Temporal dynamics of extended-spectrum β-lactamase-producing Escherichia coli and carbapenemase-producing Gram-negative bacteria in hospital wastewater.

Hospital wastewater is a reservoir for the environmental spread of clinically relevant antimicrobial-resistant bacteria and resistance genes. The aim of this study was to quantify total Escherichia coli, extended-spectrum β-lactamase (ESBL)-producing E. coli, and carbapenemase-producing organisms (CPOs) and perform whole-genome sequencing-based characterization of these bacterial isolates in hospital wastewater samples collected bimonthly in Japan from January to November 2021. Total E. coli counts were 8.1 × 103-8.8 × 104 colony-forming units (CFU)/mL. ESBL-producing E. coli were detected in the sampling months of January, March, May, and July, with the ratio of ESBL-producing E. coli to total E. coli being remarkably highest (95 %) in July. In contrast, DHA-1 Ambler class C β-lactamase (AmpC)-producing E. coli was detected in September and November, accounting for 28 % and 3 % of total E. coli counts, respectively. All 140 ESBL-producing E. coli isolates harbored the blaCTX-M genes, with blaCTX-M-14 being the most common genotype (94.3 %), the vast majority of which were associated with the human virulent B2-O25b: H4-ST131-fimH30R/non-Rx. In September, E. coli clade I-O8:H33-ST3910-fimH1074 was primarily associated with blaDHA-1. Among 26 representative CPO isolates, Aeromonas caviae (34.6 %) and A. hydrophila subsp. hydrophila (30.8 %) were dominant. The most frequently detected carbapenemase gene was blaIMP-1 (57.7 %), followed by blaGES-24 (34.6 %) and blaGES-4 (7.7 %). Estimated bacterial counts of CPOs ranged from 4.0 × 10-1 to 4.7 × 103 CFU/mL over the six sampling months. blaIMP-1-positive A. hydrophila subsp. hydrophila ST860, which was repeatedly detected over the five sampling months, accounted for the highest total number of this bacterial clone (79 %). Overall, this study provides insights into the overwhelming presence and persistence of E. coli B2-O25b:H4-ST131-H30R/non-Rx with blaCTX-M-14 and Aeromonas spp. with blaIMP-1 in hospital wastewater, and the change in the dynamics of resistance gene prevalence from blaCTX-M-positive E. coli to blaDHA-1-positive E. coli.