Colonisation with ESBL-producing and carbapenemase-producing Enterobacteriaceae, vancomycin-resistant enterococci, and meticillin-resistant Staphylococcus aureus in a long-term care facility over one year.

BACKGROUND: This study examined colonisation with and characteristics of antimicrobial-resistant organisms among residents of a long-term care facility (LTCF) over one year, including strain persistence and molecular diversity among isolates of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. METHODS: Sixty-four residents of a LTCF were recruited (51 at baseline, 13 during the year). Data on dependency levels, hospitalisations, and antimicrobial prescribing were collected. Nasal and rectal swabs and catheter urine specimens were examined quarterly, using chromogenic agars, for ESBL-producing Enterobacteriaceae, carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and meticillin-resistant S. aureus (MRSA). All ESBL-producing E. coli (ESBL-EC) were characterised by pulsed-field gel electrophoresis (PFGE) and PCR to assess for sequence type (ST) ST131, its resistance-associated H30 and H30-Rx subclones, and blaCTX-M, blaTEM, blaSHV, and blaOXA-1. RESULTS: The overall number of residents colonised, by organism, was as follows: ESBL-EC, 35 (55%); MRSA, 17 (27%); ESBL-producing K. pneumoniae (ESBL-KP), 5 (8%); VRE, 2 (3%) and CPE, 0 (0%). All 98 ESBL-EC isolates were H30-Rx ST131, with bla CTX-M-group 1. By PFGE, a group of 91 ESBL-EC (from 33 participants) had ≥85% similar profiles and resembled UK epidemic strain A/ international pulsotype PFGE812. Sequential ESBL-EC from individual residents were closely related. Six ESBL-KP isolates, from five participants, had bla CTX-M-group 1 and by PFGE were closely related. Colonisation with ESBL and MRSA was associated with location within the LTCF and previous exposure to antimicrobials. CONCLUSIONS: Among LTCF residents, colonisation with ESBL-EC and MRSA was common. All ESBL-EC were H30-Rx ST131, consistent with clonal dissemination

Defining nosocomial transmission of Escherichia coli and antimicrobial resistance genes: a genomic surveillance study

E. coli is a leading cause of bloodstream infections. Developing interventions to reduce this burden requires an understanding of the frequency of nosocomial transmission, but available evidence is limited. This study aimed to detect and characterise transmission of E. coli and associated plasmids in a hospitalised cohort. Methods: Genomic surveillance of E. coli was conducted in a prospective observational cohort study of hospitalised adult patients over 6 months in Cambridge, England. Stool samples were collected from study participants on admission, weekly and discharge. We sequenced multiple E. coli colonies (median=5) from each stool. A genetic threshold to infer E. coli transmission was defined by maximum within-host SNP diversity and the probability of drawing observed pairs of between-patient isolates at different SNP thresholds. Findings: We obtained and cultured 376 stools from 149 patients, of which 152 stools from 97 patients grew E. coli. We identified extensive diversity in the bacterial population (90 sequence types, STs), and mixed E. coli ST carriage in almost half of patients (26%, 13% and 6% patients carried 2, 3 or >4 STs, respectively). Using a 17 SNP cut-off we identified 10 clusters (defined as >2 cases) involving 20 patients. The largest cluster contained 7 patients, while 4 patients were linked to multiple clusters. Half of cases in the 10 clusters also had a strong epidemiological link to another patient in the cluster. A minority of all patients (17/149, 11%) carried extended-spectrum beta-lactamase (ESBL)-producing E.coli, the most common of which was blaCTX-M-15 (12/17, 71%). Long-read sequencing revealed that blaCTX-M- 40 was often integrated into the chromosome, with little evidence for plasmid-mediated transmission. Seven patients developed E. coli bloodstream infection, four with identical strains in those in stool; two of these had documented nosocomial acquisition. Interpretation: We provide evidence of bacterial transmission and endogenous infections during routine care by integrating genomic and epidemiological data and through determination of a genetic similarity cut-off informed by within-host diversity in the population studied. Our findings challenge single colony-based investigations, and the paradigm of plasmid spread in this setting

Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts.

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays, and higher health care costs. E. coli sequence type 131 (ST131) is a major ExPEC clonal group worldwide, with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes blaCTX-M-14, blaCTX-M-15, and blaCTX-M-27, which are often rearranged, amplified, and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding blaCTX-M genes to be fully determined. Here, we performed long-read sequencing to decipher the genome structures of six E. coli ST131 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to more fragmented assemblies created with short reads alone. The long-read assemblies highlighted diverse accessory genomes with blaCTX-M-15, blaCTX-M-14, and blaCTX-M-27 genes identified in three, one, and one isolates, respectively. One sample had no blaCTX-M gene. Two samples had chromosomal blaCTX-M-14 and blaCTX-M-15 genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: ISEcp1, IS903B, and Tn2 This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts, even between closely related isolates within a clonal group such as E. coli ST131.IMPORTANCE Drug-resistant bacteria are a major cause of illness worldwide, and a specific subtype called Escherichia coli ST131 causes a significant number of these infections. ST131 bacteria become resistant to treatments by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to that of an existing approach. A combination of methods shows that drug resistance genes on plasmids are highly mobile because they can jump into ST131's chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes

Chromogenic media for ESBL-positive Enterobacteriaceae

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.This work was supported by a grant awarded to SP from the UK Department of Health, Wellcome Trust and the Health Innovation Challenge Fund (HICF-T5-342 and WT098600). 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. The authors declare no conflicts of interest.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.diagmicrobio.2015.11.00

Transmission of methicillin-resistant Staphylococcus aureus in long-term care facilities and their related healthcare networks.

BACKGROUND: Long-term care facilities (LTCF) are potential reservoirs for methicillin-resistant Staphylococcus aureus (MRSA), control of which may reduce MRSA transmission and infection elsewhere in the healthcare system. Whole-genome sequencing (WGS) has been used successfully to understand MRSA epidemiology and transmission in hospitals and has the potential to identify transmission between these and LTCF. METHODS: Two prospective observational studies of MRSA carriage were conducted in LTCF in England and Ireland. MRSA isolates were whole-genome sequenced and analyzed using established methods. Genomic data were available for MRSA isolated in the local healthcare systems (isolates submitted by hospitals and general practitioners). RESULTS: We sequenced a total of 181 MRSA isolates from the two study sites. The majority of MRSA were multilocus sequence type (ST)22. WGS identified one likely transmission event between residents in the English LTCF and three putative transmission events in the Irish LTCF. WGS also identified closely related isolates present in colonized Irish residents and their immediate environment. Based on phylogenetic reconstruction, closely related MRSA clades were identified between the LTCF and their healthcare referral network, together with putative MRSA acquisition by LTCF residents during hospital admission. CONCLUSIONS: These data confirm that MRSA is transmitted between residents of LTCF and is both acquired and transmitted to others in referral hospitals and beyond. Our data present compelling evidence for the importance of environmental contamination in MRSA transmission, reinforcing the importance of environmental cleaning. The use of WGS in this study highlights the need to consider infection control in hospitals and community healthcare facilities as a continuum.UKCRC Translational Infection Research (TIR) Initiative, Medical Research Council (Grant ID: G1000803), Biotechnology and Biological Sciences Research Council, National Institute for Health Research, Chief Scientist Office of the Scottish Government Health Directorate, Hospital Infection Society (Major Research Grant), Wellcome Trust (Grant ID: 098051), Academy of Medical Sciences, Health Foundation, National Institute for Health Research Cambridge Biomedical Research Centr

A One Health study of the genetic relatedness of Klebsiella pneumoniae and their mobile elements in the East of England

Klebsiella pneumoniae is a human, animal and environmental commensal and a leading cause of nosocomial infections, which are often caused by multi-resistant strains that are challenging to treat. We conducted a One Health evaluation of putative sources of K. pneumoniae that are carried by, and infect hospital patients. This combined data from a six-month study on two haematology wards at Addenbrooke’s Hospital, Cambridge, in 2015 to isolate K. pneumoniae from stool, blood and the environment, and a cross-sectional survey of K. pneumoniae from 29 livestock farms, 97 meat products, the hospital sewer and 20 municipal wastewater treatment plants in the East of England between 2014 and 2015. K. pneumoniae was isolated from stools of 17/149 (11%) patients and 18/922 swabs of their environment, together with one patient bloodstream infection during the study and 4 others over a 24-month period. Each patient carried one or more lineages that was unique to them, but two broad environmental contamination events and patient-environmental transmission were identified. K. pneumoniae was isolated from cattle and poultry, hospital sewage and 12/20 wastewater plants. There was low genetic relatedness between isolates from patients/their hospital environment versus isolates from elsewhere. Identical genes encoding cephalosporin resistance were carried by isolates from different reservoirs, but were carried on different plasmids by isolates from patients/their environment versus elsewhere. We identified no patient-to-patient transmission and no evidence for livestock as a source of K. pneumoniae infecting humans, but our findings reaffirm the importance of the hospital environment as a source of K. pneumoniae associated with serious human infection.This work was 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. This project was also funded by a grant awarded to the Wellcome Trust Sanger Institute (098051). TG is a Wellcome Trust Research Training Fellow (103387/Z/13/Z). CL is a Wellcome Trust Sir Henry Postdoctoral Fellow (110243/Z/15/Z). DJ is funded by the Wellcome Trust grant 098051

Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli

ABSTRACT Objectives: E. coli O25b-ST131 has disseminated worldwide in hospitals and the community. The objective of this study was to determine the extent to which E. coli O25b-ST131 accounts for extended-spectrum beta-lactamase (ESBL)-producing E. coli from clinical samples from all sources in this region. Methods: Between January and June 2010 ESBL-producing E. coli were collected from 94 routine samples including 47 from residents of 25 nursing homes, 15 categorized as hospital acquired and 32 others. PCR was performed for detection of bla CTX-M , bla OXA-1 , bla TEM , bla SHV and for the identification of members of the E. coli O25b:ST131 clonal group. PFGE was carried out using XbaI in accordance with PulseNet protocols. Results: The majority (97%) of isolates harbored a bla CTX-M gene. E. coli O25b-ST131 accounted for 87% of all ESBLproducing E. coli and for 96% of isolates from nursing home residents. Sonuç: E. coli O25b-ST131 klonal grubu bakımevi kaynaklı olanlarda daha belirgin olmak üzere toplanan ESBL üreten E. coli suşları arasında baskındı. Conclusion Anahtar kelimeler: Escherichia coli, O25b-ST131, Direnç, Sağlık hizmeti, Bakımevi Ludden C

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 ten plants in direct receipt of hospital sewage was significantly higher than ten 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. Amongst 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.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. This work was supported by a Wellcome Trust Research Training Fellowship (to T.G. [103387/Z/13/Z]), a Wellcome Trust Sir Henry Postdoctoral Fellowship (to C.L. [110243/Z/15/Z] and F.C. [201344/Z/16/Z]), and an ERC grant (742158) (to J.C.)

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

A One Health Study of the Genetic Relatedness of Klebsiella pneumoniae and Their Mobile Elements in the East of England.

BACKGROUND: Klebsiella pneumoniae is a human, animal, and environmental commensal and a leading cause of nosocomial infections, which are often caused by multiresistant strains. We evaluate putative sources of K. pneumoniae that are carried by and infect hospital patients. METHODS: We conducted a 6-month survey on 2 hematology wards at Addenbrooke's Hospital, Cambridge, United Kingdom, in 2015 to isolate K. pneumoniae from stool, blood, and the environment. We conducted cross-sectional surveys of K. pneumoniae from 29 livestock farms, 97 meat products, the hospital sewer, and 20 municipal wastewater treatment plants in the East of England between 2014 and 2015. Isolates were sequenced and their genomes compared. RESULTS: Klebsiella pneumoniae was isolated from stool of 17/149 (11%) patients and 18/922 swabs of their environment, together with 1 bloodstream infection during the study and 4 others over a 24-month period. Each patient carried 1 or more lineages that was unique to them, but 2 broad environmental contamination events and patient-environment transmission were identified. Klebsiella pneumoniae was isolated from cattle, poultry, hospital sewage, and 12/20 wastewater treatment plants. There was low genetic relatedness between isolates from patients/their hospital environment vs isolates from elsewhere. Identical genes encoding cephalosporin resistance were carried by isolates from humans/environment and elsewhere but were carried on different plasmids. CONCLUSION: We identified no patient-to-patient transmission and no evidence for livestock as a source of K. pneumoniae infecting humans. However, our findings reaffirm the importance of the hospital environment as a source of K. pneumoniae associated with serious human infection