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

Abstract Background Healthcare-associated wastewater and asymptomatic patient reservoirs colonized by carbapenemase-producing Enterobacterales (CPE) contribute to nosocomial CPE dissemination, but the characteristics and dynamics of this remain unclear. Methods We systematically sampled wastewater sites (n = 4488 samples; 349 sites) and patients (n = 1247) across six wards over 6–12 months to understand blaKPC-associated CPE (KPC-E) diversity within these reservoirs and transmission in a healthcare setting. Up to five KPC-E-positive isolates per sample were sequenced (Illumina). Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based approaches were used to characterize antimicrobial resistance genes, insertion sequences (ISs) and Tn4401 types/target site sequences. The accessory genome was evaluated in some of the largest clusters, and those crossing reservoirs. Results Wastewater site KPC-E-positivity was substantial [101/349 sites (28.9%); 228/5601 (4.1%) patients cultured]. Thirteen KPC-E species and 109 strains were identified using genomics, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured one or more strains. Most diversity was explained by the individual niche, suggesting localized factors are important in selection and spread. Tn4401 + flanking target site sequence diversity was greater in wastewater sites (P