Search

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., A One Health genomic survey of Klebsiella pneumoniae demonstrated transmission between patients and their ward environment but no evidence to implicate livestock as a source of K. pneumoniae or their mobile genetic elements encoding antibiotic resistance.

There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1 hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq platform and a 13-hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive, and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and in detecting mecA Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. Of the test panel MRSA genomes, 168/173 (97%) passed QC metrics based on the correct species assigned, detection of mecA and ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained

We examined whether genomic surveillance of Escherichia coli in wastewater could capture the dominant E. coli lineages associated with bloodstream infection and livestock in the East of England, together with the antibiotic-resistance genes circulating in the wider E. coli population. Treated and untreated wastewater was taken from 20 municipal treatment plants in the East of England, half in direct receipt of acute hospital waste. All samples were culture positive for E. coli, and all but one were positive for extended-spectrum β-lactamase (ESBL)-producing E. coli. The most stringent wastewater treatment (tertiary including UV light) did not eradicate ESBL-E. coli in 2/3 cases. We sequenced 388 E. coli (192 ESBL, 196 non-ESBL). Multilocus sequence type (ST) diversity was similar between plants in direct receipt of hospital waste versus the remainder (93 vs 95 STs, respectively). We compared the genomes of wastewater E. coli with isolates from bloodstream infection (n=437), and livestock farms and retail meat (n=431) in the East of England. A total of 19/20 wastewater plants contained one or more of the three most common STs associated with bloodstream infection (ST131, ST73, ST95), and 14/20 contained the most common livestock ST (ST10). In an analysis of 1254 genomes (2 cryptic E. coli were excluded), wastewater isolates were distributed across the phylogeny and intermixed with isolates from humans and livestock. Ten blaCTX-M elements were identified in E. coli isolated from wastewater, together with a further 47 genes encoding resistance to the major antibiotic drug groups. Genes encoding resistance to colistin and the carbapenems were not detected. Genomic surveillance of E. coli in wastewater could be used to monitor new and circulating lineages and resistance determinants of public-health importance.

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.

BackgroundMandatory reporting of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) has occurred in England for over 15years. Epidemiological information is recorded, but routine collection of isolates for characterisation has not been routinely undertaken. Ongoing developments in whole-genome sequencing (WGS) have demonstrated its value in outbreak investigations and for determining the spread of antimicrobial resistance and bacterial population structure. Benefits of adding genomics to routine epidemiological MRSA surveillance are unknown.AimTo determine feasibility and potential utility of adding genomics to epidemiological surveillance of MRSA.MethodsWe conducted an epidemiological and genomic survey of MRSA BSI in England over a 1-year period (1 October 2012--30 September 2013).ResultsDuring the study period, 903 cases of MRSA BSI were reported; 425 isolates were available for sequencing of which, 276 (65%) were clonal complex (CC) 22. Addition of 64 MRSA genomes from published outbreak investigations showed that the study genomes could provide context for outbreak isolates and supported cluster identification. Comparison to other MRSA genome collections demonstrated variation in clonal diversity achieved through different sampling strategies and identified potentially high-risk clones e.g. USA300 and local expansion of CC5 MRSA in South West England.ConclusionsWe demonstrate the potential utility of combined epidemiological and genomic MRSA BSI surveillance to determine the national population structure of MRSA, contextualise previous MRSA outbreaks, and detect potentially high-risk lineages. These findings support the integration of epidemiological and genomic surveillance for MRSA BSI as a step towards a comprehensive surveillance programme in England.

Additional file 4: Figure S4. Heatmap analysis with annotation for disease duration in months and active GPA and inactive GPA disease stage using top 28 species with a minimum abundance of 0.5% in at least one sample in the bacterial 16S rRNA dataset. (PDF 75 kb)

The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research., Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50 single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause ofnosocomial infection and is categorized as high priority by the World Health Organizationglobal priority list of antibiotic-resistant bacteria. In the past, livestock havebeen proposed as a putative reservoir for drug-resistant E. faecium strains that infecthumans, and isolates of the same lineage have been found in both reservoirs. Weundertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestockfarms, retail meat, and wastewater treatment plants in the United Kingdom.More than 600 isolates from these sources were sequenced, and their relatednessand antibiotic resistance genes were compared with genomes of almost 800 E. faeciumisolates from patients with bloodstream infection in the United Kingdom andIreland. E. faecium was isolated from 28/29 farms; none of these isolates wereVREfm, suggesting a decrease in VREfm prevalence since the last UK livestock surveyin 2003. However, VREfm was isolated from 1% to 2% of retail meat products andwas ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstratedthat the majority of human and livestock-related isolates were geneticallydistinct, although pig isolates from three farms were more genetically related to humanisolates from 2001 to 2004 (minimum of 50 single-nucleotide polymorphisms[SNPs]). Analysis of accessory (variable) genes added further evidence for distinctniche adaptation. An analysis of acquired antibiotic resistance genes and their variantsrevealed limited sharing between humans and livestock. Our findings indicatethat the majority of E. faecium strains infecting patients are largely distinct fromthose from livestock in this setting, with limited sharing of strains and resistancegenes.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0507-0) contains supplementary material, which is available to authorized users.

Figures and descriptions of Supplemental Figures S1â S6 mentioned throughout the manuscript. (DOCX 1927Â kb)

Predominant STs of ESBL-producing E. coli isolated from clinical and environmental origins in Thailand. Graph showing the prevalence of the eight most frequently identified STs among each of the clinical or environmental Thai ESBL-producing E. coli isolates, split by source of isolation. The total adds up to 12 since four of the top eight most frequent in each category did not overlap. The presence of all isolates in each of the 12 STs is shown. Figure S2. Phylogeny of CTX-M genes identified in Thai ESBL-producing E. coli. Phylogeny of the seven CTX-M gene variants identified in the 149 study genomes. Figure S3. Phylogeny of Thai ESBL-producing E. coli in a global context. Maximum-likelihood tree of the 149 study genomes and 514 global genomes based on 222,900 SNPs in the 1983 core genes. Inner ring shows origin of isolate, middle ring shows country of isolation and outer ring indicates the study isolates (black). Scale bar indicates ~ 20,000 SNPs. Figure S4. Virulence gene comparison between ST131 and non-ST131 isolates. Graph showing the prevalence of virulence factors in ST131 (n 26) compared to non-ST131 (n 123) in the study isolates. Using Fisher’s exact test at a p value of 0.05, only those factors over-represented between the two groups are shown. (DOCX 2381 kb)

$\textbf{Background:}$ Long-term care facilities (LTCF) are potential reservoirs for methicillin-resistant $\textit{Staphylococcus aureus}$ (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. $\textbf{Methods:}$ 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). $\textbf{Results:}$ 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. $\textbf{Conclusions:}$ 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 Centre, This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13073-016-0353-5

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. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0353-5) contains supplementary material, which is available to authorized users.

The study was supported by grants from the UKCRC 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); and by Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute. MET is Clinician Scientist Fellow supported by the Academy of Medical Sciences and the Health Foundation. 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 UK MRSA database containing over 1,000 methicillin-resistant Staphylococcus aureus (MRSA) genomes drawn from England, Northern Ireland, Wales, Scotland and the Republic of Ireland over a decade. We sequenced 1,013 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 otherwise grouped based on country of origin. Phylogenetic reconstructions were used to contextualise MRSA outbreak investigations, and to detect the spread of resistance. The majority of isolates (n=783, 77%) belonged to CC22, which contains the dominant UK 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 UK and Ireland, and have shown the value of this during outbreak investigation and tracking of antimicrobial resistance. Publisher PDF

Phylogeny of CC22 MRSA in the Cambridge LTCF with bootstrap values. Bootstrap values for branches are shown in grey. (PDF 224 kb)

Phylogeny of CC5 MRSA in the Galway study. Maximum likelihood tree generated from core genome SNPs of CC5 MRSA isolates from the Galway LTCF residents (blue) and isolates from Galway University Hospital (red). Bootstrap values for branches are shown in grey. (PDF 266 kb)