Genomic epidemiology and longitudinal sampling of ward wastewater environments and patients reveals complexity of the transmission dynamics of blaKPC-carbapenemase-producing Enterobacterales in a hospital setting

2. Healthcare-associated wastewater reservoirs and asymptomatic gastrointestinal patient colonisation by carbapenemase-producing Enterobacterales (CPE) contribute to nosocomial CPE dissemination. We systematically sampled wastewater sites (n=4488 sampling events; 349 sites) and patients (n=1247) across six wards over 6-12 months in 2016 to better understand bla KPC -associated CPE (KPC-E) diversity within these niches and transmission potential in an endemic healthcare setting. Up to five isolates in KPC-E-positive samples were sequenced (Illumina). Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based typing approaches were used to characterise antimicrobial resistance genes, insertion sequences, and Tn4401 types/target site sequences. The wider accessory genome was evaluated in a subset of the largest clusters, and those crossing niches. Wastewater site KPC-E-positivity was substantial (101/349 sites [28.9%] positive); 228/5,601 (4.1%) patients cultured were CPE culture-positive over the same timeframe. At a genomic-level, 13 KPC-E species and 109 strains were identified, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured ≥1 strain. Most diversity was explained by the individual niche, suggesting localised factors are important in selection and spread. Tn4401+target site sequence diversity was greater in wastewater sites (p<0.001), which might favour Tn4401-associated transposition/evolution. 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 bla KPC dissemination dynamics. There may be substantial KPC-E colonisation of wastewater sites and patients in KPC-E-endemic healthcare settings. Niche-specific factors (e.g. microbial interactions, selection pressure) likely affect carbapenemase gene persistence and evolution, and different strains and mobile genetic elements with different transmission dynamics influence carbapenemase gene dissemination; these factors should be considered in surveillance and control strategies.