56 research outputs found
Genome-Based Analysis of Enterococcus faecium Bacteremia Associated with Recurrent and Mixed-Strain Infection.
Vancomycin-resistant Enterococcus faecium (VREfm) bloodstream infections are associated with high recurrence rates. This study used genome sequencing to accurately distinguish the frequency of relapse and reinfection in patients with recurrent E. faecium bacteremia and to investigate strain relatedness in patients with apparent VREfm and vancomycin-susceptible E. faecium (VSEfm) mixed infection. A retrospective study was performed at the Cambridge University Hospitals NHS Foundation Trust (CUH) between November 2006 and December 2012. We analyzed the genomes of 44 E. faecium isolates from 21 patients (26 VREfm isolates from 12 patients with recurrent bacteremia and 18 isolates from 9 patients with putative VREfm/VSEfm mixed infection). Phenotypic antibiotic susceptibility was determined using a Vitek2 instrument. Genomes were compared with those of a further 263 E. faecium isolates associated with bacteremia in patients at CUH over the same time period. Pairwise comparison of core genomes indicated that 10 (71%) episodes of recurrent VREfm bacteremia were due to reinfection with a new strain, with reinfection being more likely with increasing time between the two positive cultures. The majority (78%) of patients with a mixed VREfm and VSEfm infection had unrelated strains. More than half (59%) of study isolates were closely related to another isolate associated with bacteremia from CUH. This included 60% of isolates associated with reinfection, indicating acquisition in the hospital. This study provides the first high-resolution insights into recurrence and mixed infection by E. faecium and demonstrates that reinfection with a new strain, often acquired from the hospital, is a driver of recurrence
Defining metrics for whole-genome sequence analysis of MRSA in clinical practice.
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
Whole-genome sequencing reveals transmission of vancomycin-resistant Enterococcus faecium in a healthcare network.
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-
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Evaluation of a fully automated bioinformatics tool to predict antibiotic resistance from MRSA genomes.
OBJECTIVES: The genetic prediction of phenotypic antibiotic resistance based on analysis of WGS data is becoming increasingly feasible, but a major barrier to its introduction into routine use is the lack of fully automated interpretation tools. Here, we report the findings of a large evaluation of the Next Gen Diagnostics (NGD) automated bioinformatics analysis tool to predict the phenotypic resistance of MRSA. METHODS: MRSA-positive patients were identified in a clinical microbiology laboratory in England between January and November 2018. One MRSA isolate per patient together with all blood culture isolates (total n = 778) were sequenced on the Illumina MiniSeq instrument in batches of 21 clinical MRSA isolates and three controls. RESULTS: The NGD system activated post-sequencing and processed the sequences to determine susceptible/resistant predictions for 11 antibiotics, taking around 11 minutes to analyse 24 isolates sequenced on a single sequencing run. NGD results were compared with phenotypic susceptibility testing performed by the clinical laboratory using the disc diffusion method and EUCAST breakpoints. Following retesting of discrepant results, concordance between phenotypic results and NGD genetic predictions was 99.69%. Further investigation of 22 isolate genomes associated with persistent discrepancies revealed a range of reasons in 12 cases, but no cause could be found for the remainder. Genetic predictions generated by the NGD tool were compared with predictions generated by an independent research-based informatics approach, which demonstrated an overall concordance between the two methods of 99.97%. CONCLUSIONS: We conclude that the NGD system provides rapid and accurate prediction of the antibiotic susceptibility of MRSA
Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand.
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
Comparison of two chromogenic media for the detection of vancomycin-resistant enterococcal carriage by nursing home residents.
We compared ChromID VRE and Brilliance VRE media for the detection of vancomycin-resistant enterococci (VRE). Using a panel of 28 enterococcal isolates, 10 vanA Enterococcus faecium and three vanA Enterococcus faecalis isolates grew as per manufacturers' instructions whilst growth of two vanC and eight vancomycin-susceptible enterococci was inhibited on both media. Important differences were noted in the selectivity and chromogenic properties of the two media for vanA Enterococcus raffinosus and vanB E. faecium. The two media were further evaluated using 295 stool samples from nursing home residents, 34 of which grew VRE (11.5%). ChromID and Brilliance had comparable sensitivity, which was increased markedly by prolonging incubation to 48 hours (from 29% to 82%, and from 41% to 85%, respectively) and by a pre-enrichment step (to 97% and 100%, respectively). Brilliance VRE agar had higher selectivity at 48 hours, and after pre-enrichment.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. TG is a Wellcome Trust Research Training Fellow. MET is a Clinician Scientist Fellow supported by the Academy of Medical Sciences and the Health Foundation. SJP and MET are supported by the NIHR Cambridge Biomedical Research Centre.This is the final version of the article It first appeared from Elsevier via https://dx.doi.org/10.1016/j.diagmicrobio.2016.04.02
Longitudinal genomic surveillance of multidrug-resistant Escherichia coli carriage in a long-term care facility in the United Kingdom.
BACKGROUND: Residents of long-term care facilities (LTCF) may have high carriage rates of multidrug-resistant pathogens, but are not currently included in surveillance programmes for antimicrobial resistance or healthcare-associated infections. Here, we describe the value derived from a longitudinal epidemiological and genomic surveillance study of drug-resistant Escherichia coli in a LTCF in the United Kingdom (UK). METHODS: Forty-five of 90 (50%) residents were recruited and followed for six months in 2014. Participants were screened weekly for carriage of extended-spectrum beta-lactamase (ESBL) producing E. coli. Participants positive for ESBL E. coli were also screened for ESBL-negative E. coli. Phenotypic antibiotic susceptibility of E. coli was determined using the Vitek2 instrument and isolates were sequenced on an Illumina HiSeq2000 instrument. Information was collected on episodes of clinical infection and antibiotic consumption. RESULTS: Seventeen of 45 participants (38%) carried ESBL E. coli. Twenty-three of the 45 participants (51%) had 63 documented episodes of clinical infection treated with antibiotics. Treatment with antibiotics was associated with higher risk of carrying ESBL E. coli. ESBL E. coli was mainly sequence type (ST)131 (16/17, 94%). Non-ESBL E. coli from these 17 cases was more genetically diverse, but ST131 was found in eight (47%) cases. Whole-genome analysis of 297 ST131 E. coli from the 17 cases demonstrated highly related strains from six participants, indicating acquisition from a common source or person-to-person transmission. Five participants carried highly related strains of both ESBL-positive and ESBL-negative ST131. Genome-based comparison of ST131 isolates from the LTCF study participants with ST131 associated with bloodstream infection at a nearby acute hospital and in hospitals across England revealed sharing of highly related lineages between the LTCF and a local hospital. CONCLUSIONS: This study demonstrates the power of genomic surveillance to detect multidrug-resistant pathogens and confirm their connectivity within a healthcare network
Duration of exposure to multiple antibiotics is associated with increased risk of VRE bacteraemia: a nested case-control study.
BACKGROUND: VRE bacteraemia has a high mortality and continues to defy control. Antibiotic risk factors for VRE bacteraemia have not been adequately defined. We aimed to determine the risk factors for VRE bacteraemia focusing on duration of antibiotic exposure. METHODS: A retrospective matched nested case-control study was conducted amongst hospitalized patients at Cambridge University Hospitals NHS Foundation Trust (CUH) from 1 January 2006 to 31 December 2012. Cases who developed a first episode of VRE bacteraemia were matched 1:1 to controls by length of stay, year, specialty and ward type. Independent risk factors for VRE bacteraemia were evaluated using conditional logistic regression. RESULTS: Two hundred and thirty-five cases were compared with 220 controls. Duration of exposure to parenteral vancomycin, fluoroquinolones and meropenem was independently associated with VRE bacteraemia. Compared with patients with no exposure to vancomycin, those who received courses of 1-3 days, 4-7 days or >7 days had a stepwise increase in risk of VRE bacteraemia [conditional OR (cOR) 1.2 (95% CI 0.4-3.8), 3.8 (95% CI 1.2-11.7) and 6.6 (95% CI 1.9-22.8), respectively]. Other risk factors were: presence of a central venous catheter (CVC) [cOR 8.7 (95% CI 2.6-29.5)]; neutropenia [cOR 15.5 (95% CI 4.2-57.0)]; hypoalbuminaemia [cOR 8.5 (95% CI 2.4-29.5)]; malignancy [cOR 4.4 (95% CI 1.6-12.0)]; gastrointestinal disease [cOR 12.4 (95% CI 4.2-36.8)]; and hepatobiliary disease [cOR 7.9 (95% CI 2.1-29.9)]. CONCLUSIONS: Longer exposure to vancomycin, fluoroquinolones or meropenem was associated with VRE bacteraemia. Antimicrobial stewardship interventions targeting high-risk antibiotics are required to complement infection control procedures against VRE bacteraemia
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A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing
Abstract: Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories
Detection of vancomycin-resistant Enterococcus faecium hospital-adapted lineages in municipal wastewater treatment plants indicates widespread distribution and release into the environment.
Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infection. Reservoirs of VREfm are largely assumed to be nosocomial although there is a paucity of data on alternative sources. Here, we describe an integrated epidemiological and genomic analysis of E. faecium associated with bloodstream infection and isolated from wastewater. Treated and untreated wastewater from 20 municipal treatment plants in the East of England, United Kingdom was obtained and cultured to isolate E. faecium, ampicillin-resistant E. faecium (AREfm), and VREfm. VREfm was isolated from all 20 treatment plants and was released into the environment by 17/20 plants, the exceptions using terminal ultraviolet light disinfection. Median log10 counts of AREfm and VREfm in untreated wastewater from 10 plants in direct receipt of hospital sewage were significantly higher than 10 plants that were not. We sequenced and compared the genomes of 423 isolates from wastewater with 187 isolates associated with bloodstream infection at five hospitals in the East of England. Among 481 E. faecium isolates belonging to the hospital-adapted clade, we observed genetic intermixing between wastewater and bloodstream infection, with highly related isolates shared between a major teaching hospital in the East of England and 9/20 plants. We detected 28 antibiotic resistance genes in the hospital-adapted clade, of which 23 were represented in bloodstream, hospital sewage, and municipal wastewater isolates. We conclude that our findings are consistent with widespread distribution of hospital-adapted VREfm beyond acute healthcare settings with extensive release of VREfm into the environment in the East of England
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