Carbapenem-resistant (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large carbapenemase (KPC)-producing outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing isolates ( = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening ( = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis ( = 268 isolates) identified the spread of a KPC-producing outbreak clone (strain A, sequence type 216 [ST216]; = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other lineages and species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of in, including pathogenic lineages, are of concern.

Conclusion: Slow drainage rates and sink designs with the drain directly underneath the tap increase the risk of CRE present in waste traps and drains contaminating the ward environment.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, blaKPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of blaKPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-blaKPC and blaKPC plasmids and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the commonest transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC has historically been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the UK, blaKPC has caused a large-scale, persistent outbreak focused on hospitals in North-West England. This outbreak has evolved to be polyclonal and poly-species, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterisation. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 (97%) isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments amongst plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates) and IncR replicons (252/604 isolates). The subset of reconstructed plasmid sequences also highlighted modular exchange amongst non-blaKPC and blaKPC plasmids, and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales, for the implementation of adequate surveillance approaches, and for control.IMPORTANCEAntimicrobial resistance is a major threat to the management of infections, and resistance to carbapenems, one of the “last line” antibiotics available for managing drug-resistant infections, is a significant problem. This study used large-scale whole genome sequencing over a five-year period in the UK to highlight the complexity of genetic structures facilitating the spread of an important carbapenem resistance gene (blaKPC) amongst a number of bacterial species that cause disease in humans. In contrast to a recent pan-European study from 2012-2013(1), which demonstrated the major role of spread of clonal blaKPC-Klebsiella pneumoniae lineages in continental Europe, our study highlights the substantial plasticity in genetic mechanisms underpinning the dissemination of blaKPC. This genetic flux has important implications for: the surveillance of drug resistance (i.e. making surveillance more difficult); detection of outbreaks and tracking hospital transmission; generalizability of surveillance findings over time and for different regions; and for the implementation and evaluation of control interventions.

BackgroundHealthcare-associated wastewater reservoirs and asymptomatic gastrointestinal patient colonisation by carbapenemase-producing Enterobacterales (CPE) can be important in nosocomial CPE dissemination and infection. We characterised these niches and within-niche diversity in a blaKPC-associated CPE (KPC-E) endemic healthcare setting, to better understand transmission potential.MethodsWe systematically sampled wastewater sites and patients across three units (six wards) over 6-12 months in 2016 in a KPC-E endemic hospital. We used Illumina sequencing to characterise up to five isolates per sample. Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based typing approaches were used to characterise antimicrobial resistance gene and insertion sequence profiles, and Tn4401 types/target site sequences. The wider accessory genome was evaluated in a subset of the largest clusters, and those crossing niches.FindingsWastewater site KPC-E-positivity was substantial (101/349 sites [28.9%] positive, 319/4,488 [7.1%] sampling events positive); 183/4,425 (4.1%) of patients were CPE culture-positive over the same timeframe. 13 species and 109 KPC-E strains were observed across niches, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured ≥1 strain. Most diversity was explained by the individual niche, suggesting highly localised factors are important in selection and spread. Tn4401+target site sequence (TSS) diversity was greater in wastewater sites (p<0.001), suggesting these might favour Tn4401-associated transposition/evolution and dissemination. Shower/bath and sluice/mop-associated sites were more likely to be KPC-E-positive (Adjusted Odds Ratio [95% CI]: 2.69 [1.44-5.01], p=0.0019 and 2.60 [1.04-6.52], p=0.0410, respectively). Different strains had different transmission and blaKPC dissemination dynamics.InterpretationThere may be substantial KPC-E colonisation of wastewater sites and patients in KPC-E-endemic healthcare settings. Niche-specific factors, and different strains with different transmission dynamics influence carbapenemase gene dissemination. New transmission models incorporating complex, multi-level dynamics are needed to better quantify CPE dissemination to inform interventions and reduce transmission.FundingThis study was supported by the National Institute for Health Research, UK.RESEARCH IN CONTEXTEvidence before this studyWe searched PubMed to identify previous studies using whole genome sequencing (WGS) to characterise in-hospital dissemination and persistence of carbapenemase-producing Enterobacterales (CPEs) or the presence of CPEs in hospital wastewater reservoirs. In our search (01/Jan/1996-30/Sep/2021) we used the terms ((((CPE) OR (KPC)) AND (healthcare-associated OR nosocomial OR hospital-associated)) AND (genom*) AND ((environment OR patient))). We had no restrictions on language. We found sixty-six studies, but these were limited by: (i) evaluating single species or lineages only (n=34), (ii) including patients or environmental outbreaks only, and not both (n=23), (iii) sampling only small numbers (n<50) of patients or environmental sites (n=26), or (iv) performing no evaluation of within-niche diversity (n=64). 13 studies were not relevant (e.g. investigating non-hospital locations, ex vivo or in vivo evolution), two studies did not use WGS, and one study was not primary research.Added value of this studyWe provide a comprehensive assessment of healthcare-associated CPE dissemination and persistence in hospital wastewater reservoirs through systematic sampling of a large number of environmental sites (n=349 sites, n=4,488 samples) and patients (n=4,425) over 6-12 months in a single hospital. For all CPE-positive environmental samples (n=319) and a subset of CPE-positive patient samples (n=97/399 [24.3%] samples from 76/183 [41.5%] CPE-positive patients), we sampled up to five CPE isolates per sample to capture within-niche diversity, and used short-read Illumina WGS (n=1,732 isolates successfully sequenced) to characterise within-niche diversity, changes in colonisation over time, and genetic overlap between patient and environmental niches.Implications of all the available evidenceThrough a detailed and resource-intensive study we captured the dynamics of seeding and dissemination of important carbapenemase genes amongst patients and environmental reservoirs within a hospital. We found differential colonisation and dissemination dynamics for different species and lineages within and between niches. Improved approaches incorporating variable within-niche diversity, accessory distances and horizontal gene transfer in transmission evaluations are required to better understand CPE dissemination and persistence, in order to direct interventions limiting transmission.