register interest

Professor Angela B Brueggemann

Research Area: Genetics and Genomics
Technology Exchange: Bioinformatics, Computational biology and Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease and Genetics & Genomics
Keywords: Streptococcus pneumoniae, evolution, whole genome sequencing, vaccines and antimicrobial resistance
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The main research focus of the Brueggemann group is Streptococcus pneumoniae (the ‘pneumococcus’), a bacterium that is a major cause of diseases such as pneumonia and meningitis worldwide. Current research involves using high-throughput genotyping and whole genome sequencing techniques and unique collections of isolates to understand pneumococcal evolution, especially evolutionary changes related to antimicrobial and vaccine selective pressures. Molecular epidemiology and population biology provide the foundation for all aspects of the group’s research.

Our work has particular relevance to: i) understanding how antimicrobial resistance determinants evolve and spread; ii) long-term effectiveness of the existing pneumococcal conjugate vaccines; iii) the design of future vaccines; and iv) the development of novel antimicrobials.

Name Department Institution Country
Professor Karl Kristinsson Landspitali-University Hospital Iceland
Professor Ásgeir Haraldsson Landspitali-University Hospital Iceland
Dr Helga Erlendsdóttir Landspitali-University Hospital Iceland
Dr Chris Farmer (MPLS) Mathematical Institute Oxford University, Mathematical Institute United Kingdom
Professor Stephen Bentley Wellcome Trust Sanger Institute United Kingdom
Professor Julian Parkhill Wellcome Trust Sanger Institute United Kingdom
Professor Keith Klugman Bill & Melinda Gates Foundation United States
Professor Regine Hakenbeck University Kaiserslautern Germany
Dr Bernard Beall Centers for Disease Control and Prevention United States
Dr Anne von Gottberg National Institute for Communicable Diseases South Africa
Professor Andrew Pollard Jenner Institute Oxford University, Centre for Clinical Vaccinology and Tropical Medicine United Kingdom
Professor Martin Maiden Jenner Institute Oxford University, Zoology United Kingdom
Brueggemann AB, Harrold CL, Rezaei Javan R, van Tonder AJ, McDonnell AJ, Edwards BA. 2017. Pneumococcal prophages are diverse, but not without structure or history. Sci Rep, 7 pp. 42976. | Show Abstract | Read more

Bacteriophages (phages) infect many bacterial species, but little is known about the diversity of phages among the pneumococcus, a leading global pathogen. The objectives of this study were to determine the prevalence, diversity and molecular epidemiology of prophages (phage DNA integrated within the bacterial genome) among pneumococci isolated over the past 90 years. Nearly 500 pneumococcal genomes were investigated and RNA sequencing was used to explore prophage gene expression. We revealed that every pneumococcal genome contained prophage DNA. 286 full-length/putatively full-length pneumococcal prophages were identified, of which 163 have not previously been reported. Full-length prophages clustered into four major groups and every group dated from the 1930-40 s onward. There was limited evidence for genes shared between prophage clusters. Prophages typically integrated in one of five different sites within the pneumococcal genome. 72% of prophages possessed the virulence genes pblA and/or pblB. Individual prophages and the host pneumococcal genetic lineage were strongly associated and some prophages persisted for many decades. RNA sequencing provided clear evidence of prophage gene expression. Overall, pneumococcal prophages were highly prevalent, demonstrated a structured population, possessed genes associated with virulence, and were expressed under experimental conditions. Pneumococcal prophages are likely to play a more important role in pneumococcal biology and evolution than previously recognised.

van Tonder AJ, Bray JE, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Hoffmann S, Bentley SD, Haraldsson Á, Erlendsdóttir H et al. 2016. Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes. Microb Genom, 2 (10), pp. 000090. | Show Abstract | Read more

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Bentley SD et al. 2016. Correction for van Tonder et al., Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci. J Clin Microbiol, 54 (6), pp. 1670. | Read more

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Bentley SD et al. 2015. Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci. J Clin Microbiol, 53 (7), pp. 2271-2285. | Show Abstract | Read more

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

Bogaardt C, van Tonder AJ, Brueggemann AB. 2015. Genomic analyses of pneumococci reveal a wide diversity of bacteriocins - including pneumocyclicin, a novel circular bacteriocin. BMC Genomics, 16 (1), pp. 554. | Show Abstract | Read more

BACKGROUND: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the 'pneumococcus'). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure. RESULTS: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the 'bacteriocin/immunity region' of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes. CONCLUSIONS: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

van Tonder AJ, Mistry S, Bray JE, Hill DM, Cody AJ, Farmer CL, Klugman KP, von Gottberg A, Bentley SD, Parkhill J et al. 2014. Defining the estimated core genome of bacterial populations using a Bayesian decision model. PLoS Comput Biol, 10 (8), pp. e1003788. | Show Abstract | Read more

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance.

Hanieh S, Hamaluba M, Kelly DF, Metz JA, Wyres KL, Fisher R, Pradhan R, Shakya D, Shrestha L, Shrestha A et al. 2014. Streptococcus pneumoniae carriage prevalence in Nepal: evaluation of a method for delayed transport of samples from remote regions and implications for vaccine implementation. PLoS One, 9 (6), pp. e98739. | Show Abstract | Read more

BACKGROUND: Pneumococcal disease is a significant cause of morbidity and mortality in young children in Nepal, and currently available pneumococcal conjugate vaccines offer moderate coverage of invasive disease isolates. METHODS: A prevalence study of children aged 1.5 to 24 months in urban and rural Nepal was conducted. In the urban group, nasopharyngeal swabs (NPS) were transported using silica desiccant packages (SDP) with delayed processing (2 weeks), or skim-milk-tryptone-glucose-glycerin (STGG) with immediate processing (within 8 hours). Pneumococcal nasopharyngeal carriage prevalence, serogroup/type distribution and isolate genotypes (as defined by multilocus sequence typing) were determined. RESULTS: 1101 children were enrolled into the study: 574 in the urban group and 527 in the rural group. Overall carriage prevalence based on culture from specimens transported and stored in STGG was 58.7% (337/574), compared to 40.9% (235/574) in SDP. There was concordance of detection of pneumococcus in 67% of samples. Using the SDP method, pneumococcal carriage prevalence was higher in the rural population (69.2%; 364/526) compared to the urban population (40.9%; 235/574). Serogroup/type distribution varied with geographical location. Over half of the genotypes identified in both the urban and rural pneumococcal populations were novel. CONCLUSION: The combination of delayed culture and transport using SDP underestimates the prevalence of pneumococcal carriage; however, in remote areas, this method could still provide a useful estimate of carriage prevalence and serogroup/type distribution. Vaccine impact is unpredictable in a setting with novel genotypes and limited serotype coverage as described here. Consequently, continued surveillance of pneumococcal isolates from carriage and disease in Nepali children following the planned introduction of pneumococcal conjugate vaccines introduction will be essential.

Domenech A, Ardanuy C, Grau I, Calatayud L, Pallares R, Fenoll A, Brueggemann AB, Liñares J. 2014. Evolution and genetic diversity of the Spain23F-ST81 clone causing adult invasive pneumococcal disease in Barcelona (1990-2012). J Antimicrob Chemother, 69 (4), pp. 924-931. | Show Abstract | Read more

OBJECTIVES: We aimed to analyse the clinical epidemiology and genetic diversity of invasive pneumococcal disease (IPD) episodes attributed to the Spain(23F)-ST81 (PMEN1) clone. METHODS: Fifty-eight (2.7%) of 2117 invasive pneumococci isolated from adult patients during the 1990-2012 period shared a PFGE pattern related to the PMEN1 clone. The genotype was confirmed by multilocus sequence typing. The pbp2x, pbp1a, pbp2b and pspA genes were PCR-amplified and sequenced. Polymorphisms in the pspC gene were identified by PCR restriction fragment length polymorphism. The presence of transposons with erythromycin and tetracycline resistance determinants was detected by PCR. RESULTS: The prevalence of the PMEN1 clone increased from 0.8% in 1991 to 6.2% in 2001, and decreased to 0% in 2010-12, concomitant with the introduction of the seven-valent pneumococcal conjugate vaccine for children. A total of 93.1% of patients had pneumonia, meningitis or peritonitis; 87.9% of patients had associated underlying diseases, mainly cancer, chronic obstructive pulmonary disease and diabetes. Two closely related sequence types (STs) (ST81, n = 52; ST85, n = 6) were detected, with different serotypes: 23F (n = 42), 19A (n = 9) and 19F (n = 6). All the isolates were resistant to penicillin, co-trimoxazole and chloramphenicol. All the isolates also shared the same pbp1a allele, whereas multiple alleles of pbp2b, pbp2x, pspA and pspC were detected. Of the isolates, 89.7% were tetracycline resistant and 60.3% (n = 35) were macrolide resistant, and resistance was associated with different Tn916-like transposons. CONCLUSIONS: Adult IPD caused by this clone was mainly detected in patients with underlying conditions, and genetic variability was observed among PMEN1 isolates collected in our area over the past 20 years.

Brueggemann AB, Muroki BM, Kulohoma BW, Karani A, Wanjiru E, Morpeth S, Kamau T, Sharif S, Scott JAG. 2013. Population genetic structure of Streptococcus pneumoniae in Kilifi, Kenya, prior to the introduction of pneumococcal conjugate vaccine PLoS ONE, 8 (11), | Show Abstract | Read more

Background: The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Kenya in 2011. Introduction of any PCV will perturb the existing pneumococcal population structure, thus the aim was to genotype pneumococci collected in Kilifi before PCV10. Methods and Findings: Using multilocus sequence typing (MLST), we genotyped > 1100 invasive and carriage pneumococci from children, the largest collection genotyped from a single resource-poor country and reported to date. Serotype 1 was the most common serotype causing invasive disease and was rarely detected in carriage; all serotype 1 isolates were members of clonal complex (CC) 217. There were temporal fluctuations in the major circulating sequence types (STs); and although 1-3 major serotype 1, 14 or 23F STs co-circulated annually, the two major serotype 5 STs mainly circulated independently. Major STs/CCs also included isolates of serotypes 3, 12F, 18C and 19A and each shared ≤2 MLST alleles with STs that circulate widely elsewhere. Major CCs associated with non-PCV10 serotypes were predominantly represented by carriage isolates, although serotype 19A and 12F CCs were largely invasive and a serotype 10A CC was equally represented by invasive and carriage isolates. Conclusions: Understanding the pre-PCV10 population genetic structure in Kilifi will allow for the detection of changes in prevalence of the circulating genotypes and evidence for capsular switching post-vaccine implementation. © 2013 Brueggemann et al.

Wyres KL, van Tonder A, Lambertsen LM, Hakenbeck R, Parkhill J, Bentley SD, Brueggemann AB. 2013. Evidence of antimicrobial resistance-conferring genetic elements among pneumococci isolated prior to 1974. BMC Genomics, 14 (1), pp. 500. | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements. RESULTS: Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes. CONCLUSIONS: These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.

Brueggemann AB, Muroki BM, Kulohoma BW, Karani A, Wanjiru E, Morpeth S, Kamau T, Sharif S, Scott JA. 2013. Population genetic structure of Streptococcus pneumoniae in Kilifi, Kenya, prior to the introduction of pneumococcal conjugate vaccine. PLoS One, 8 (11), pp. e81539. | Show Abstract | Read more

BACKGROUND: The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Kenya in 2011. Introduction of any PCV will perturb the existing pneumococcal population structure, thus the aim was to genotype pneumococci collected in Kilifi before PCV10. METHODS AND FINDINGS: Using multilocus sequence typing (MLST), we genotyped >1100 invasive and carriage pneumococci from children, the largest collection genotyped from a single resource-poor country and reported to date. Serotype 1 was the most common serotype causing invasive disease and was rarely detected in carriage; all serotype 1 isolates were members of clonal complex (CC) 217. There were temporal fluctuations in the major circulating sequence types (STs); and although 1-3 major serotype 1, 14 or 23F STs co-circulated annually, the two major serotype 5 STs mainly circulated independently. Major STs/CCs also included isolates of serotypes 3, 12F, 18C and 19A and each shared ≤ 2 MLST alleles with STs that circulate widely elsewhere. Major CCs associated with non-PCV10 serotypes were predominantly represented by carriage isolates, although serotype 19A and 12F CCs were largely invasive and a serotype 10A CC was equally represented by invasive and carriage isolates. CONCLUSIONS: Understanding the pre-PCV10 population genetic structure in Kilifi will allow for the detection of changes in prevalence of the circulating genotypes and evidence for capsular switching post-vaccine implementation.

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Liñares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP et al. 2013. Pneumococcal capsular switching: a historical perspective. J Infect Dis, 207 (3), pp. 439-449. | Show Abstract | Read more

BACKGROUND: Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. METHODS: Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. RESULTS: We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥ 58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. CONCLUSIONS: Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Liñares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP et al. 2012. The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success. Genome Biol, 13 (11), pp. R103. | Show Abstract | Read more

BACKGROUND: Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species. RESULTS: We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described. CONCLUSIONS: These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

Domenech A, Ardanuy C, Balsalobre L, Marti S, Calatayud L, De la Campa AG, Brueggemann AB, Liñares J. 2012. Pneumococci can persistently colonize adult patients with chronic respiratory disease. J Clin Microbiol, 50 (12), pp. 4047-4053. | Show Abstract | Read more

Streptococcus pneumoniae plays an important role in causing acute exacerbations in patients with chronic respiratory disease. However, few data are available regarding pneumococcal persistence in adult patients with chronic respiratory diseases. Fifty pneumococci recovered from sputum samples (1995 to 2010) from 13 adult patients with ≥ 3 episodes of acute exacerbation or pneumonia, with the same serotype and pulsed-field gel electrophoresis (PFGE) pattern, were studied. Multilocus sequence typing (MLST) loci, penicillin-binding protein (PBP) genes (pbp2x, pbp1a, pbp2b), and the quinolone-resistant determining regions (QRDRs) of parC, parE, and gyrA were PCR amplified and sequenced. The average time between the first and last episode was 582 days (standard deviation [SD], ± 362). All but two patients received multiple courses of β-lactam treatment, and all persistent strains were resistant to penicillin; however, the PBP sequences were stable over time apart from one variable nucleotide in pbp2x, observed among pneumococci isolated from three patients. In contrast, 7/11 patients treated with fluoroquinolones had fluoroquinolone-resistant pneumococci. In three patients, the initially fluoroquinolone-susceptible strain developed resistance after fluoroquinolone therapy, and in the remaining four patients, the persistent strain was fluoroquinolone resistant from the first episode. QRDR changes involved in fluoroquinolone resistance were frequently observed in persistent strains after fluoroquinolone treatment; however, the PBP sequences and MLST genotypes of these strains were stable over time.

Golubchik T, Brueggemann AB, Street T, Gertz RE, Spencer CC, Ho T, Giannoulatou E, Link-Gelles R, Harding RM, Beall B 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.

Harrison OB, Brueggemann AB, Caugant DA, van der Ende A, Frosch M, Gray S, Heuberger S, Krizova P, Olcen P, Slack M et al. 2011. Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review. Microbiology, 157 (Pt 8), pp. 2181-2195. | Show Abstract | Read more

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

Beall BW, Gertz RE, Hulkower RL, Whitney CG, Moore MR, Brueggemann AB. 2011. Shifting genetic structure of invasive serotype 19A pneumococci in the United States. J Infect Dis, 203 (10), pp. 1360-1368. | Show Abstract | Read more

BACKGROUND: Following 7-valent conjugate vaccine introduction in the United States in 2000, invasive serotype (sero19A) pneumococcal disease (IPD) emerged rapidly. Sero19A IPD incidence increased slightly during 2005-2008 (from 2.3 cases to 2.5 cases per 100,000 population), whereas sero19A penicillin resistance (defined as a minimum inhibitor concentration [MIC] ≥2 μg/mL) increased significantly (from 28.7% to 43.7%). To better understand changes, we characterized sero19A isolates recovered during 2004-2008. METHODS: We performed antimicrobial susceptibility testing on all 2767 sero19A IPD isolates identified through the Centers for Disease Control Active Bacterial Core surveillance during 2004-2008. We genotyped 1804 (96.3%) of 1874 sero19A isolates recovered during 2005-2007 and all 148 year 2008 sero19A isolates from children <5 years of age. RESULTS: Resistant clonal complex (CC) 320/271(19A) increased from 20.9% (115 of 550) to 32.9% (208 of 633; P < .001) of IPD isolates during 2005-2007, which paralleled increased sero19A penicillin resistance (from 28.7% [163 of 567 isolates] to 39.5% [261 of 661 isolates]; P < .001). Total IPD due to 320/271(19A) increased during 2005-2007 and increased from 2.1 to 3.6 cases per 100,000 population during 2005-2008 in children <5 years of age. The penicillin-susceptible/intermediate, putative vaccine-escape CC695(19A) increased from 7.5% (41 of 550) to 13.6% (85 of 633) of sero19A isolates during 2005-2007 (P = .002). CONCLUSIONS: Sero19A rates may have plateaued; however, clonal shifts are increasing resistance. Increased IPD caused by CC320/271(19A) and CC695(19A) could reflect additional selective advantages in addition to resistance.

Obando I, Muñoz-Almagro C, Arroyo LA, Tarrago D, Sanchez-Tatay D, Moreno-Perez D, Dhillon SS, Esteva C, Hernandez-Bou S, Garcia-Garcia JJ et al. 2008. Pediatric parapneumonic empyema, Spain. Emerg Infect Dis, 14 (9), pp. 1390-1397. | Show Abstract | Read more

Pediatric parapneumonic empyema (PPE) has been increasing in several countries including Spain. Streptococcus pneumoniae is a major PPE pathogen; however, antimicrobial pretreatment before pleural fluid (PF) sampling frequently results in negative diagnostic cultures, thus greatly underestimating the contribution of pneumococci, especially pneumococci susceptible to antimicrobial agents, to PPE. The study aim was to identify the serotypes and genotypes that cause PPE by using molecular diagnostics and relate these data to disease incidence and severity. A total of 208 children with PPE were prospectively enrolled; blood and PF samples were collected. Pneumococci were detected in 79% of culture-positive and 84% of culture-negative samples. All pneumococci were genotyped by multilocus sequence typing. Serotypes were determined for 111 PPE cases; 48% were serotype 1, of 3 major genotypes previously circulating in Spain. Variance in patient complication rates was statistically significant by serotype. The recent PPE increase is principally due to nonvaccine serotypes, especially the highly invasive serotype 1.

Sánchez-Tatay D, Arroyo LA, Tarragó D, Lirola MJ, Porras A, Fenoll A, Hausdorff WP, Brueggemann AB, Obando I. 2008. Antibiotic susceptibility and molecular epidemiology of nasopharyngeal pneumococci from Spanish children. Clin Microbiol Infect, 14 (8), pp. 797-801. | Show Abstract | Read more

Nasopharyngeal pneumococci were collected from 635 Spanish children aged 6 months to 6 years attending four primary healthcare centres (n = 276) or two hospital emergency rooms (n = 359); 36% of the children had received >/=1 dose of pneumococcal conjugate vaccine (PCV7). Overall, the carriage rate of Streptococcus pneumoniae was 31%, with no significant differences in carriage rates according to setting. Colonization with vaccine serotypes was significantly associated with the absence of PCV7 immunization (29.4% vs. 5.9%, p <0.001). Forty-seven per cent of all isolates were penicillin- and/or erythromycin-non-susceptible; 13 international antibiotic-resistant clones were represented among non-susceptible pneumococci and were similarly distributed among vaccine and non-vaccine serotypes.

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.

Moore MR, Gertz RE, Woodbury RL, Barkocy-Gallagher GA, Schaffner W, Lexau C, Gershman K, Reingold A, Farley M, Harrison LH et al. 2008. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J Infect Dis, 197 (7), pp. 1016-1027. | Show Abstract | Read more

BACKGROUND: Serotype 19A invasive pneumococcal disease (IPD) increased annually in the United States after the introduction of the 7-valent conjugate vaccine (PCV7). To understand this increase, we characterized serotype 19A isolates recovered during 2005. METHODS: IPD cases during 1998-2005 were identified through population-based surveillance. We performed susceptibility testing and multilocus sequence typing on 528 (95%) of 554 serotype 19A isolates reported in 2005. RESULTS: The incidence of IPD due to serotype 19A increased from 0.8 to 2.5 cases per 100,000 population between 1998 and 2005 (P < .05), whereas the overall incidence of IPD decreased from 24.4 to 13.8 cases per 100,000 population (P < .05). Simultaneously, the incidence of IPD due to penicillin-resistant 19A isolates increased from 6.7% to 35% (P < .0001). Of 151 penicillin-resistant 19A isolates, 111 (73.5%) belonged to the rapidly emerging clonal complex 320, which is related to multidrug-resistant Taiwan(19F)-14. The remaining penicillin-resistant strains were highly related to other clones of PCV7 serotypes or to isolates within major 19A clonal complex 199 (CC199). In 1999, only CC199 and 3 minor clones were apparent among serotype 19A isolates. During 2005, 11 multiple-isolate clonal sets were detected, including capsular switch variants of a serotype 4 clone. CONCLUSIONS: PCV7 ineffectiveness against serotype 19A, antibiotic resistance, clonal expansion and emergence, and capsular switching have contributed to the genetic diversity of 19A and to its emergence as the predominant invasive pneumococcal serotype in the United States.

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.

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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.

Obando I, Arroyo LA, Sánchez-Tatay D, Tarragó D, Moreno D, Hausdorff WP, Brueggemann AB. 2007. Molecular epidemiology of paediatric invasive pneumococcal disease in southern Spain after the introduction of heptavalent pneumococcal conjugate vaccine. Clin Microbiol Infect, 13 (3), pp. 347-348. | Read more

Jefferies JM, Johnston CH, Kirkham LA, Cowan GJ, Ross KS, Smith A, Clarke SC, Brueggemann AB, George RC, Pichon B et al. 2007. Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks. J Infect Dis, 196 (6), pp. 936-944. | Show Abstract | Read more

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

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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.

Brueggemann AB. 2006. Antibiotic resistance mechanisms among pediatric respiratory and enteric pathogens: A current update. Pediatr Infect Dis J, 25 (10), pp. 969-973. | Show Abstract | Read more

Antibiotic resistance is a continually increasing problem that has, to a greater or lesser extent, affected virtually every area of the world. The scientific literature is abundant with papers related to antibiotics and antibiotic resistance. Many excellent papers and reviews have been published during the past few years, and the literature base continues to expand at rapid speed. This review is meant to provide a recent update on antibiotic resistance among respiratory and enteric pathogens, with a focus on infections in children. Not a small task, but this paper is not meant to be exhaustive. Rather, the intention is to highlight the key antibiotics and antibiotic resistance mechanisms that are currently the most relevant to pediatrics. The most recently published literature is used wherever possible, and the reader is encouraged to explore specific topics of interest further by reviewing the referenced literature.

Obando I, Arroyo LA, Sánchez-Tatay D, Moreno D, Hausdorff WP, Brueggemann AB. 2006. Molecular typing of pneumococci causing parapneumonic empyema in Spanish children using multilocus sequence typing directly on pleural fluid samples. Pediatr Infect Dis J, 25 (10), pp. 962-963. | Read more

Harnden A, Grant C, Harrison T, Perera R, Brueggemann AB, Mayon-White R, Mant D. 2006. Whooping cough in school age children with persistent cough: prospective cohort study in primary care. BMJ, 333 (7560), pp. 174-177. | Show Abstract | Read more

OBJECTIVE: To estimate the proportion of school age children with a persistent cough who have evidence of a recent Bordetella pertussis infection. DESIGN: Prospective cohort study (October 2001 to March 2005). SETTING: General practices in Oxfordshire, England. PARTICIPANTS: 172 children aged 5-16 years who presented to their general practitioner with a cough lasting 14 days or more who consented to have a blood test. MAIN OUTCOME MEASURES: Serological evidence of a recent Bordetella pertussis infection; symptoms at presentation; duration and severity of cough; sleep disturbance (parents and child). RESULTS: 64 (37.2%, 95% confidence interval 30.0% to 44.4%) children had serological evidence of a recent Bordetella pertussis infection; 55 (85.9%) of these children had been fully immunised. At presentation, children with whooping cough were more likely than others to have whooping (odds ratio 2.85, 95% confidence interval 1.39 to 5.82), vomiting (4.35, 2.04 to 9.25), and sputum production (2.39, 1.14 to 5.02). Children with whooping cough were also more likely to still be coughing two months after the start of their illness (85% v 48%; P = 0.001), continue to have more than five coughing episodes a day (P = 0.049), and cause sleep disturbance for their parents (P = 0.003). CONCLUSIONS: For school age children presenting to primary care with a cough lasting two weeks or more, a diagnosis of whooping cough should be considered even if the child has been immunised. Making a secure diagnosis of whooping cough may prevent inappropriate investigations and treatment.

Polgreen PM, Beekmann SE, Chen YY, Doern GV, Pfaller MA, Brueggemann AB, Herwaldt LA, Diekema DJ. 2006. Epidemiology of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus in a rural state. Infect Control Hosp Epidemiol, 27 (3), pp. 252-256. | Show Abstract | Read more

BACKGROUND: Most data on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) isolates come from large tertiary care centers. Infection control personnel need to understand the epidemiology of MRSA and VRE across the continuum of care, including small rural hospitals, to develop effective control strategies. OBJECTIVE: To describe the epidemiology of MRSA and VRE in Iowa. SETTING: Fifteen hospitals in Iowa. Methods Between July 1998 and June 2001, a total of 1,968 S. aureus isolates and 1,845 Enterococcus isolates from patients infected with these pathogens were examined. Multivariate models were developed to evaluate patient and institutional risk factors for MRSA infection and VRE infection. RESULTS: The proportion of S. aureus isolates resistant to methicillin was 31%, and the proportion of Enterococcus isolates resistant to vancomycin was 6%. Independent risk factors for MRSA infection included residence in a long-term care facility, age of more than 60 years, hospitalization in a hospital with less than 200 short-term care beds, and acquiring the infection in the hospital. Independent risk factors for VRE infection included use of a central venous catheter, residence in a long-term care facility, acquisition of infection in the hospital, and hospitalization in a hospital with more than 200 short-term care beds. CONCLUSIONS: In Iowa, the epidemiology of MRSA differ from those of VRE. MRSA has become established in small rural hospitals. Effective MRSA control strategies may require inclusion of all hospitals in a state or region.

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.

Rose PW, Harnden A, Brueggemann AB, Perera R, Mant D. 2005. Antibiotics for acute infective conjunctivitis in children - Reply LANCET, 366 (9495), pp. 1431-1431. | Read more

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.

Wise R. 2005. Antibiotics for acute infective conjunctivitis in children. Lancet, 366 (9495), pp. 1431-1432. | Read more

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.

Reinert RR, Muckel S, Al-Lahham A, Spratt BG, Brueggemann AB, Hakenbeck R, Lütticken R. 2003. Characterization of German penicillin non-susceptible serotype 23F pneumococci using multilocus sequence typing. J Med Microbiol, 52 (Pt 11), pp. 981-987. | Show Abstract | Read more

Three nationwide multicentre studies (n = 5071) showed an increase in antibiotic resistance in pneumococci in Germany. Serotype 23F was the predominant serotype (n = 45, 22.4 %), followed by 6B (n = 30, 14.9 %) and 9V (n = 19, 9.5 %). Multilocus sequence typing was used to characterize 45 serotype 23F strains with reduced penicillin susceptibility. The Spanish(23F)-1 clone [profile 4-4-2-4-4-1-1, sequence type (ST) 81] contributes significantly to the emergence of penicillin resistance in Germany (n = 21, 46.7 % of all penicillin non-susceptible serotype 23F isolates). Isolates of ST 277 (profile 7-13-8-6-6-12-8), which has been found previously in the Netherlands, are also observed, particularly in western Germany (n = 8, 17.8 %). A high proportion of strains (n = 11, 24.4 %) have sequence types that have not been reported to date from other countries (STs 353-362). The major penicillin-resistant clones are present in Germany, a country with relatively low levels of beta-lactam resistance.

Brueggemann AB, Spratt BG. 2003. Geographic distribution and clonal diversity of Streptococcus pneumoniae serotype 1 isolates. J Clin Microbiol, 41 (11), pp. 4966-4970. | Show Abstract | Read more

Serotype 1 pneumococci are a major cause of serious disease and have been associated with outbreaks but are rarely carried. The high attack rate and lack of coverage of this serotype by the heptavalent conjugate vaccine prompted the characterization of a geographically diverse collection of 166 serotype 1 isolates from recent cases of invasive disease. The isolates were resolved by multilocus sequence typing into 16 clones, which clustered into three major lineages with very different geographic distributions. Lineage A isolates were exclusively from Europe and North America, lineage B isolates were predominantly from Africa and Israel, and lineage C isolates were mainly from Chile. There was no clear association between the presence of individual clones within a country and the prevalence of serotype 1 disease.

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

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.

Diekema DJ, Messer SA, Brueggemann AB, Coffman SL, Doern GV, Herwaldt LA, Pfaller MA. 2002. Epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of Iowa organisms study. J Clin Microbiol, 40 (4), pp. 1298-1302. | Show Abstract | Read more

Bloodstream infections due to Candida species cause significant morbidity and mortality. Surveillance for candidemia is necessary to detect trends in species distribution and antifungal resistance. We performed prospective surveillance for candidemia at 16 hospitals in the State of Iowa from 1 July 1998 through 30 June 2001. Using U.S. Census Bureau and Iowa Hospital Association data to estimate a population denominator, we calculated the annual incidence of candidemia in Iowa to be 6.0 per 100,000 of population. Candida albicans was the most common species detected, but 43% of candidemias were due to species other than C. albicans. Overall, only 3% of Candida species were resistant to fluconazole. However, Candida glabrata was the most commonly isolated species other than C. albicans and demonstrated some resistance to azoles (fluconazole MIC at which 90% of the isolates tested are inhibited, 32 microg/ml; 10% resistant, 10% susceptible dose dependent). C. glabrata was more commonly isolated from older patients (P = 0.02) and caused over 25% of candidemias among persons 65 years of age or older. The investigational triazoles posaconazole, ravuconazole, and voriconazole had excellent in vitro activity overall against Candida species. C. albicans is the most important cause of candidemia and remains highly susceptible to available antifungal agents. However, C. glabrata has emerged as an important and potentially antifungal resistant cause of candidemia, particularly among the elderly.

Brueggemann AB, Coffman SL, Rhomberg P, Huynh H, Almer L, Nilius A, Flamm R, Doern GV. 2002. Fluoroquinolone resistance in Streptococcus pneumoniae in United States since 1994-1995. Antimicrob Agents Chemother, 46 (3), pp. 680-688. | Show Abstract | Read more

The in vitro activities of ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin against a large collection of clinical isolates of Streptococcus pneumoniae (n = 4,650) obtained over a 5-year period, 1994-1995 through 1999-2000, were assessed as part of a longitudinal multicenter U.S. surveillance study of antimicrobial resistance. Three sampling periods were used during this investigation, the winter seasons of 1994-1995, 1997-1998, and 1999-2000; and 1,523, 1,596 and 1,531 isolates were collected during these three periods, respectively. The overall rank order of activity of the four fluoroquinolones examined in this study was moxifloxacin > gatifloxacin > levofloxacin = ciprofloxacin, in which moxifloxacin (MIC at which 90% of isolates are inhibited [MIC(90)], 0.25 microg/ml; modal MIC, 0.12 microg/ml) was twofold more active than gatifloxacin (MIC(90), 0.5 microg/ml; modal MIC, 0.25 microg/ml), which in turn was fourfold more active than either levofloxacin (MIC(90), 1 microg/ml; modal MIC, 1 microg/ml) or ciprofloxacin (MIC(90), 2 microg/ml; modal MIC, 1 microg/ml). Changes in the in vitro activities of fluoroquinolones against S. pneumoniae strains in the United States over the 5-year period of the survey were assessed by comparing the MIC frequency distributions of the study drugs against the isolates obtained during the three sampling periods encompassing this investigation. These comparisons revealed no evidence of changes in the in vitro activities of the fluoroquinolones. In addition, the percentages of isolates in the three sampling periods for which MICs were above the resistance breakpoints were compared. Low percentages of resistant strains were detected, and there was no evidence of resistance rate changes over time. For example, by use of a ciprofloxacin MIC of > or = 4 microg/ml to define resistance, the proportions of isolates from the three sampling periods for which MICs were at or above this breakpoint were 1.2, 1.6, and 1.4%, respectively. A total of 164 unique isolates (n = 58 from 1994-1995, 65 from 1997-1998, and 42 from 1999-2000) were examined for evidence of mutations in the quinolone resistance-determining regions (QRDRs) of the parC and the gyrA genes. Forty-nine isolates harbored at least one mutation in the QRDRs of one or both genes (1994-1995, n = 15; 1997-1998, n = 19; 1999-2000, n = 15). Among the 4,650 isolates of S. pneumoniae examined in the study, we estimated that 0.3% had mutations in both the parC and gyrA loci. The majority of mutations (67.3% of the mutations in 49 isolates with mutations) were amino acid substitutions in the parC locus only. Four isolates had a mutation in the gyrA locus only, and 12 isolates had mutations in both genes (8.2 and 24.5% of isolates with mutations, respectively). There was no significant difference in the number of isolates with parC and/or gyrA mutations detected during each study period. Finally, because of the magnitude of the study, we had reasonably large numbers of pneumococcal isolates with genotypically defined mechanisms of fluoroquinolone resistance and were thus able to determine the effects of specific resistance mutations on the activities of different fluoroquinolones. In general, isolates with mutations in parC only were resistant to ciprofloxacin but remained susceptible to levofloxacin, gatifloxacin, and moxifloxacin, whereas isolates with mutations in gyrA only and isolates with mutations in both parC and gyrA were resistant to all four fluoroquinolones tested.

Dickson EW, Heard SO, Tarara TE, Weers JG, Brueggemann AB, Doern GV. 2002. Liquid ventilation with perflubron in the treatment of rats with pneumococcal pneumonia. Crit Care Med, 30 (2), pp. 393-395. | Show Abstract | Read more

OBJECTIVE: To determine the efficacy of liquid ventilation using a medical-grade perfluorocarbon (perflubron) combined with parenteral or intratracheal antibiotics in a rat model of pneumonia. DESIGN: Prospective, laboratory investigation. SETTING: Experimental laboratory in a university medical center. SUBJECTS: Wistar rats (n = 112). INTERVENTIONS: One day after intratracheal inoculation with Streptococcus pneumoniae, rats received one of five experimental treatments or no treatment (control): modified liquid ventilation (MLV), intramuscular ampicillin, MLV plus intramuscular ampicillin, MLV with intratracheal ampicillin, or MLV plus ampicillin PulmoSpheres. MEASUREMENTS AND MAIN RESULTS: Animals receiving MLV plus intramuscular ampicillin, MLV with intratracheal ampicillin, or MLV plus ampicillin PulmoSpheres had significantly improved 10-day survival rates (85%, 72%, and 72%, respectively) compared with all other groups (0% to 25%). CONCLUSIONS: MLV in combination with either intramuscular, intratracheal, or PulmoSpheres ampicillin improved survival as compared with MLV alone or the same dose of antibiotics delivered intramuscularly.

Richter SS, Heilmann KP, Coffman SL, Huynh HK, Brueggemann AB, Pfaller MA, Doern GV. 2002. The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in the United States, 1994-2000. Clin Infect Dis, 34 (3), pp. 330-339. | Show Abstract | Read more

The genetic relatedness of 672 penicillin-resistant isolates of Streptococcus pneumoniae (PRSP) recovered during national surveillance studies conducted in the United States during the periods of 1994-1995, 1997-1998, and 1999-2000 was determined by use of pulsed-field gel electrophoresis (PFGE). Overall, 104 different PFGE types were elucidated. For all study periods combined, the 12 most prevalent PFGE types included >75% of all isolates, and 5 types were closely related to widespread clones (Spain(23F)-1, France(9V)-3, Spain(6B)-2, Tennessee(23F)-4, and Taiwan(19F)-14). From 1994-1995 to 1999-2000, 3 major PFGE types (not closely related to 16 recognized clones) increased in prevalence. Multidrug resistance was identified among 96%-100% of the isolates in 9 of 12 predominant PFGE types. The prevalence of erythromycin resistance increased within 4 major PFGE types. These observations support the hypothesis that the dominant factor in the emergence of PRSP in the United States during the 1990s has been human-to-human spread of relatively few clonal groups that harbor resistance determinants to multiple classes of antibiotics.

Doern GV, Heilmann KP, Huynh HK, Rhomberg PR, Coffman SL, Brueggemann AB. 2001. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999--2000, including a comparison of resistance rates since 1994--1995. Antimicrob Agents Chemother, 45 (6), pp. 1721-1729. | Show Abstract | Read more

A total of 1,531 recent clinical isolates of Streptococcus pneumoniae were collected from 33 medical centers nationwide during the winter of 1999--2000 and characterized at a central laboratory. Of these isolates, 34.2% were penicillin nonsusceptible (MIC > or = 0.12 microg/ml) and 21.5% were high-level resistant (MIC > or = 2 microg/ml). MICs to all beta-lactam antimicrobials increased as penicillin MICs increased. Resistance rates among non-beta-lactam agents were the following: macrolides, 25.2 to 25.7%; clindamycin, 8.9%; tetracycline, 16.3%; chloramphenicol, 8.3%; and trimethoprim-sulfamethoxazole (TMP-SMX), 30.3%. Resistance to non-beta-lactam agents was higher among penicillin-resistant strains than penicillin-susceptible strains; 22.4% of S. pneumoniae were multiresistant. Resistance to vancomycin and quinupristin-dalfopristin was not detected. Resistance to rifampin was 0.1%. Testing of seven fluoroquinolones resulted in the following rank order of in vitro activity: gemifloxacin > sitafloxacin > moxifloxacin > gatifloxacin > levofloxacin = ciprofloxacin > ofloxacin. For 1.4% of strains, ciprofloxacin MICs were > or = 4 microg/ml. The MIC(90)s (MICs at which 90% of isolates were inhibited) of two ketolides were 0.06 microg/ml (ABT773) and 0.12 microg/ml (telithromycin). The MIC(90) of linezolid was 2 microg/ml. Overall, antimicrobial resistance was highest among middle ear fluid and sinus isolates of S. pneumoniae; lowest resistance rates were noted with isolates from cerebrospinal fluid and blood. Resistant isolates were most often recovered from children 0 to 5 years of age and from patients in the southeastern United States. This study represents a continuation of two previous national studies, one in 1994--1995 and the other in 1997--1998. Resistance rates with S. pneumoniae have increased markedly in the United States during the past 5 years. Increases in resistance from 1994--1995 to 1999--2000 for selected antimicrobial agents were as follows: penicillin, 10.6%; erythromycin, 16.1%; tetracycline, 9.0%; TMP-SMX, 9.1%; and chloramphenicol, 4.0%, the increase in multiresistance was 13.3%. Despite awareness and prevention efforts, antimicrobial resistance with S. pneumoniae continues to increase in the United States.

Dunne WM, Kehl KS, Holland-Staley CA, Brueggemann AB, Pfaller MA, Doern GV. 2001. Comparison of results generated by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence PCR used to characterize penicillin-resistant pneumococci from the United States. J Clin Microbiol, 39 (5), pp. 1791-1795. | Show Abstract | Read more

One hundred forty-seven isolates of Streptococcus pneumoniae with high-level penicillin resistance collected during a national surveillance program in the United States were characterized by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence (BOX element) PCR. The results generated by each method were compared by frequency of association to examine whether relationships existed between the various typing methods and statistically to determine association with the geographic source of the isolate or the age of the patient from whom the isolate was obtained. When the data were examined by pairwise analysis of individual strain classifications produced by each typing method, no statistically significant relationships between strain type, geographic location, or patient age were identified, suggesting that distinct clones of penicillin-resistant S. pneumoniae have been widely distributed throughout the United States. However, we did observed shared expression of two or three typing markers at a high frequency (>50%) among clusters of strains, indicating a certain level of concordance between the various typing methods used to classify penicillin-resistant S. pneumoniae.

Brueggemann AB, Pfaller MA, Doern GV. 2001. Use of penicillin MICs to predict in vitro activity of other beta-lactam antimicrobial agents against Streptococcus pneumoniae. J Clin Microbiol, 39 (1), pp. 367-369. | Show Abstract | Read more

Linear regression analysis was used to compare penicillin MICs determined with 3,129 recent clinical isolates of Streptococcus pneumoniae to MICs obtained with nine other beta-lactam antimicrobial agents. A strong correlation between penicillin MICs and those obtained with other beta-lactams was demonstrated. It may be possible to test penicillin and use MICs obtained with penicillin to predict MICs of other beta-lactam antimicrobials for Streptococcus pneumoniae.

Brueggemann AB, Doern GV. 2000. Resistance among Streptococcus pneumoniae: patterns, mechanisms, interpreting the breakpoints. Am J Manag Care, 6 (23 Suppl), pp. S1189-S1196. | Show Abstract

Streptococcus pneumoniae is a frequent cause of respiratory tract infections. In the United States and worldwide, antimicrobial resistance of S pneumoniae has complicated the management of infections caused by this organism. In the United States, antimicrobial resistance with S pneumoniae has evolved almost entirely during the 1990s. Resistance currently exists at high rates with beta-lactams, macrolides, tetracyclines, chloramphenicol, and trimethoprim/sulfamethoxazole. Multiresistant strains--strains that are resistant to penicillin plus at least 2 other antimicrobial classes--are also increasing in prevalence. Fluoro-quinolone resistance remains at low levels in the United States. Control of the problem of antimicrobial resistance will require more judicious and appropriate use of antimicrobials, the development of new agents with novel targets of action, and strategies for preventing disease from occurring in the first place. In addition, the pursuit of an understanding of resistance mechanisms and pharmacodynamics as they relate to clinical outcome must be an ongoing effort, and that knowledge must be applied to the development of more effective approaches for the treatment of infections caused by S pneumoniae.

Bishai W, Brueggemann AB, Nicolau DP, Steinberg I, Spiritus E, Tanigawa C, Koenig S, Chaisson RE, Browne BA, Yoder DM. 2000. Roundtable discussion. Optimizing the use of antimicrobial therapy. Am J Manag Care, 6 (23 Suppl), pp. S1222-S1235.

Winokur PL, Brueggemann A, DeSalvo DL, Hoffmann L, Apley MD, Uhlenhopp EK, Pfaller MA, Doern GV. 2000. Animal and human multidrug-resistant, cephalosporin-resistant salmonella isolates expressing a plasmid-mediated CMY-2 AmpC beta-lactamase. Antimicrob Agents Chemother, 44 (10), pp. 2777-2783. | Show Abstract | Read more

Salmonella spp. are important food-borne pathogens that are demonstrating increasing antimicrobial resistance rates in isolates obtained from food animals and humans. In this study, 10 multidrug-resistant, cephalosporin-resistant Salmonella isolates from bovine, porcine, and human sources from a single geographic region were identified. All isolates demonstrated resistance to cephamycins and extended-spectrum cephalosporins as well as tetracycline, chloramphenicol, streptomycin, and sulfisoxazole. Molecular epidemiological analyses revealed eight distinct chromosomal DNA patterns, suggesting that clonal spread could not entirely explain the distribution of this antimicrobial resistance phenotype. However, all isolates encoded an AmpC-like beta-lactamase, CMY-2. Eight isolates contained a large nonconjugative plasmid that could transform Escherichia coli. Transformants coexpressed cephalosporin, tetracycline, chloramphenicol, streptomycin, and sulfisoxazole resistances. Plasmid DNA revealed highly related restriction fragments though plasmids appeared to have undergone some evolution over time. Multidrug-resistant, cephalosporin-resistant Salmonella spp. present significant therapeutic problems in animal and human health care and raise further questions about the association between antimicrobial resistance, antibiotic use in animals, and transfer of multidrug-resistant Salmonella spp. between animals and man.

Diekema DJ, Brueggemann AB, Doern GV. 2000. Antimicrobial-drug use and changes in resistance in Streptococcus pneumoniae. Emerg Infect Dis, 6 (5), pp. 552-556. | Show Abstract | Read more

Resistance of Streptococcus pneumoniae to antimicrobial drugs is increasing. To investigate the relationship between antimicrobial use and susceptibility of S. pneumoniae isolates at 24 U.S. medical centers, we obtained data on outpatient antimicrobial-drug use for the regions surrounding 23 of these centers. We found an association between decreased penicillin susceptibility and use of beta-lactam antimicrobial drugs.

Brueggemann AB, Doern GV, Huynh HK, Wingert EM, Rhomberg PR. 2000. In vitro activity of ABT-773, a new ketolide, against recent clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Antimicrob Agents Chemother, 44 (2), pp. 447-449. | Show Abstract | Read more

The in vitro activity of ABT-773 was evaluated against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates. ABT-773 was the most active antimicrobial tested against S. pneumoniae. ABT-773 and azithromycin were equivalent in activity against H. influenzae and M. catarrhalis and more active than either clarithromycin or erythromycin.

Richter SS, Winokur PL, Brueggemann AB, Huynh HK, Rhomberg PR, Wingert EM, Doern GV. 2000. Molecular characterization of the beta-lactamases from clinical isolates of Moraxella (Branhamella) catarrhalis obtained from 24 U.S. medical centers during 1994-1995 and 1997-1998. Antimicrob Agents Chemother, 44 (2), pp. 444-446. | Show Abstract | Read more

The beta-lactamases from 403 Moraxella (Branhamella) catarrhalis clinical isolates obtained during 1994-1995 and 1997-1998 U.S. multicenter surveillance studies were characterized by isoelectric focusing. The overall prevalences of the BRO-1 and BRO-2 enzymes among beta-lactamase-positive isolates were estimated to be 97.5 and 2.5%, respectively. The minimum inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing isolates were </=1 microg/ml; however, numerous beta-lactamase-positive isolates for which the ampicillin MICs were </=1 microg/ml produced the BRO-1 enzyme (88. 1%).

Brueggemann AB, Doern GV. 2000. Resistance among Streptococcus pneumoniae: Patterns, mechanisms, interpreting the breakpoints American Journal of Managed Care, 6 (23 SUPPL.), | Show Abstract

Streptococcus pneumoniae is a frequent cause of respiratory tract infections. In the United States and worldwide, antimicrobial resistance of S pneumoniae has complicated the management of infections caused by this organism. In the United States, antimicrobial resistance with S pneumoniae has evolved almost entirely during the 1990s. Resistance currently exists at high rates with β-lactams, macrolides, tetracyclines, chloramphenicol, and trimethoprim/sulfamethoxazole. Multiresistant strains-strains that are resistant to penicillin plus at least 2 other antimicrobial classes-are also increasing in prevalence. Fluoro-quinolone resistance remains at low levels in the United States. Control of the problem of antimicrobial resistance will require more judicious and appropriate use of antimicrobials, the development of new agents with novel targets of action, and strategies for preventing disease from occurring in the first place. In addition, the pursuit of an understanding of resistance mechanisms and pharmacodynamics as they relate to clinical outcome must be an ongoing effort, and that knowledge must be applied to the development of more effective approaches for the treatment of infections caused by S pneumoniae.

Doern GV, Brueggemann AB, Huynh H, Wingert E. 1999. Antimicrobial resistance with Streptococcus pneumoniae in the United States, 1997 98. Emerg Infect Dis, 5 (6), pp. 757-765. | Show Abstract | Read more

From November 1997 to April 1998, 1,601 clinical isolates of Streptococcus pneumoniae were obtained from 34 U.S. medical centers. The overall rate of strains showing resistance to penicillin was 29. 5%, with 17.4% having intermediate resistance. Multidrug resistance, defined as lack of susceptibility to penicillin and at least two other non-ss-lactam classes of antimicrobial drugs, was observed in 16.0% of isolates. Resistance to all 10 ss-lactam drugs examined in this study was directly related to the level of penicillin resistance. Penicillin resistance rates were highest in isolates from middle ear fluid and sinus aspirates of children ambulatory-care settings. Twenty-four of the 34 medical centers in this study had participated in a similar study 3 years before. In 19 of these 24 centers, penicillin resistance rates increased 2.9% to 39.2%. Similar increases were observed with rates of resistance to other antimicrobial drugs.

Shortridge VD, Doern GV, Brueggemann AB, Beyer JM, Flamm RK. 1999. Prevalence of macrolide resistance mechanisms in Streptococcus pneumoniae isolates from a multicenter antibiotic resistance surveillance study conducted in the United States in 1994-1995. Clin Infect Dis, 29 (5), pp. 1186-1188. | Show Abstract | Read more

Two main mechanisms of macrolide resistance have been described in erythromycin-resistant Streptococcus pneumoniae (ERSP): a ribosomal methylase, ErmAM, and a macrolide efflux pump, MefE. In this study, we examined the prevalence of these mechanisms in 114 clinical isolates of ERSP from a 30-center study conducted in the United States between November 1994 and April 1995. The isolates were screened by polymerase chain reaction for the presence of known macrolide resistance genes. Seventy (61%) ERSP contained the macrolide efflux gene (mefE), whereas 36 isolates (32%) contained the biosomal methylase gene (ermAM). Isolates that were ermAM-positive had constitutive macrolide resistance. The minimum inhibitory concentrations (for which 90% of isolates were susceptible) of clarithromycin for the efflux-positive strains were much lower than those for the ermAM-positive strains (4 microg/mL vs. >128 microg/mL, respectively). The efflux mechanism is the predominant form of macrolide resistance in the United States.

Richter SS, Brueggemann AB, Huynh HK, Rhomberg PR, Wingert EM, Flamm R, Doern GV. 1999. A 1997-1998 national surveillance study: Moraxella catarrhalis and Haemophilus influenzae antimicrobial resistance in 34 US institutions. Int J Antimicrob Agents, 13 (2), pp. 99-107. | Show Abstract | Read more

From November 1, 1997 to April 30, 1998, 726 Moraxella catarrhalis isolates and 1529 Haemophilus influenzae isolates were obtained from 34 medical centres throughout the United States. Rates of beta-lactamase production were 94.6% among M. catarrhalis and 31.1% among H. influenzae strains. Susceptibility rates of M. catarrhalis isolates to selected antimicrobial agents were greater than 99% for amoxycillin-clavulanate, cefixime, cefpodoxime, cefuroxime, cefaclor, loracarbef, clarithromycin, azithromycin, chloramphenicol and tetracycline, 97.8% for cefprozil, 50.4% for trimethoprim-sulphamethoxazole and 28.1% for ampicillin. Of the antimicrobials tested against H. influenzae, the only agents with susceptibility rates below 96% were loracarbef (87.6%), cefprozil (83.4%), cefaclor (82.7%), trimethoprim-sulphamethoxazole (67.3%) and ampicillin (64.7%). The clarithromycin susceptibility rate was 67.4% but this agent was not tested in the presence of its 14-OH metabolite.

Murphy TF, Sethi S, Klingman KL, Brueggemann AB, Doern GV. 1999. Simultaneous respiratory tract colonization by multiple strains of nontypeable haemophilus influenzae in chronic obstructive pulmonary disease: implications for antibiotic therapy. J Infect Dis, 180 (2), pp. 404-409. | Show Abstract | Read more

Nontypeable Haemophilus influenzae often causes exacerbations of chronic obstructive pulmonary disease (COPD), and these exacerbations are frequently treated with oral antibiotics. The goals of this study were to determine the frequency of the simultaneous presence of multiple strains of H. influenzae in sputum and to measure the MICs of antibiotics for the isolates. In a prospective study, adults with COPD were seen monthly. Sputum cultures were obtained, and individual colonies were subjected to genomic DNA typing and MIC determinations. Multiple strains of H. influenzae were present simultaneously in the sputum of 26.3% of adults with COPD. In 64.5% of these, MICs of >/=1 antibiotic varied by >/=4-fold among the strains. Therefore, multiple strains of H. influenzae are frequently present simultaneously in the sputum of adults with COPD, and the antimicrobial susceptibility of different strains in the same sputum sometimes differs.

Doern GV, Brueggemann AB, Pfaller MA, Jones RN. 1999. Assessment of laboratory performance with Streptococcus pneumoniae antimicrobial susceptibility testing in the United States: a report from the College of American Pathologists Microbiology Proficiency Survey Program. Arch Pathol Lab Med, 123 (4), pp. 285-289. | Show Abstract | Read more

OBJECTIVE: To assess the performance of clinical microbiology laboratories in the United States when conducting in vitro susceptibility tests with Streptococcus pneumoniae. METHODS: The results of a nationwide College of American Pathologists Proficiency Survey test sample, in which susceptibility testing of an isolate of S. pneumoniae was performed, were assessed with respect to precision and accuracy. RESULTS: Wide variability was noted among participating laboratories with both minimum inhibitory concentration procedures and disk diffusion susceptibility tests when both methods were applied to S. pneumoniae. Despite this high degree of variation, categorical interpretive errors were uncommon. Numerous laboratories reported results for antimicrobial agents that are not recommended by the National Committee for Clinical Laboratory Standards for tests with S. pneumoniae. CONCLUSIONS: Current susceptibility testing practices with S. pneumoniae in the United States indicate limited precision and a tendency for laboratories to test and report results obtained with antimicrobial agents of questionable therapeutic value against this organism. Continued efforts to standardize susceptibility testing of S. pneumoniae in the United States are warranted. In addition, modifications of existing interpretive criteria may be necessary.

Doern GV, Pfaller MA, Erwin ME, Brueggemann AB, Jones RN. 1998. The prevalence of fluoroquinolone resistance among clinically significant respiratory tract isolates of Streptococcus pneumoniae in the United States and Canada--1997 results from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis, 32 (4), pp. 313-316. | Show Abstract | Read more

As part of the SENTRY antimicrobial resistance surveillance program, a total of 1100 clinically significant respiratory tract isolates of Streptococcus pneumoniae were tested for susceptibility to six fluoroquinolone antimicrobial agents: ciprofloxacin, levofloxacin, gatifloxacin, grepafloxacin, sparfloxacin, and trovafloxacin. Isolates were obtained during the 5-month period, February to June, 1997 from 27 United States medical center laboratories and seven laboratories in Canadian health care institutions. All testing was performed in a single center. Of 1100 test strains, 3 (0.3%), all from different U.S. centers, were fluoroquinolone resistant. Among the remaining 1097 fluoroquinolone-susceptible isolates, the rank order of activity among the six agents tested in this study was grepafloxacin (modal MIC = 0.25 microgram/mL) = trovafloxacin (modal MIC = 0.25 microgram/mL) = sparfloxacin (0.25 microgram/mL) > gatifloxacin (0.5 microgram/mL) > levofloxacin (1 microgram/mL) = ciprofloxacin (1 microgram/mL). Fluoroquinolone resistance is currently uncommon among respiratory tract isolates of S. pneumoniae in North America, but there exist clear differences between the in vitro activities of different fluoroquinolones for this organism.

Doern GV, Brueggemann AB, Blocker M, Dunne M, Holley HP, Kehl KS, Duval J, Kugler K, Putnam S, Rauch A, Pfaller MA. 1998. Clonal relationships among high-level penicillin-resistant Streptococcus pneumoniae in the United States. Clin Infect Dis, 27 (4), pp. 757-761. | Show Abstract | Read more

During the past 6 to 7 years, the problem of antimicrobial resistance in Streptococcus pneumoniae has grown dramatically in the United States. Currently, approximately 26.5% of pneumococcal isolates express intermediate levels of resistance to penicillin; approximately 17.5% are highly penicillin resistant. We studied whether clonal relationships exist among current isolates of high-level penicillin-resistant S. pneumoniae (PRSP) in the United States. One hundred forty-seven PRSP isolates recovered in a 30-center surveillance study in the United States during 1994-1995 were characterized with respect to serotype, antimicrobial susceptibility pattern, and pulsed-field gel electrophoresis (PFGE) profile. Only six serotypes were observed among the 147 PRSP isolates examined in this study: 6A, 6B, 9A, 14, 19F, and 23F. One hundred three (70.1%) of the 147 strains were characterized by one of only nine PFGE types; 76 (51.7%) of the 147 isolates were characterized by only four PFGE profiles. Currently in the United States, most PRSP strains are represented by relatively few clonal groups.

Heard SO, Wagle M, Vijayakumar E, McLean S, Brueggemann A, Napolitano LM, Edwards LP, O'Connell FM, Puyana JC, Doern GV. 1998. Influence of triple-lumen central venous catheters coated with chlorhexidine and silver sulfadiazine on the incidence of catheter-related bacteremia. Arch Intern Med, 158 (1), pp. 81-87. | Show Abstract | Read more

OBJECTIVE: To evaluate the efficacy of triple-lumen central venous catheters coated with a combination product of chlorhexidine and silver sulfadiazine (CSS) in reducing the incidence of local catheter infection and catheter-related bacteremia. DESIGN: Randomized, controlled trial. SETTING: The surgical intensive care units in a university hospital. PATIENTS: All patients who needed central venous catheterization were randomized to receive either an uncoated triple-lumen catheter (n = 157) or a catheter coated with CSS (n = 151). MAIN OUTCOME MEASURE: Catheters were removed when no longer needed or suspected as a cause of infection. The tip and a 5-cm segment of the intradermal portion of the catheter were cultured semiquantitatively. Blood cultures were obtained when clinically indicated. The remaining segment of catheters coated with CSS were cut and incubated on an agar plate with strains of Staphylococcus aureus and Enterococcus. Zone of inhibition was determined 24 hours later. Data were analyzed by survival and logistic multivariate regression methods. RESULTS: Catheters coated with CSS were effective in reducing the rate of significant bacterial growth on either the tip or intradermal segment (40%) compared with control catheters (52%; P = .04). However, there was no difference in the incidence of catheter-related bacteremia (3.8% [uncoated] vs 3.3% [coated]; P = .81). In vitro activity of catheters with CSS against S aureus was evident up to 25 days but activity against Enterococcus dissipated more quickly over time and was absent by day 4. The most common colonizing organisms were coagulase-negative staphylococcus and enterococcus. Variables that were associated with a significant amount of growth on the tip or intradermal segment were a duration of catheterization of longer than 7 days, jugular insertion site, and the absence of a CSS coating. The use of a guidewire when the catheter was removed was associated with a lower risk of significant bacterial growth. CONCLUSIONS: The use of CSS reduces the incidence of significant bacterial growth on either the tip or intradermal segments of coated triple-lumen catheters but has no effect on the incidence of catheter-related bacteremia. In this patient population, catheters coated with CSS provide no additional benefit over uncoated catheters.

Dickson EW, Heard SO, Otto S, Tarara TE, Brueggemann AB, Doem GV. 1998. Efficacy of a novel perfluorocarbon-based intrapulmonary drug delivery system Critical Care Medicine, 26 (1 SUPPL.), | Show Abstract

Introduction: We sought to determine the efficacy of a novel intrapulmonary perfluorocarbon (PFC) based antibiotic delivery system in a rat model of pneumococcal pneumonia. Methods: Wistar rats (400-500 g) were inoculated with 8x10' type 3 pneumococcus via direct intratracheal (IT) injection. Twenty-four h after inoculation, rats received no treatment (control, n=15), 10 mg IM ampicillin (AMP, n =10), 10 ml lavage with an AMP containing (1 mg/ml) microparticulate dispersion in PFC (AMPPFC; Alliance Pharmaceutical Corp.; n=ll). Animals were monitored daily for survival for 10 days. AMP concentrations, were determined in serum and lung homogenates using a bioassay. The log-rank test with Bonferroni's correction was used to determine differences in mortality among groups. The null hypothesis was rejected for a critical p < 0.017. Results: Mortality was significantly higher in controls than AMPPFC (100% vs 18%, p < 0.017). There was a trend favoring treatment with AMPPFC compared to AMP (18% vs 70% mortality, p < 0.019). The table shows serum and lung antibiotic concentrations (n=2) Time (h) AMP (jig/ml) AMPPFC Qig/ml) Serum Lung Serum Lung 1 15 2 15 500 2 1 1 2 120 3 0 0 1 60 4 0 0 2 50 8 0 0 0 15 24 0 0 0 10 48 0 0 0 3 72 0 0 0 2 Conclusions: PFC-based IT antibiotic microparticulate formüations may be more effect than systemic antibiotic therapy alone in the treatment of severe pneumonia.

Dickson EW, Heard SO, Chu B, Fraire A, Brueggemann AB, Doern GV. 1998. Partial liquid ventilation with perfluorocarbon in the treatment of rats with lethal pneumococcal pneumonia. Anesthesiology, 88 (1), pp. 218-223. | Show Abstract | Read more

BACKGROUND: Partial liquid ventilation with perfluorocarbon is a new therapeutic strategy to treat various lung disorders. The current study was undertaken to determine the efficacy of partial liquid ventilation with a perfluorocarbon (FC-77) in the treatment of pneumococcal pneumonia in rats. METHODS: Male Wistar rats (weight, 275-300 g; n, 75) were infected via direct intratracheal inoculation with ca 10(9) colony-forming units of viable Streptococcus pneumoniae, serotype 3, and 24 h after infection were placed into one of five groups, each containing 15 rats. The groups were (1) no treatment, (2) one intramuscular injection of penicillin G benzathine (200,000 U), (3) partial liquid ventilation with FC-77, (4) partial liquid ventilation with FC-77 and a single intramuscular dose of penicillin G benzathine (200,000 U), and (5) gas ventilation. Animals were observed every 24 h for survival. RESULTS: All untreated or gas-ventilated animals or animals that received only partial liquid ventilation were dead by 7 days. Those receiving only partial liquid ventilation survived longer than untreated controls, but ultimately all succumbed by day 7. Survival was 40% for penicillin-treated rats compared with controls (P < 0.05) and 80% for animals treated with both partial liquid ventilation and penicillin versus antibiotic alone (P < 0.05). CONCLUSIONS: These observations suggest that partial liquid ventilation with perfluorocarbon in combination with antibiotic administration may be an effective therapeutic modality in pneumococcal pneumonia.

Doern GV, Brueggemann AB, Perla R, Daly J, Halkias D, Jones RN, Saubolle MA. 1997. Multicenter laboratory evaluation of the bioMérieux Vitek antimicrobial susceptibility testing system with 11 antimicrobial agents versus members of the family Enterobacteriaceae and Pseudomonas aeruginosa. J Clin Microbiol, 35 (8), pp. 2115-2119. | Show Abstract

A four-center study in which a total of 1,082 recent clinical isolates of members of the family Enterobacteriaceae and Pseudomonas aeruginosa were examined versus 11 antimicrobial agents with the bioMérieux Vitek susceptibility test system (Hazelwood, Mo.) and the GNS-F6 card was conducted. In addition, a challenge set consisting of the same 200 organisms was examined in each of the four participating laboratories. Results obtained with the Vitek system were compared to MICs determined by a standardized broth microdilution method. For purposes of comparison, susceptibility categories (susceptible, intermediate, or resistant) were assigned on the basis of the results of both methods. The result of the broth microdilution test was considered definitive. The total category error rate with the Vitek system and the recent clinical isolates (11,902 organism-antimicrobial comparisons) was 4.5%, i.e., 1.7% very major errors, 0.9% major errors, and 1.9% minor errors. The total category error rate calculated from tests performed with the challenge set (i.e., 8,800 organism-antimicrobial comparisons) was 5.9%, i.e., 2.2% very major errors, 1.1% major errors, and 2.6% minor errors. Very major error rates higher than the totals were noted with Enterobacter cloacae versus ampicillin-sulbactam, aztreonam, ticarcillin, and ticarcillin-clavulanate and with P. aeruginosa versus mezlocillin, ticarcillin, and ticarcillin-clavulanate. Major error rates higher than the averages were observed with Proteus mirabilis versus imipenem and with Klebsiella pneumoniae versus ofloxacin. Excellent overall interlaboratory reproducibility was observed with the Vitek system. The importance of inoculum size as a primary determinant in the accuracy of susceptibility test results with the Vitek system was clearly demonstrated in this study. Specifically, when an inoculum density fourfold higher than that recommended by the manufacturer was used, high rates of false resistance results were obtained with cell wall-active antimicrobial agents versus both the Enterobacteriaceae and P. aeruginosa.

Brueggemann AB, Kugler KC, Doern GV. 1997. In vitro activity of BAY 12-8039, a novel 8-methoxyquinolone, compared to activities of six fluoroquinolones against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Antimicrob Agents Chemother, 41 (7), pp. 1594-1597. | Show Abstract

The in vitro activity of a novel 8-methoxyquinolone, BAY 12-8039, against recent clinical isolates of Streptococcus pneumoniae (n = 404), Haemophilus influenzae (n = 330), and Moraxella catarrhalis (n = 250) was evaluated. Activity was compared to those of six other fluoroquinolones: ciprofloxacin, clinafloxacin, levofloxacin, ofloxacin, sparfloxacin and trovafloxacin. BAY 12-8039 and clinafloxacin had the highest levels of activity against S. pneumoniae, both with a MIC at which 90% of the isolates were inhibited (MIC90) of 0.06 microg/ml. Trovafloxacin and sparfloxacin were the next most active agents versus S. pneumoniae (MIC90s = 0.12 microg/ml). No differences in activity against penicillin-susceptible, -intermediate, or -resistant strains of S. pneumoniae were noted for any of the fluoroquinolones tested. MIC90s for the seven fluoroquinolones ranged from 0.008 to 0.06 microg/ml versus H. influenzae and from 0.008 to 0.12 microg/ml for M. catarrhalis. The MICs for two strains of S. pneumoniae and one strain of H. influenzae were noted to be higher than those for the general population of organisms for all of the fluoroquinolones tested. Finally, the activity of BAY 12-8039 versus S. pneumoniae was found to be diminished when MIC determinations were performed with incubation of agar dilution plates or broth microdilution trays in 5 to 7% CO2 versus ambient air.

Doern GV, Brueggemann AB, Dunne WM, Jenkins SG, Halstead DC, McLaughlin JC. 1997. Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system. J Clin Microbiol, 35 (5), pp. 1290-1292. | Show Abstract

Blood culture records from 1994 to 1995 from five U.S. medical centers all using the Difco ESP continuous monitoring blood culture system were reviewed retrospectively. Among a total of 7,362 isolates of bacteria and yeasts, only 0.1% of possibly significant isolates would have been missed had blood cultures been routinely incubated for 4 days instead of the 5 days recommended by the manufacturer. Conversely, numerous contaminants, detected only on day 5, would have been eliminated by a 4-day incubation period.

Doern GV, Brueggemann AB, Pierce G. 1997. Assessment of the oxacillin disk screening test for determining penicillin resistance in Streptococcus pneumoniae. Eur J Clin Microbiol Infect Dis, 16 (4), pp. 311-314. | Show Abstract | Read more

The 1 microgram oxacillin disk diffusion screening test was performed on 1516 recent clinical isolates of Streptococcus pneumoniae obtained in a 1994-1995 U.S. surveillance study and the results compared to penicillin MICs determined using a standardized broth microdilution method. The oxacillin disk screening method failed to distinguish penicillin-resistant strains from those that were intermediately susceptible. Furthermore, a high percentage (11.1%) of penicillin-susceptible strains, for which MICs of penicillin were usually 0.06 or 0.03 microgram/ml, yielded zone diameters of < or = 19 mm with the oxacillin screen test and thus would have been falsely categorized as being resistant to penicillin.

Doern GV, Brueggemann AB, Pierce G, Holley HP, Rauch A. 1997. Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of beta-lactamase-positive strains resistant to amoxicillin-clavulanate: results of a national multicenter surveillance study. Antimicrob Agents Chemother, 41 (2), pp. 292-297. | Show Abstract

A total of 1,537 clinical isolates of Haemophilus influenzae were recovered in 30 U.S. medical center laboratories between 1 November 1994 and 30 April 1995 and were characterized in a central laboratory with respect to serotype and beta-lactamase production and the in vitro activities of 15 oral antimicrobial agents. Overall, 36.4% of the isolates were found to produce beta-lactamase. The rank order of activity of six cephalosporins on the basis of MICs was cefixime > cefpodoxime > cefuroxime > loracarbef > or = cefaclor > cefprozil. On the basis of current National Committee for Clinical Laboratory Standards (NCCLS) breakpoints ages of isolates found to be resistant or intermediate to these agents were as follows: 0.1, 0.3, 6.4, 16.3, 18.3, and 29.8, respectively (National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. M7-A4, 1995). Azithromycin was, on a weight basis, the most potent of the macrolides tested in this study, followed by erythromycin and then clarithromycin. Azithromycin was typically fourfold more active than erythromycin, which was, in turn, slightly more active than clarithromycin. However, when compared on the basis of the frequency of resistance determined by using current NCCLS breakpoints, there was essentially no difference between azithromycin and clarithromycin, i.e., 0.5 and 1.9%, respectively (P = 0.086). Interpretive breakpoints for erythromycin MIC tests versus H. influenzae have not been developed. Resistance to other non- beta-lactam agents was variable, as follows: trimethoprim-sulfamethoxazole, 9.0%; chloramphenicol, 0.2%; tetracycline, 1.3%; and rifampin, 0.3%. Two conspicuous findings in this study were the identification of 39 strains H. influenzae that were beta-lactamase negative but ampicillin intermediate or resistant (BLNAR) and, even more surprisingly, 17 beta-lactamase-positive isolates that were resistant to amoxicillin-clavulanate (BLPACR). Strains of H. influenzae in the first group have heretofore been very uncommon; organisms in the second group have not previously been described in the literature. The percentages of all study isolates comprised of BLNAR and BLPACR organisms were 2.5 and 1.1, respectively. Overall resistance to ampicillin was thus 38.9%, and that to amoxicillin-clavulanate was 4.5%.

Doern GV, Brueggemann AB, Pierce G, Hogan T, Holley HP, Rauch A. 1996. Prevalence of antimicrobial resistance among 723 outpatient clinical isolates of Moraxella catarrhalis in the United States in 1994 and 1995: results of a 30-center national surveillance study. Antimicrob Agents Chemother, 40 (12), pp. 2884-2886. | Show Abstract

Seven hundred twenty-three isolates of Moraxella catarrhalis obtained from outpatients with a variety of infections in 30 medical centers in the United States between 1 November 1994 and 30 April 1995 were characterized in a central laboratory. The overall rate of beta-lactamase production was 95.3%. When the National Committee for Clinical Laboratory Standards MIC interpretive breakpoints for Haemophilus influenzae were applied, percentages of strains found to be susceptible to selected oral antimicrobial agents were as follows: azithromycin, clarithromycin, and erythromycin, 100%; tetracycline and chloramphenicol, 100%; amoxicillin-clavulanate, 100%; cefixime, 99.3%; cefpodoxime, 99.0%; cefaclor, 99.4%; loracarbef, 99.0%; cefuroxime, 98.5%; cefprozil, 94.3%; and trimethoprim-sulfamethoxazole, 93.5%.

Doern GV, Pierce G, Brueggemann AB. 1996. In vitro activity of sanfetrinem (GV104326), a new trinem antimicrobial agent, versus Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Diagn Microbiol Infect Dis, 26 (1), pp. 39-42. | Show Abstract | Read more

Sanfetrinem, formerly GV104326, is a new trinem antimicrobial agent with extensive in vitro activity for a variety of different bacteria. The activity of sanfetrinem was determined using a broth microdilution MIC method versus a large number of clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis obtained in a recent 30-center United States surveillance study. The sanfetrinem MIC50 and MIC90 values for these three organism groups were 0.015 and 0.5 microgram/ml, 0.25, and 0.5 microgram/ml, and 0.015 and 0.03, respectively.

Doern GV, Brueggemann A, Holley HP, Rauch AM. 1996. Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a 30-center national surveillance study. Antimicrob Agents Chemother, 40 (5), pp. 1208-1213. | Show Abstract

A total of 1,527 clinically significant outpatient isolates of Streptococcus pneumoniae were prospectively collected in 30 different U.S. medical centers between November 1994 and April 1995. Overall, 23.6% of strains were not susceptible to penicillin, with 14.1% intermediate and 9.5% high-level resistant. The frequencies of recovery of intermediate and high-level resistant strains varied considerably between different medical centers and in different geographic areas. In general, intermediate and high-level penicillin resistance was most common with isolates of S. pneumoniae recovered from pediatric patients. The in vitro activities of 22 other antimicrobial agents were assessed against this collection of isolates. Ampicillin was consistently 1 twofold dilution less active than penicillin. Amoxicillin and amoxicillin-clavulanate were essentially equivalent to penicillin in activity. The rank order of activity for cephalosporins was cefotaxime = ceftriaxone > or = cefpodoxime > or = cefuroxime > cefprozil > or = cefixime > cefaclor = loracarbef > cefadroxil = cephalexin. The National Committee for Clinical Laboratory Standards [Performance Standards for Antimicrobial Susceptibility Testing, Sixth Information Supplement (M100-S6), 1995] has established MIC breakpoints for resistance (i.e., > or = 2 micrograms/ml) with three cephalosporins versus S. pneumoniae, namely, cefotaxime, ceftriaxone, and cefuroxime. The overall percentages of strains resistant to these three antimicrobial agents were 3, 5, and 12, respectively. The overall frequency of resistance was 10% with all three macrolides examined in this study, clarithromycin, erythromycin, and azithromycin. The overall percentages of chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole resistance were 4.3, 7.5, and 18, respectively. The resistance percentages among the cephalosporins, macrolides, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole were consistently higher among penicillin-intermediate strains than among susceptible isolates and even higher still among organisms expressing high-level penicillin resistance. Multiply resistant strains represented 9.1% of the organisms examined in this study. Finally, rifampin resistance was uncommon (i.e., 0.5%), and vancomycin resistance was not detected. The quinopristin-dalfopristin combination was consistently active at concentrations of 0.25 to 4 micrograms/ml, but rates of resistance could not be determined in the absence of established interpretive criteria for MIC results.

Doern GV, Ferraro MJ, Brueggemann AB, Ruoff KL. 1996. Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States. Antimicrob Agents Chemother, 40 (4), pp. 891-894. | Show Abstract

Three hundred fifty-two blood culture isolates of viridans group streptococci obtained from 43 U.S. medical centers during 1993 and 1994 were characterized. Included were 48 isolates of "Streptococcus milleri," 219 S. mitis isolates, 29 S. salivarius isolates, and 56 S. sanguis isolates. High-level penicillin resistance (MIC, > or = 4.0 micrograms/ml) was noted among 13.4% of the strains; for 42.9% of the strains, penicillin MICs were 0.25 to 2.0 micrograms/ml (i.e., intermediate resistance). In general, amoxicillin was slightly more active than penicillin. The rank order of activity for five cephalosporins versus viridans group streptococci was cefpodoxime = ceftriaxone > cefprozil = cefuroxime > cephalexin. The percentages of isolates resistant (MIC, > or = 2 micrograms/ml) to these agents were 15, 17, 18, 20, and 96, respectively. The rates of resistance to erythromycin, tetracycline, and trimethoprim-sulfamethoxazole were 12 to 38%. Resistance to either chloramphenicol or ofloxacin was uncommon (i.e., < 1%). In general, among the four species, S. mitis was the most resistant and "S. milleri" was the most susceptible.

Doern GV, Jones RN, Gerlach EH, Washington JA, Biedenbach DJ, Brueggemann A, Erwin ME, Knapp C, Raymond J. 1995. Multicenter clinical laboratory evaluation of a beta-lactamase disk assay employing a novel chromogenic cephalosporin, S1. J Clin Microbiol, 33 (6), pp. 1665-1667. | Show Abstract

S1, a new chromogenic cephalosporin (International BioClinical, Inc., Portland, Oreg.), was used to detect beta-lactamase production among a variety of commonly encountered bacteria in a four-center collaborative study. Results of an S1 disk assay were compared with those obtained by a nitrocefin-based disk procedure (Cefinase; Becton-Dickinson Microbiology Systems, Cockeysville, Md.), with repetitive testing of five quality control organisms and with individual tests of recent clinical isolates of Neisseria gonorrhoeae (162 strains), Haemophilus influenzae (162 strains), Moraxella catarrhalis (155 strains), Staphylococcus aureus (161 strains), and Bacteroides fragilis (164 strains). The performances of the two beta-lactamase disk assays were comparable for the first three species cited above. However, the S1 assay appeared to be a more sensitive procedure than the Cefinase assay when applied to S. aureus and B. fragilis, with respect to both total numbers of positive results and length of time to a definitive positive endpoint.

Brueggemann AB, Harrold CL, Rezaei Javan R, van Tonder AJ, McDonnell AJ, Edwards BA. 2017. Pneumococcal prophages are diverse, but not without structure or history. Sci Rep, 7 pp. 42976. | Show Abstract | Read more

Bacteriophages (phages) infect many bacterial species, but little is known about the diversity of phages among the pneumococcus, a leading global pathogen. The objectives of this study were to determine the prevalence, diversity and molecular epidemiology of prophages (phage DNA integrated within the bacterial genome) among pneumococci isolated over the past 90 years. Nearly 500 pneumococcal genomes were investigated and RNA sequencing was used to explore prophage gene expression. We revealed that every pneumococcal genome contained prophage DNA. 286 full-length/putatively full-length pneumococcal prophages were identified, of which 163 have not previously been reported. Full-length prophages clustered into four major groups and every group dated from the 1930-40 s onward. There was limited evidence for genes shared between prophage clusters. Prophages typically integrated in one of five different sites within the pneumococcal genome. 72% of prophages possessed the virulence genes pblA and/or pblB. Individual prophages and the host pneumococcal genetic lineage were strongly associated and some prophages persisted for many decades. RNA sequencing provided clear evidence of prophage gene expression. Overall, pneumococcal prophages were highly prevalent, demonstrated a structured population, possessed genes associated with virulence, and were expressed under experimental conditions. Pneumococcal prophages are likely to play a more important role in pneumococcal biology and evolution than previously recognised.

van Tonder AJ, Bray JE, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Hoffmann S, Bentley SD, Haraldsson Á, Erlendsdóttir H et al. 2016. Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes. Microb Genom, 2 (10), pp. 000090. | Show Abstract | Read more

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Bentley SD et al. 2015. Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci. J Clin Microbiol, 53 (7), pp. 2271-2285. | Show Abstract | Read more

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

Bogaardt C, van Tonder AJ, Brueggemann AB. 2015. Genomic analyses of pneumococci reveal a wide diversity of bacteriocins - including pneumocyclicin, a novel circular bacteriocin. BMC Genomics, 16 (1), pp. 554. | Show Abstract | Read more

BACKGROUND: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the 'pneumococcus'). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure. RESULTS: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the 'bacteriocin/immunity region' of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes. CONCLUSIONS: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

van Tonder AJ, Mistry S, Bray JE, Hill DM, Cody AJ, Farmer CL, Klugman KP, von Gottberg A, Bentley SD, Parkhill J et al. 2014. Defining the estimated core genome of bacterial populations using a Bayesian decision model. PLoS Comput Biol, 10 (8), pp. e1003788. | Show Abstract | Read more

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance.

Brueggemann AB, Muroki BM, Kulohoma BW, Karani A, Wanjiru E, Morpeth S, Kamau T, Sharif S, Scott JAG. 2013. Population genetic structure of Streptococcus pneumoniae in Kilifi, Kenya, prior to the introduction of pneumococcal conjugate vaccine PLoS ONE, 8 (11), | Show Abstract | Read more

Background: The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Kenya in 2011. Introduction of any PCV will perturb the existing pneumococcal population structure, thus the aim was to genotype pneumococci collected in Kilifi before PCV10. Methods and Findings: Using multilocus sequence typing (MLST), we genotyped > 1100 invasive and carriage pneumococci from children, the largest collection genotyped from a single resource-poor country and reported to date. Serotype 1 was the most common serotype causing invasive disease and was rarely detected in carriage; all serotype 1 isolates were members of clonal complex (CC) 217. There were temporal fluctuations in the major circulating sequence types (STs); and although 1-3 major serotype 1, 14 or 23F STs co-circulated annually, the two major serotype 5 STs mainly circulated independently. Major STs/CCs also included isolates of serotypes 3, 12F, 18C and 19A and each shared ≤2 MLST alleles with STs that circulate widely elsewhere. Major CCs associated with non-PCV10 serotypes were predominantly represented by carriage isolates, although serotype 19A and 12F CCs were largely invasive and a serotype 10A CC was equally represented by invasive and carriage isolates. Conclusions: Understanding the pre-PCV10 population genetic structure in Kilifi will allow for the detection of changes in prevalence of the circulating genotypes and evidence for capsular switching post-vaccine implementation. © 2013 Brueggemann et al.

Wyres KL, van Tonder A, Lambertsen LM, Hakenbeck R, Parkhill J, Bentley SD, Brueggemann AB. 2013. Evidence of antimicrobial resistance-conferring genetic elements among pneumococci isolated prior to 1974. BMC Genomics, 14 (1), pp. 500. | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements. RESULTS: Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes. CONCLUSIONS: These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Liñares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP et al. 2013. Pneumococcal capsular switching: a historical perspective. J Infect Dis, 207 (3), pp. 439-449. | Show Abstract | Read more

BACKGROUND: Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. METHODS: Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. RESULTS: We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥ 58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. CONCLUSIONS: Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Liñares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP et al. 2012. The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success. Genome Biol, 13 (11), pp. R103. | Show Abstract | Read more

BACKGROUND: Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species. RESULTS: We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described. CONCLUSIONS: These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

Beall BW, Gertz RE, Hulkower RL, Whitney CG, Moore MR, Brueggemann AB. 2011. Shifting genetic structure of invasive serotype 19A pneumococci in the United States. J Infect Dis, 203 (10), pp. 1360-1368. | Show Abstract | Read more

BACKGROUND: Following 7-valent conjugate vaccine introduction in the United States in 2000, invasive serotype (sero19A) pneumococcal disease (IPD) emerged rapidly. Sero19A IPD incidence increased slightly during 2005-2008 (from 2.3 cases to 2.5 cases per 100,000 population), whereas sero19A penicillin resistance (defined as a minimum inhibitor concentration [MIC] ≥2 μg/mL) increased significantly (from 28.7% to 43.7%). To better understand changes, we characterized sero19A isolates recovered during 2004-2008. METHODS: We performed antimicrobial susceptibility testing on all 2767 sero19A IPD isolates identified through the Centers for Disease Control Active Bacterial Core surveillance during 2004-2008. We genotyped 1804 (96.3%) of 1874 sero19A isolates recovered during 2005-2007 and all 148 year 2008 sero19A isolates from children <5 years of age. RESULTS: Resistant clonal complex (CC) 320/271(19A) increased from 20.9% (115 of 550) to 32.9% (208 of 633; P < .001) of IPD isolates during 2005-2007, which paralleled increased sero19A penicillin resistance (from 28.7% [163 of 567 isolates] to 39.5% [261 of 661 isolates]; P < .001). Total IPD due to 320/271(19A) increased during 2005-2007 and increased from 2.1 to 3.6 cases per 100,000 population during 2005-2008 in children <5 years of age. The penicillin-susceptible/intermediate, putative vaccine-escape CC695(19A) increased from 7.5% (41 of 550) to 13.6% (85 of 633) of sero19A isolates during 2005-2007 (P = .002). CONCLUSIONS: Sero19A rates may have plateaued; however, clonal shifts are increasing resistance. Increased IPD caused by CC320/271(19A) and CC695(19A) could reflect additional selective advantages in addition to resistance.

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