Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study

BACKGROUND: A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster. METHODS AND FINDINGS: We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998-22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation. CONCLUSIONS: Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases

Genomic surveillance reveals low prevalence of livestock-associated methicillin-resistant Staphylococcus aureus in the East of England

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging problem in many parts of the world. LA-MRSA has been isolated previously from animals and humans in the United Kingdom (UK), but the prevalence is unknown. The aim of this study was to determine the prevalence and to describe the molecular epidemiology of LA-MRSA isolated in the East of England (broadly Cambridge and the surrounding area). We accessed whole genome sequence data for 2,283 MRSA isolates from 1,465 people identified during a 12-month prospective study between 2012 and 2013 conducted in the East of England, United Kingdom. This laboratory serves four hospitals and 75 general practices. We screened the collection for multilocus sequence types (STs) and for host specific resistance and virulence factors previously associated with LA-MRSA. We identified 13 putative LA-MRSA isolates from 12 individuals, giving an estimated prevalence of 0.82% (95% CI 0.47% to 1.43%). Twelve isolates were mecC-MRSA (ten CC130, one ST425 and one ST1943) and single isolate was ST398. Our data demonstrate a low burden of LA-MRSA in the East of England, but the detection of mecC-MRSA and ST398 indicates the need for vigilance. Genomic surveillance provides a mechanism to detect and track the emergence and spread of MRSA clones of human importance.Supported by grants from the UKCRC Translational Infection Research (TIR) Initiative, and the Medical Research Council (Grant Number G1000803) with contributions to the Grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, and the Chief Scientist Ofce of the Scottish Government Health Directorate (to Prof. Peacock); a Hospital Infection Society Major Research Grant, and by Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute. Tis work was supported by the Wellcome Trust 201344/Z/16/Z. M.E.T. is a Clinician Scientist Fellow, supported by the Academy of Medical Sciences and the Health Foundation, and by the NIHR Cambridge Biomedical Research Centre

Longitudinal genomic surveillance of MRSA in the UK reveals transmission patterns in hospitals and the community

Genome sequencing has provided snapshots of the transmission of methicillin-resistant Staphylococcus aureus (MRSA) during suspected outbreaks in isolated hospital wards. Scale-up to populations is now required to establish the full potential of this technology for surveillance. We prospectively identified all individuals over a 12-month period who had at least one MRSA-positive sample processed by a routine diagnostic microbiology laboratory in the East of England, which received samples from three hospitals and 75 general practitioner (GP) practices. We sequenced at least 1 MRSA isolate from 1465 individuals (2282 MRSA isolates) and recorded epidemiological data. An integrated epidemiological and phylogenetic analysis revealed 173 transmission clusters containing between 2 and 44 cases and involving 598 people (40.8%). Of these, 118 clusters (371 people) involved hospital contacts alone, 27 clusters (72 people) involved community contacts alone, and 28 clusters (157 people) had both types of contact. Community- and hospital-associated MRSA lineages were equally capable of transmission in the community, with instances of spread in households, long-term care facilities, and GP practices. Our study provides a comprehensive picture of MRSA transmission in a sampled population of 1465 people and suggests the need to review existing infection control policy and practice.This work was supported by grants from the UK Clinical Research Collaboration Translational Infection Research Initiative and the Medical Research Council (grant no. G1000803) with contributions to the grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research (NIHR) on behalf of the Department of Health, and the Chief Scientist Office of the Scottish Government Health Directorate (to S.J.P.); by a Hospital Infection Society Major Research Grant; by Wellcome Trust grant no. 098051 awarded to the Wellcome Trust Sanger Institute; and by Wellcome Trust 201344/Z/16/Z awarded to F.C. M.S.T. is a Wellcome Trust Clinical PhD fellow. M.E.T. is a Clinician Scientist Fellow, supported by the Academy of Medical Sciences and the Health Foundation and by the NIHR Cambridge Biomedical Research Centr

Transient silencing of antibiotic resistance by mutation represents a significant potential source of unanticipated therapeutic failure

Sporadic literature reports describe isolates of pathogenic bacteria that harbor an antibiotic resistance determinant but remain susceptible to the corresponding antibiotic as a consequence of a genetic defect. Such strains represent a source from which antibiotic resistance may reemerge to cause treatment failure in patients. Here, we report a systematic investigation into the prevalence and nature of this phenomenon, which we term silencing of antibiotic resistance by mutation (SARM). Instances of SARM were detected among 1,470 Staphylococcus aureus isolates through side-by-side comparison of antibiotic resistance genotype (as determined by whole-genome sequencing) versus phenotype (as assessed through susceptibility testing). Of the isolates analyzed, 152 (10.3%) harbored a silenced resistance gene, including 46 (3.1%) that exhibited SARM to currently deployed antistaphylococcal drugs. SARM resulted from diverse mutational events but most commonly through frameshift mutation of resistance determinants as a result of point deletion in poly(A) tracts. The majority (∼90%) of SARM strains reverted to antibiotic resistance at frequencies of ≥10−9; thus, while appearing antibiotic sensitive in the clinical microbiology laboratory, most S. aureus isolates exhibiting SARM will revert to antibiotic resistance at frequencies achievable in patients. In view of its prevalence in a major pathogen, SARM represents a significant potential threat to the therapeutic efficacy of antibiotics

Detection of livestock-associated meticillin-resistant Staphylococcus aureus CC398 in retail pork, United Kingdom, February 2015

No abstract available

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

Microevolution of extensively drug-resistant tuberculosis in Russia.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72%belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted. © 2012 by Cold Spring Harbor Laboratory Press

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant staphylococcus aureus pandemic

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens