Sandra Reuter

Roary: rapid large-scale prokaryote pan genome analysis

Summary: A typical prokaryote population sequencing study can now consist of hundreds or thousands of isolates. Interrogating these datasets can provide detailed insights into the genetic structure of prokaryotic genomes. We introduce Roary, a tool that rapidly builds large-scale pan genomes, identifying the core and accessory genes. Roary makes construction of the pan genome of thousands of prokaryote samples possible on a standard desktop without compromising on the accuracy of results. Using a single CPU Roary can produce a pan genome consisting of 1000 isolates in 4.5 hours using 13 GB of RAM, with further speedups possible using multiple processors. Availability and implementation: Roary is implemented in Perl and is freely available under an open source GPLv3 license from http://sanger-pathogens.github.io/Roary Contact: roary@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance.

Parallel independent evolution of pathogenicity within the genus Yersinia.

Significance Our past understanding of pathogen evolution has been fragmented because of tendencies to study human clinical isolates. To understand the evolutionary trends of pathogenic bacteria though, we need the context of their nonpathogenic relatives. Our unique and detailed dataset allows description of the parallel evolution of two key human pathogens: the causative agents of plague and Yersinia diarrhea. The analysis reveals an emerging pattern where few virulence-related functions are found in all pathogenic lineages, representing key “foothold” moments that mark the emergence of these pathogens. Functional gene loss and metabolic streamlining are equally complementing the evolution of Yersinia across the pathogenic spectrum. The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

A pilot study of rapid whole-genome sequencing for the investigation of a Legionella outbreak

Objectives Epidemiological investigations of Legionnaires’ disease outbreaks rely on the rapid identification and typing of clinical and environmental Legionella isolates in order to identify and control the source of infection. Rapid bacterial whole-genome sequencing (WGS) is an emerging technology that has the potential to rapidly discriminate outbreak from non-outbreak isolates in a clinically relevant time frame. Methods We performed a pilot study to determine the feasibility of using bacterial WGS to differentiate outbreak from non-outbreak isolates collected during an outbreak of Legionnaires’ disease. Seven Legionella isolates (three clinical and four environmental) were obtained from the reference laboratory and sequenced using the Illumina MiSeq platform at Addenbrookes Hospital, Cambridge. Bioinformatic analysis was performed blinded to the epidemiological data at the Wellcome Trust Sanger Institute. Results We were able to distinguish outbreak from non-outbreak isolates using bacterial WGS, and to confirm the probable environmental source. Our analysis also highlighted constraints, which were the small number of Legionella pneumophila isolates available for sequencing, and the limited number of published genomes for comparison. Conclusions We have demonstrated the feasibility of using rapid WGS to investigate an outbreak of Legionnaires’ disease. Future work includes building larger genomic databases of L pneumophila from both clinical and environmental sources, developing automated data interpretation software, and conducting a cost–benefit analysis of WGS versus current typing methods.

Rapid Whole-Genome Sequencing for Investigation of a Suspected Tuberculosis Outbreak

ABSTRACT Two Southeast Asian students attending the same school in the United Kingdom presented with pulmonary tuberculosis. An epidemiological investigation failed to link the two cases, and drug resistance profiles of the Mycobacterium tuberculosis isolates were discrepant. Whole-genome sequencing of the isolates found them to be genetically identical, suggesting a missed transmission event.

Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes

Objectives Genome sequencing will be increasingly used in the clinical setting to tailor antimicrobial prescribing and inform infection control outbreaks. A recent technological innovation that could reduce the delay between pathogen sampling and data generation is single molecule sequencing. An example of this technology, which is undergoing evaluation through an early access programme, is the Oxford Nanopore MinION. Methods We undertook a feasibility study on six clinically significant pathogens, comparing the MinION to the Illumina MiSeq and PacBio RSII platforms. Genomic DNA was prepared and sequenced using the MinION as instructed by the manufacturer, and Illumina MiSeq and PacBio sequencing was performed using established methods. Results An evaluation of the accuracy of the MinION based on sequencing of an MRSA isolate showed that error rates were higher in the MinION reads, but provided an even coverage across the entire genome length. The MinION detected all of the expected carbapenemases and ESBL genes in five Gram-negative isolates and the mecA gene in an MRSA isolate. Conclusions The MinION can detect the presence of acquired resistance genes, but improvements in accuracy are needed so that antimicrobial resistance associated with mutations in chromosomal genes can be identified.

'Add, stir and reduce': Yersinia spp. as model bacteria for pathogen evolution

Pathogenic species in the Yersinia genus have historically been targets for research aimed at understanding how bacteria evolve into mammalian pathogens. The advent of large-scale population genomic studies has greatly accelerated the progress in this field, and Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica have once again acted as model organisms to help shape our understanding of the evolutionary processes involved in pathogenesis. In this Review, we highlight the gene gain, gene loss and genome rearrangement events that have been identified by genomic studies in pathogenic Yersinia species, and we discuss how these findings are changing our understanding of pathogen evolution. Finally, as these traits are also found in the genomes of other species in the Enterobacteriaceae, we suggest that they provide a blueprint for the evolution of enteropathogenic bacteria.

Rapid single-colony whole-genome sequencing of bacterial pathogens

Objectives As a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates. Methods We developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of blaNDM-1, a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis. Results We validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests. Conclusions This advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology.

Building a genomic framework for prospective MRSA surveillance in the United Kingdom and the Republic of Ireland.

The correct interpretation of microbial sequencing data applied to surveillance and outbreak investigation depends on accessible genomic databases to provide vital genetic context. Our aim was to construct and describe a United Kingdom MRSA database containing over 1000 methicillin-resistant Staphylococcus aureus (MRSA) genomes drawn from England, Northern Ireland, Wales, Scotland, and the Republic of Ireland over a decade. We sequenced 1013 MRSA submitted to the British Society for Antimicrobial Chemotherapy by 46 laboratories between 2001 and 2010. Each isolate was assigned to a regional healthcare referral network in England and was otherwise grouped based on country of origin. Phylogenetic reconstructions were used to contextualize MRSA outbreak investigations and to detect the spread of resistance. The majority of isolates (n = 783, 77%) belonged to CC22, which contains the dominant United Kingdom epidemic clone (EMRSA-15). There was marked geographic structuring of EMRSA-15, consistent with widespread dissemination prior to the sampling decade followed by local diversification. The addition of MRSA genomes from two outbreaks and one pseudo-outbreak demonstrated the certainty with which outbreaks could be confirmed or refuted. We identified local and regional differences in antibiotic resistance profiles, with examples of local expansion, as well as widespread circulation of mobile genetic elements across the bacterial population. We have generated a resource for the future surveillance and outbreak investigation of MRSA in the United Kingdom and Ireland and have shown the value of this during outbreak investigation and tracking of antimicrobial resistance.

Whole-genome sequencing reveals transmission of vancomycin-resistant Enterococcus faecium in a healthcare network

BackgroundBacterial whole-genome sequencing (WGS) has the potential to identify reservoirs of multidrug-resistant organisms and transmission of these pathogens across healthcare networks. We used WGS to define transmission of vancomycin-resistant enterococci (VRE) within a long-term care facility (LTCF), and between this and an acute hospital in the United Kingdom (UK).MethodsA longitudinal prospective observational study of faecal VRE carriage was conducted in a LTCF in Cambridge, UK. Stool samples were collected at recruitment, and then repeatedly until the end of the study period, discharge or death. Selective culture media were used to isolate VRE, which were subsequently sequenced and analysed. We also analysed the genomes of 45 Enterococcus faecium bloodstream isolates collected at Cambridge University Hospitals NHS Foundation Trust (CUH).ResultsForty-five residents were recruited during a 6-month period in 2014, and 693 stools were collected at a frequency of at least 1 week apart. Fifty-one stool samples from 3/45 participants (7 %) were positive for vancomycin-resistant E. faecium. Two residents carried multiple VRE lineages, and one carried a single VRE lineage. Genome analyses based on single nucleotide polymorphisms (SNPs) in the core genome indicated that VRE carried by each of the three residents were unrelated. Participants had extensive contact with the local healthcare network. We found that VRE genomes from LTCF residents and hospital-associated bloodstream infection were interspersed throughout the phylogenetic tree, with several instances of closely related VRE strains from the two settings.ConclusionsA proportion of LTCF residents are long-term carriers of VRE. Evidence for genetic relatedness between these and VRE associated with bloodstream infection in a nearby acute NHS Trust indicate a shared bacterial population.