Impacts of chemicals and microbiota from a hospital on the emergence of resistance in sewer systems

Hospital wastewaters are composed of antibiotic resistant bacteria (ARB) and a cocktail of chemical agents, including antibiotics (ATB) exerting selective pressures. The aim of this study was to clarify the role of ARB and chemicals originating from hospitals in the emergence / dissemination of antimicrobial resistance in mixed municipal wastewater. As part of a field approach, domestic wastewater, hospital wastewater and mixed wastewater (domestic + hospital) were sampled and compared in terms of ATB concentrations and antibiotic resistance. Controlled microcosms were also conducted to assess the contribution of the microbiological versus chemical component of the hospital wastewater exposome. They consisted in mixing domestic wastewaters with either full hospital effluent, its chemical constituents, or its microbiota. They were incubated for 96 hours at 21°C and 100 rpm. Antibiotic resistance was monitored by: (i) assessment of percentage of ARB to ciprofloxacin and cefotaxime, (ii) quantification of antibiotic resistance genes (ARGs) by qPCR, (iii) determination of the sequence variants of ARGs from various antibiotic classes (fluoroquinolones, beta-lactamases, polymyxins, and others) by multiplex amplicon sequencing. The proportions of ARB to ciprofloxacin were similar in microcosms that received full hospital effluent or only its chemical component, suggesting that selective pressure rather than community coalescence/immigration was the main factor giving rise to ARB in mixed municipal wastewaters. Some ARG variants were unique to each wastewater type, whilst others were shared. Their profiles in the microcosms suggested that hospital-derived variants could persist and coexist along domestic wastewater variants in some instances (e.g., blaMIR, mdtg). In other cases, hospital variants were not maintained in the final microcosm communities (e.g., blaFOX). This research sheds light on the complex interplay between ARGs and chemical agents form hospitals in municipal wastewater, providing insights into the potential dissemination routes and persistence patterns.