Antimicrobial resistance gene/vector transmission across human, animal and environmental reservoirs

Project Overview

Antimicrobial resistance (AMR) is a complex problem, and developing an accurate understanding of the extent and mechanisms of its spread is a major challenge. Several bacteria in the Enterobacteriaceae family, including Escherichia coli, Klebsiella spp., Enterobacter spp. and Citrobacter spp., are major causes of human invasive disease, and are able to asymptomatically colonise the gastrointestinal tracts of humans and animals, and various environmental niches. The last ten years have seen the alarming spread of multiple families of drug resistance genes in these species, which are transmitted both within and between species. This makes Enterobacteriaceae infections increasingly difficult to treat and therefore more lethal.

With the REHAB project (REsistance in Human and Animal Biota) we investigate the contribution of humans, animals and the environment in the dissemination of AMR amongst Enterobacteriaceae on an unprecedented scale. We use whole genome sequencing (WGS) to identify opportunities for halting the spread of AMR. We will sequence ~200 environmental metagenomes (pools of DNA from different bacteria) and ~1000 bacterial isolates (individual bacterial cells). The aim of this studentship is to develop new methods for the rapid assembly and classification of these sequencing datasets, statistically investigate source attribution of AMR, and study genetic evolution of AMR vectors.

Nicola De Maio is a mathematician and statistical geneticist based at the University of Oxford Nuffield Department of Medicine, with expertise in the development of evolutionary models and methods. Nicole Stoesser is an Academic Clinical Lecturer in Infectious diseases and Microbiology and a co-PI on the REHAB project. Derrick Crook is the lead of the Modernising Medical Microbiology (MMM) Project, an international consortium focused on the use of WGS to understand the genomic epidemiology of important pathogens. He is also professor of microbiology at the University of Oxford and director of the National Infection Service at Public Health England. The REHAB project is a multi-disciplinary study also involving lead scientists from the Centre of Ecology and Hydrology (Prof Mark Bailey, Drs Dan Read/Mike Bowes); the Animal and Plant Health Agency (Drs Muna Anjum/Richard Smith); Public Health England (Prof Neil Woodford); and Thames Water.

The MMM consortium is supported by a multi-million pound funding portfolio, including from the Wellcome Trust, the National Environmental Research Council, Medical Research Council and the Bill and Melinda Gates Foundation. We have a strong record of publishing in the top internationally recognized journals, and our research has impacted on the delivery of public health and microbiology in Britain and beyond. For more information visit www.modmedmicro.ac.uk.

Training Opportunities

The Modernising Medical Microbiology consortium provides an excellent research environment in which to develop new skills and train among world-leading scientists in their field. Based at the John Radcliffe Hospital, the University of Oxford team consists of a community of research groups led by Profs Derrick Crook, Tim Peto, Sarah Walker and Drs David Clifton, Kate Dingle, Phil Fowler, Zam Iqbal, Danny Wilson and David Wyllie. We have specialist expertise in microbiology, genomics, statistics, epidemiology and bioinformatics, and our work focuses on understanding the causes of infectious disease in populations. Training is provided by weekly supervisory meetings, weekly Modernising Medical Microbiology work-in-progress meetings, journal clubs, seminar series, and external opportunities including attending national and international conferences. The department and university run numerous training courses, including several targeted at DPhil students through the Doctoral Training Centre programmes, while the Department for Continuing Education and the Language Centre offer further opportunities for personal development to research students at Oxford.

Theme

Genetics & Genomics and Immunology & Infectious Disease

Admissions

Project reference number: 841

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Derrick Crook Experimental Medicine Division Oxford University, John Radcliffe Hospital GBR derrick.crook@ndcls.ox.ac.uk
Dr Nicola De Maio University of Oxford GBR nicola.demaio@ndm.ox.ac.uk
Dr Nicole Stoesser Experimental Medicine Division Oxford University, John Radcliffe Hospital GBR nicole.stoesser@ndm.ox.ac.uk

De Maio N, Wu CH, O'Reilly KM, Wilson D. 2015. New Routes to Phylogeography: A Bayesian Structured Coalescent Approximation. PLoS Genet., 11 (8), pp. e1005421. Read abstract | Read more

Phylogeographic methods aim to infer migration trends and the history of sampled lineages from genetic data. Applications of phylogeography are broad, and in the context of pathogens include the reconstruction of transmission histories and the origin and emergence of outbreaks. Phylogeographic inference based on bottom-up population genetics models is computationally expensive, and as a result faster alternatives based on the evolution of discrete traits have become popular. In this paper, we show that inference of migration rates and root locations based on discrete trait models is extremely unreliable and sensitive to biased sampling. To address this problem, we introduce BASTA (BAyesian STructured coalescent Approximation), a new approach implemented in BEAST2 that combines the accuracy of methods based on the structured coalescent with the computational efficiency required to handle more than just few populations. We illustrate the potentially severe implications of poor model choice for phylogeographic analyses by investigating the zoonotic transmission of Ebola virus. Whereas the structured coalescent analysis correctly infers that successive human Ebola outbreaks have been seeded by a large unsampled non-human reservoir population, the discrete trait analysis implausibly concludes that undetected human-to-human transmission has allowed the virus to persist over the past four decades. As genomics takes on an increasingly prominent role informing the control and prevention of infectious diseases, it will be vital that phylogeographic inference provides robust insights into transmission history. Hide abstract

Stoesser N, Sheppard AE, Pankhurst L, De Maio N, Moore CE, Sebra R, Turner P, Anson LW, Kasarskis A, Batty EM, Kos V, Wilson DJ, Phetsouvanh R, Wyllie D, Sokurenko E, Manges AR, Johnson TJ, Price LB, Peto TE, Johnson JR, Didelot X, Walker AS, Crook DW, Modernizing Medical Microbiology Informatics Group (MMMIG). 2016. Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131. MBio, 7 (2), pp. e02162. Read abstract | Read more

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

Sheppard AE, Stoesser N, Wilson DJ, Sebra R, Kasarskis A, Anson LW, Giess A, Pankhurst LJ, Vaughan A, Grim CJ, Cox HL, Yeh AJ, Modernising Medical Microbiology (MMM) Informatics Group, Sifri CD, Walker AS, Peto TE, Crook DW, Mathers AJ. 2016. Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC. Antimicrob. Agents Chemother., 60 (6), pp. 3767-78. Read abstract | Read more

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

Stoesser N, Xayaheuang S, Vongsouvath M, Phommasone K, Elliott I, Del Ojo Elias C, Crook DW, Newton PN, Buisson Y, Lee SJ, Dance DA. 2015. Colonization with Enterobacteriaceae producing ESBLs in children attending pre-school childcare facilities in the Lao People's Democratic Republic. J. Antimicrob. Chemother., 70 (6), pp. 1893-7. Read abstract | Read more

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

Stoesser N, Sheppard AE, Moore CE, Golubchik T, Parry CM, Nget P, Saroeun M, Day NP, Giess A, Johnson JR, Peto TE, Crook DW, Walker AS, Modernizing Medical Microbiology Informatics Group. 2015. Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses. J. Clin. Microbiol., 53 (7), pp. 2122-31. Read abstract | Read more

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

Stoesser N, Sheppard AE, Shakya M, Sthapit B, Thorson S, Giess A, Kelly D, Pollard AJ, Peto TE, Walker AS, Crook DW. 2015. Dynamics of MDR Enterobacter cloacae outbreaks in a neonatal unit in Nepal: insights using wider sampling frames and next-generation sequencing. J. Antimicrob. Chemother., 70 (4), pp. 1008-15. Read abstract | Read more

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

Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. 2016. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis, 16 (3), pp. 285-6. | Read more

Stoesser N, Sheppard AE, Peirano G, Sebra R, Lynch T, Anson L, Kasarskis A, Motyl MR, Crook DW, Pitout JD. 2016. Complete Sequencing of Plasmids Containing blaOXA-163 and blaOXA-48 in Escherichia coli Sequence Type 131. Antimicrob. Agents Chemother., 60 (11), pp. 6948-6951. Read abstract | Read more

OXA-48-like enzymes have emerged as important extended-spectrum β-lactamases/carbapenemases in Escherichia coli sequence type 131 (ST131). We report the structures of the first fully sequenced blaOXA-163 plasmid and of two other blaOXA-48 plasmids in this lineage. blaOXA-163 was located on a 71-kb IncN plasmid with other resistance genes. blaOXA-48 was present on IncL/M plasmids, genetically similar to other blaOXA-48 plasmid sequences, and consistent with interspecies/interlineage spread. The presence of blaOXA-48-like genes on epidemic plasmids in ST131 is of concern. Hide abstract

Stoesser N, Giess A, Batty EM, Sheppard AE, Walker AS, Wilson DJ, Didelot X, Bashir A, Sebra R, Kasarskis A, Sthapit B, Shakya M, Kelly D, Pollard AJ, Peto TE, Crook DW, Donnelly P, Thorson S, Amatya P, Joshi S. 2014. Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting. Antimicrob. Agents Chemother., 58 (12), pp. 7347-57. Read abstract | Read more

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

De Maio N, Schrempf D, Kosiol C. 2015. PoMo: An Allele Frequency-Based Approach for Species Tree Estimation. Syst. Biol., 64 (6), pp. 1018-31. Read abstract | Read more

Incomplete lineage sorting can cause incongruencies of the overall species-level phylogenetic tree with the phylogenetic trees for individual genes or genomic segments. If these incongruencies are not accounted for, it is possible to incur several biases in species tree estimation. Here, we present a simple maximum likelihood approach that accounts for ancestral variation and incomplete lineage sorting. We use a POlymorphisms-aware phylogenetic MOdel (PoMo) that we have recently shown to efficiently estimate mutation rates and fixation biases from within and between-species variation data. We extend this model to perform efficient estimation of species trees. We test the performance of PoMo in several different scenarios of incomplete lineage sorting using simulations and compare it with existing methods both in accuracy and computational speed. In contrast to other approaches, our model does not use coalescent theory but is allele frequency based. We show that PoMo is well suited for genome-wide species tree estimation and that on such data it is more accurate than previous approaches. Hide abstract