Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data.

BACKGROUND: DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. METHODS: In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. FINDINGS: We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), and linezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. INTERPRETATION: To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. FUNDING: Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Dissemination of carbapenemase-producing Enterobacterales in Ireland from 2012 to 2017: a retrospective genomic surveillance study.

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli.

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission.

Understanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.

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

BACKGROUND: Escherichia coli is a leading cause of bloodstream infections. Developing interventions to reduce E coli infections requires an understanding of the frequency of nosocomial transmission, but the available evidence is scarce. We aimed to detect and characterise transmission of E coli and associated plasmids in a hospital setting. METHODS: In this prospective observational cohort study, patients were admitted to two adult haematology wards at the Cambridge University Hospitals NHS Foundation Trust in England. Patients aged 16 years and older who were treated for haematological malignancies were included. Stool samples were collected from study participants on admission, once per week, and at discharge. We sequenced multiple E coli isolates (both extended spectrum ß-lactamase [ESBL]-producing and non-ESBL-producing) from each stool sample. A genetic threshold to infer E coli transmission was defined by maximum within-host single nucleotide polymorphism (SNP) diversity and the probability of drawing observed pairs of between-patient isolates at different SNP thresholds. Putative transmission clusters were identified when sequences were less than the genetic threshold. Epidemiological links for each transmission event were investigated. We sequenced all E coli positive blood samples from the two adult haematology wards. FINDINGS: We recruited 174 (51%) of 338 adult patients admitted to the wards between May 13 and Nov 13, 2015. We obtained and cultured 376 stool samples from 149 patients, of which 152 samples from 97 (65%) patients grew E coli. Whole-genome sequencing was done on 970 isolates. We identified extensive diversity in the bacterial population (90 sequence types) and mixed E coli sequence type carriage. 24 (26%) patients carried two sequence types, 12 (13%) carried three, and six (6%) patients carried four or more sequence types. Using a 17 SNP cutoff we identified ten clusters in 20 patients. The largest cluster contained seven patients, whereas four patients were included in multiple clusters. Strong epidemiological links were found between patients in seven clusters. 17 (11%) of 149 patients had stool samples positive for ESBL-producing E coli, the most common of which was associated with bla CTX-M-15 (12 [71%] of 17). Long-read sequencing revealed that bla CTX-M-15 was often integrated into the chromosome, with little evidence for plasmid transmission. Seven patients developed E coli bloodstream infection, four with identical strains to those in their stool; two of these had documented nosocomial acquisition. INTERPRETATION: We provide evidence of bacterial transmission and endogenous infection during routine care by integrating genomic and epidemiological data and by determining a genetic cutoff informed by within-host diversity in the studied population. Our findings challenge single colony-based investigations, and the widely accepted notion of plasmid spread. FUNDING: UK Department of Health, Wellcome Trust, UK National Institute for Health Research.

SARS-CoV-2 variants, spike mutations and immune escape.

Although most mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome are expected to be either deleterious and swiftly purged or relatively neutral, a small proportion will affect functional properties and may alter infectivity, disease severity or interactions with host immunity. The emergence of SARS-CoV-2 in late 2019 was followed by a period of relative evolutionary stasis lasting about 11 months. Since late 2020, however, SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations, in the context of 'variants of concern', that impact virus characteristics, including transmissibility and antigenicity, probably in response to the changing immune profile of the human population. There is emerging evidence of reduced neutralization of some SARS-CoV-2 variants by postvaccination serum; however, a greater understanding of correlates of protection is required to evaluate how this may impact vaccine effectiveness. Nonetheless, manufacturers are preparing platforms for a possible update of vaccine sequences, and it is crucial that surveillance of genetic and antigenic changes in the global virus population is done alongside experiments to elucidate the phenotypic impacts of mutations. In this Review, we summarize the literature on mutations of the SARS-CoV-2 spike protein, the primary antigen, focusing on their impacts on antigenicity and contextualizing them in the protein structure, and discuss them in the context of observed mutation frequencies in global sequence datasets.

Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B.1.1.7): an exploratory analysis of a randomised controlled trial.

BACKGROUND: A new variant of SARS-CoV-2, B.1.1.7, emerged as the dominant cause of COVID-19 disease in the UK from November, 2020. We report a post-hoc analysis of the efficacy of the adenoviral vector vaccine, ChAdOx1 nCoV-19 (AZD1222), against this variant. METHODS: Volunteers (aged ≥18 years) who were enrolled in phase 2/3 vaccine efficacy studies in the UK, and who were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 or a meningococcal conjugate control (MenACWY) vaccine, provided upper airway swabs on a weekly basis and also if they developed symptoms of COVID-19 disease (a cough, a fever of 37·8°C or higher, shortness of breath, anosmia, or ageusia). Swabs were tested by nucleic acid amplification test (NAAT) for SARS-CoV-2 and positive samples were sequenced through the COVID-19 Genomics UK consortium. Neutralising antibody responses were measured using a live-virus microneutralisation assay against the B.1.1.7 lineage and a canonical non-B.1.1.7 lineage (Victoria). The efficacy analysis included symptomatic COVID-19 in seronegative participants with a NAAT positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to vaccine received. Vaccine efficacy was calculated as 1 - relative risk (ChAdOx1 nCoV-19 vs MenACWY groups) derived from a robust Poisson regression model. This study is continuing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Participants in efficacy cohorts were recruited between May 31 and Nov 13, 2020, and received booster doses between Aug 3 and Dec 30, 2020. Of 8534 participants in the primary efficacy cohort, 6636 (78%) were aged 18-55 years and 5065 (59%) were female. Between Oct 1, 2020, and Jan 14, 2021, 520 participants developed SARS-CoV-2 infection. 1466 NAAT positive nose and throat swabs were collected from these participants during the trial. Of these, 401 swabs from 311 participants were successfully sequenced. Laboratory virus neutralisation activity by vaccine-induced antibodies was lower against the B.1.1.7 variant than against the Victoria lineage (geometric mean ratio 8·9, 95% CI 7·2-11·0). Clinical vaccine efficacy against symptomatic NAAT positive infection was 70·4% (95% CI 43·6-84·5) for B.1.1.7 and 81·5% (67·9-89·4) for non-B.1.1.7 lineages. INTERPRETATION: ChAdOx1 nCoV-19 showed reduced neutralisation activity against the B.1.1.7 variant compared with a non-B.1.1.7 variant in vitro, but the vaccine showed efficacy against the B.1.1.7 variant of SARS-CoV-2. FUNDING: UK Research and Innovation, National Institute for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.

Quantifying acquisition and transmission of Enterococcus faecium using genomic surveillance.

Nosocomial acquisition and transmission of vancomycin-resistant Enterococcus faecium (VREfm) is the driver for E. faecium carriage in hospitalized patients, which, in turn, is a risk factor for invasive infection in immunocompromised patients. In the present study, we provide a comprehensive picture of E. faecium transmission in an entire sampled patient population using a sequence-driven approach. We prospectively identified and followed 149 haematology patients admitted to a hospital in England for 6 months. Patient stools (n = 376) and environmental swabs (n = 922) were taken at intervals and cultured for E. faecium. We sequenced 1,560 isolates (1,001 stool, 559 environment) and focused our genomic analyses on 1,477 isolates (95%) in the hospital-adapted clade A1. Of 101 patients who provided two or more stool samples, 40 (40%) developed E. faecium carriage after admission based on culture, compared with 64 patients (63%) based on genomic analysis (73% VREfm). Half of 922 environmental swabs (447, 48%) were positive for VREfm. Network analysis showed that, of 111 patients positive for the A1 clade, 67 had strong epidemiological and genomic links with at least one other patient and/or their direct environment, supporting nosocomial transmission. Six patients (3.4%) developed an invasive E. faecium infection from their own gut-colonizing strain, which was preceded by nosocomial acquisition of the infecting isolate in half of these. Two informatics approaches (subtype categorization to define phylogenetic clusters and the development of an SNP cut-off for transmission) were central to our analyses, both of which will inform the future translation of E. faecium sequencing into routine outbreak detection and investigation. In conclusion, we showed that carriage and environmental contamination by the hospital-adapted E. faecium lineage were hyperendemic in our study population and that improved infection control measures will be needed to reduce hospital acquisition rates.

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones.

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum ß-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short- and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that Clade C was the most prevalent (686 out of 794 isolates, 86 %) of the three major ST131 clades circulating worldwide (A with fimH41, B with fimH22, C with fimH30), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in c. 1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1-mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10), highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.

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.

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.

Genomic surveillance of Escherichia coli in municipal wastewater treatment plants as an indicator of clinically relevant pathogens and their resistance genes.

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 ß-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.

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.

One Health Genomic Surveillance of Escherichia coli Demonstrates Distinct Lineages and Mobile Genetic Elements in Isolates from Humans versus Livestock.

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli isolates (including 155 extended-spectrum ß-lactamase [ESBL]-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1,517 E. coli bacteria associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, suggesting that E. coli causing serious human infection had not directly originated from livestock. In contrast, we observed highly related isolates from the same animal species on different farms. Screening all 1,948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions in human and livestock isolates. Overall, we identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that within the confines of our sampling framework, there was limited evidence that antimicrobial-resistant pathogens associated with serious human infection had originated from livestock in our region.IMPORTANCE The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1,517 patients with bloodstream infections were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was limited overlap in the mobile elements carrying these genes. Within the limitations of sampling, our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock in our region.

Genomic Surveillance of Enterococcus faecium Reveals Limited Sharing of Strains and Resistance Genes between Livestock and Humans in the United Kingdom.

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.IMPORTANCE 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.

Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages.

There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.

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

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.

Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing.

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (blaOXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.