Metagenomic survey and whole genome sequencing of antimicrobial resistant bacteria in the sewage of a Japanese hospital

Background The dissemination of antimicrobial-resistant bacteria (ARB) and the transfer of antimicrobial resistance genes (ARGs) are a threat to public health. Antibiotics are indispensable therapeutic agents essential for the treatment of infectious diseases; however, inappropriate use of antibiotics leads to the emergence of ARB. It is established that hospitals are closely involved in the spread of antimicrobial resistance (AMR), which impedes antibiotic treatment and subsequently increases mortality. In addition, excrement of patients or healthy carriers of ARB are discharged from the hospital sewage through the wastewater treatment plant (WWTP) into the rivers, causing an AMR burden on the environment.Method Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of the extended spectrum β-lactamase (ESBL)-producing organisms (EPOs). A comparative genome analysis was also performed between EPO isolates from sewage and clinical isolates.Results Metagenomic analysis showed that the hospital sewage tanks had bacterial flora corresponding to the human gut. During the study period, the hospital was relocated to a newly constructed building with new sewage tanks; however, the presence of ARB/ARGs in the new hospital sewage tanks became markedly equivalent to that of the old hospital within one month. The ESBL blaCTX−M and carbapenemase blaIMP genes were not much detected in the original hospital sewage samples by metagenome analysis, but selection on CHROMagar ESBL increased the sensitivity to detect those β-lactamase genes. Comparative genome analysis between sewage and clinical EPO isolates revealed partial similarity; however, most EPO isolates exhibited a notable difference (≥ 50) in single nucleotide variations based on core-genome phylogeny. This result suggests that only some of the sewage EPO isolates were originated from the clinical patient. Therapeutic agents in the hospital sewage were analyzed and the concentration of levofloxacin and clarithromycin was 0.0325 and 0.0135 µg/mL, respectively.Conclusions Whole genome analysis between sewage and clinical isolates suggested that healthy or asymptomatic carriers may be involved in the contamination of hospital sewage. Moreover, the hospital sewage tank may serve as a hotspot for the horizontal transfer of ARGs under the selective pressure of antimicrobial agents. Therefore, ARB monitoring in hospital sewage is expected to detect the presence of carriers prior to nosocomial ARB outbreaks. In addition, hospital wastewater should be treated suitably to bring ARB below detectable levels to reduce the environmental AMR burden.