106 research outputs found

    Are commercial providers a viable option for clinical bacterial sequencing?

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    Bacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice

    The Emergence of Successful Streptococcus pyogenes Lineages through Convergent Pathways of Capsule Loss and Recombination Directing High Toxin Expression.

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    Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s, respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (streptolysin O) loci conferring high expression of these toxins underpinned the success of these lineages, extension to other emm genotype lineages is unreported. The emergent emm89 lineage was characterized by five regions of homologous recombination additional to nga-slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesized that other leading genotypes may have undergone similar recombination events. We analyzed a longitudinal data set of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin genotype. We identified multiple associations between recombination at this locus and inactivating mutations within hasAB, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule and high expression of nga and slo may underpin the success of many emergent S. pyogenes lineages of different genotypes, triggering new pandemics, and could change the way S. pyogenes causes disease.IMPORTANCE Streptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for the majority of human infection in high-income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time

    Defining metrics for whole-genome sequence analysis of MRSA in clinical practice.

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    Bacterial sequencing will become increasingly adopted in routine microbiology laboratories. Here, we report the findings of a technical evaluation of almost 800 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, in which we sought to define key quality metrics to support MRSA sequencing in clinical practice. We evaluated the accuracy of mapping to a generic reference versus clonal complex (CC)-specific mapping, which is more computationally challenging. Focusing on isolates that were genetically related (50 bp apart to identify same-species contamination for MRSA. These metrics were combined into a quality-control (QC) flowchart to determine whether sequence runs and individual clinical isolates passed QC, which could be adapted by future automated analysis systems to enable rapid hands-off sequence analysis by clinical laboratories

    Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages.

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    Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation

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

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    BACKGROUND: Bacterial 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). METHODS: A 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). RESULTS: Forty-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. CONCLUSIONS: A 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.We gratefully acknowledge the contribution of the staff at the LTCF in sample collection, and thank the patients who agreed to participate. We thank Kirsty Ambridge and Angela Kidney for technical assistance. We are grateful for assistance from the library construction, sequencing and core informatics teams at the Wellcome Trust Sanger Institute. This publication presents independent research supported by the Health Innovation Challenge Fund (WT098600, HICF-T5-342), a parallel funding partnership between the Department of Health and Wellcome Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the Department of Health or Wellcome Trust. MET is a Clinician Scientist Fellow supported by the Academy of Medical Sciences, The Health Foundation and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It was first available from BioMed Central via http://dx.doi.org/10.1186/s13073-015-0259-

    Systematic Surveillance Detects Multiple Silent Introductions and Household Transmission of Methicillin-Resistant Staphylococcus aureus USA300 in the East of England.

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    BACKGROUND: The spread of USA300 methicillin-resistant Staphylococcus aureus (MRSA) across the United States resulted in an epidemic of infections. In Europe, only sporadic cases or small clusters of USA300 infections are described, and its prevalence in England is unknown. We conducted prospective surveillance for USA300 in the east of England. METHODS: We undertook a 12-month prospective observational cohort study of all individuals with MRSA isolated from community and hospital samples submitted to a microbiology laboratory. At least 1 MRSA isolate from each individual underwent whole-genome sequencing. USA300 was identified on the basis of sequence analysis, and phylogenetic comparisons were made between these and USA300 genomes from the United States. RESULTS: Between April 2012 and April 2013, we sequenced 2283 MRSA isolates (detected during carriage screening and in clinical samples) from 1465 individuals. USA300 was isolated from 24 cases (1.6%). Ten cases (42%) had skin and soft tissue infection, and 2 cases had invasive disease. Phylogenetic analyses identified multiple introductions and household transmission of USA300. CONCLUSIONS: Use of a diagnostic laboratory as a sentinel for surveillance has identified repeated introductions of USA300 in eastern England in 2012-2013, with evidence for limited transmission. Our results show how systematic surveillance could provide an early warning of strain emergence and dissemination.This work was supported by grants from the UK Clinical Research Collaboration Translational Infection Research 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 Office of the Scottish Government Health Directorate (to Prof. Peacock); by a Healthcare Infection Society Major Research Grant (to Prof. Peacock), and by Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute. MST is a Wellcome Trust Clinical PhD Fellow. MET is a Clinician Scientist Fellow, supported by the Academy of Medical Sciences and the Health Foundation, and by the National Institute for Health Research Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/infdis/jiw16

    Within-host evolution of Enterococcus faecium during longitudinal carriage and transition to bloodstream infection in immunocompromised patients.

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    BACKGROUND: Enterococcus faecium is a leading cause of hospital-acquired infection, particularly in the immunocompromised. Here, we use whole genome sequencing of E. faecium to study within-host evolution and the transition from gut carriage to invasive disease. METHODS: We isolated and sequenced 180 E. faecium from four immunocompromised patients who developed bloodstream infection during longitudinal surveillance of E. faecium in stool and their immediate environment. RESULTS: A phylogenetic tree based on single nucleotide polymorphisms (SNPs) in the core genome of the 180 isolates demonstrated several distinct clones. This was highly concordant with the population structure inferred by Bayesian methods, which contained four main BAPS (Bayesian Analysis of Population Structure) groups. The majority of isolates from each patient resided in a single group, but all four patients also carried minority populations in stool from multiple phylogenetic groups. Bloodstream isolates from each case belonged to a single BAPS group, which differed in all four patients. Analysis of 87 isolates (56 from blood) belonging to a single BAPS group that were cultured from the same patient over 54 days identified 30 SNPs in the core genome (nine intergenic, 13 non-synonymous, eight synonymous), and 250 accessory genes that were variably present. Comparison of these genetic variants in blood isolates versus those from stool or environment did not identify any variants associated with bloodstream infection. The substitution rate for these isolates was estimated to be 128 (95% confidence interval 79.82 181.77) mutations per genome per year, more than ten times higher than previous estimates for E. faecium. Within-patient variation in vancomycin resistance associated with vanA was common and could be explained by plasmid loss, or less often by transposon loss. CONCLUSIONS: These findings demonstrate the diversity of E. faecium carriage by individual patients and significant within-host diversity of E. faecium, but do not provide evidence for adaptive genetic variation associated with invasion

    Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand.

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    BACKGROUND: Tackling multidrug-resistant Escherichia coli requires evidence from One Health studies that capture numerous potential reservoirs in circumscribed geographic areas. METHODS: We conducted a survey of extended β-lactamase (ESBL)-producing E. coli isolated from patients, canals and livestock wastewater in eastern Thailand between 2014 and 2015, and analyzed isolates using whole genome sequencing. RESULTS: The bacterial collection of 149 isolates consisted of 84 isolates from a single hospital and 65 from the hospital sewer, canals and farm wastewater within a 20 km radius. E. coli ST131 predominated the clinical collection (28.6%), but was uncommon in the environment. Genome-based comparison of E. coli from infected patients and their immediate environment indicated low genetic similarity overall between the two, although three clinical-environmental isolate pairs differed by ≤ 5 single nucleotide polymorphisms. Thai E. coli isolates were dispersed throughout a phylogenetic tree containing a global E. coli collection. All Thai ESBL-positive E. coli isolates were multidrug resistant, including high rates of resistance to tobramycin (77.2%), gentamicin (77.2%), ciprofloxacin (67.8%) and trimethoprim (68.5%). ESBL was encoded by six different CTX-M elements and SHV-12. Three isolates from clinical samples (n = 2) or a hospital sewer (n = 1) were resistant to the carbapenem drugs (encoded by NDM-1, NDM-5 or GES-5), and three isolates (clinical (n = 1) and canal water (n = 2)) were resistant to colistin (encoded by mcr-1); no isolates were resistant to both carbapenems and colistin. CONCLUSIONS: Tackling ESBL-producing E. coli in this setting will be challenging based on widespread distribution, but the low prevalence of resistance to carbapenems and colistin suggests that efforts are now required to prevent these from becoming ubiquitous
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