study. J Infect, 56 (3), pp. 163-170. | Show Abstract | Read more

OBJECTIVES: To compare characteristics of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible S. aureus (MSSA) bacteraemia detected on admission to a UK hospital and to determine whether these organisms are community-acquired. METHODS: Consecutive cases of MRSA bacteraemia admitted to general medicine between 2003 and 2006 were identified and compared to MSSA age-matched and unmatched controls (35, 35 and 34 patients, respectively). Demographics, MRSA risk factors, previous health-care contact and clinical presentation were compared using patient notes. Multi-locus sequence typing was performed. RESULTS: 34/35 strains of admission MRSA bacteraemia were the health-care associated Sequence Types (ST)-22 (77%) or ST-36 (21%), whereas 20 different MSSA strains were identified. No MRSA cases fitted the CDC definition of community-acquired MRSA. Compatible with health-care associated acquisition, after matching for age MRSA cases had significantly higher levels of previous hospital exposure than MSSA controls, and more co-morbidities. Notably, 63% of MRSA cases were admitted from their own home, as opposed to secondary care facilities. Clinical presentation of MRSA and MSSA bacteraemias was similar. CONCLUSIONS: MRSA strains associated with health-care were responsible for almost all cases of MRSA bacteraemia on admission to hospital during the period studied. Despite this the majority of cases with MRSA admission bacteraemia were admitted from their own homes. Further research is needed into the determinants of MRSA bacteraemia among patients outside hospital.

Walker S, Peto TE, O'Connor L, Crook DW, Wyllie D. 2008. Are there better methods of monitoring MRSA control than bacteraemia surveillance? An observational database study. PLoS One, 3 (6), pp. e2378. | Show Abstract | Read more

BACKGROUND: Despite a substantial burden of non-bacteraemic methicillin resistant Staphylococcus aureus (MRSA) disease, most MRSA surveillance schemes are based on bacteraemias. Using bacteraemia as an outcome, trends at hospital level are difficult to discern, due to random variation. We investigated rates of nosocomial bacteraemic and non-bacteraemic MRSA infection as surveillance outcomes. METHODS AND FINDINGS: We used microbiology and patient administration system data from an Oxford hospital to estimate monthly rates of first nosocomial MRSA bacteraemia, and nosocomial MRSA isolation from blood/respiratory/sterile site specimens ("sterile sites") or all clinical samples (screens excluded) in all patients admitted from the community for at least 2 days between April 1998 and June 2006. During this period there were 441 nosocomial MRSA bacteraemias, 1464 MRSA isolations from sterile sites, and 3450 isolations from clinical specimens (8% blood, 15% sterile site, 10% respiratory, 59% surface swabs, 8% urine) in over 2.6 million patient-days. The ratio of bacteraemias to sterile site and all clinical isolations was similar over this period (around 3 and 8-fold lower respectively), during which rates of nosocomial MRSA bacteraemia increased by 27% per year to July 2003 before decreasing by 18% per year thereafter (heterogeneity p<0.001). Trends in sterile site and all clinical isolations were similar. Notably, a change in rate of all clinical MRSA isolations in December 2002 could first be detected with conventional statistical significance by August 2003 (p = 0.03). In contrast, when monitoring MRSA bacteraemia, identification of probable changes in trend took longer, first achieving p<0.05 in July 2004. CONCLUSIONS: MRSA isolation from all sites of suspected infection, including bacteraemic and non-bacteraemic isolation, is a potential new surveillance method for MRSA control. It occurs about 8 times more frequently than bacteraemia, allowing robust statistical determination of changing rates over substantially shorter times or smaller areas than using bacteraemia as an outcome.

Walker AS, Spiegelhalter D, Crook DW, Wyllie D, Morris J, Peto TE. 2008. Fairness of financial penalties to improve control of Clostridium difficile. BMJ, 337 (nov20 2), pp. a2097. | Read more

Genome Evolution in Bacterial Infectious Diseases

Why do bacteria cause disease? Are there genetic differences between bacteria that affect disease severity? Does natural selection act on bacteria within the body to promote disease or attenuate infection?We are using statistical genetics and evolutionary biology to understand how mutations in the genome and changes in gene expression affect virulence - the severity or frequency of infection.Bacterial diseases are leading causes of mortality worldwide, exerting a profound effect onglobal ...

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