The emergence and spread of antimicrobial resistance (AMR) has become a persistent problem globally. In this study, an ozone treatment facility was established for an advanced hospital wastewater treatment in a core hospital facility in an urban area in Japan to evaluate the inactivation of antimicrobial-resistant bacteria and antimicrobials. Metagenomic DNA-seq analysis and the isolation of potential extended-spectrum β-lactamase (ESBL)-producing bacteria suggested that ozone exposure for at least 20 min is required for the adequate inactivation of DNA and ESBL-producing bacteria. Escherichia coli and Klebsiella species were markedly susceptible to 20-min ozone exposure, whereas Raoultella ornithinolytica and Pseudomonas putida were isolated even after an 80-min exposure. These ozone-resistant bacteria might play a pivotal role as AMR reservoirs in the environment. Nine antimicrobials (ampicillin, cefdinir, cefpodoxime, ciprofloxacin, levofloxacin, clarithromycin, chlortetracycline, minocycline, and vancomycin) were detected at 373 ng/L to 27 μg/L in the hospital wastewater, and these were removed (96–100% removal) after a 40-min treatment. These results facilitate a comprehensive understanding of the AMR risk posed by hospital wastewater and provides insights for devising strategies to eliminate or mitigate the burden of antimicrobial-resistant bacteria and the flow of antimicrobials into the environment. To the best of our knowledge, this is the first report on the implementation of a batch-type, plant-scale ozone treatment system in a hospital facility to execute and evaluate the inactivation of drug-resistant bacteria and antimicrobials.

Antimicrobial resistance (AMR) is becoming a global concern. Recently, research has emerged to evaluate the human and environmental health implications of wastewater from medical facilities and to identify acceptable wastewater treatment methods. In this study, a disinfection wastewater treatment system using an ozone-based continuous flow system was installed in a general hospital located in Japan. The effectiveness of antimicrobial-resistant bacteria (ARB) and antimicrobials in mitigating the environmental impact of hospital wastewater was evaluated. Metagenomic analysis was conducted to characterize the microorganisms in the wastewater before and after treatment. The results demonstrated that ozone treatment enables effective inactivation of general gut bacteria, including Bacteroides, Prevotella, Escherichia coli, Klebsiella, DNA molecules, and ARGs, as well as antimicrobials. Azithromycin and doxycycline removal rates were >99% immediately after treatment, and levofloxacin and vancomycin removal rates remained between 90% and 97% for approximately one month. Clarithromycin was more readily removed than the other antimicrobials (81–91%), and no clear removal trend was observed for ampicillin. Our findings provide a better understanding of the environmental management of hospital wastewater and enhance the effectiveness of disinfection wastewater treatment systems at medical facilities for mitigating the discharge of pollutants into aquatic environments.

Background: Excrement containing antimicrobial-resistant bacteria (ARB) is discharged from the hospital sewage through wastewater treatment plants (WWTP) into rivers, increasing the antimicrobial resistance (AMR) burden on the environment. Purpose: We illustrate the contamination of hospital sewage tanks with ARB harboring antimicrobial resistance genes (ARGs) using comprehensive metagenomic sequencing. During the study period, we moved to a new hospital building constructed for renovation. Therefore, we investigated the difference in bacterial flora in the sewage tanks for each building with different departments, and the change in bacterial flora over time in new sewage tanks. Furthermore, we performed a comparative genome analysis of extended spectrum β-lactamase (ESBL)-producing organisms (EPOs) from hospital sewage and clinical samples. Residual antibiotics in the sewage tank were also measured. Methods: Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of EPOs. Results: The bacterial composition of new sewage tanks was comparable with that of old tanks within 1 month after relocation and was instantly affected by excrement. The bacterial composition of sewage tanks in the old and new buildings, containing rooms where seriously ill patients were treated, was similar. Selection on CHROMagar ESBL allowed detection of EPOs harboring bla_CTX-M and carbapenemase genes in all sewage tanks. One of the sewage Escherichia coli strain comprising ST393 harboring bla_CTX-M-27 corresponded to the clinical isolates based on core genome analysis. Moreover, the levels of levofloxacin and clarithromycin in the hospital sewage were 0.0325 and 0.0135 μg/mL, respectively. Conclusion: Hospital sewage was contaminated with many ARB species, ARGs and residual antibiotics, which can cause a burden on WWTP sewage treatment. The bacterial flora in the sewage tank was rapidly affected, especially by the ward with seriously ill patients. AMR monitoring of hospital sewage may help detect carriers prior to nosocomial ARB-associated outbreaks and control the outbreaks.

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 bla_CTX−M and carbapenemase bla_IMP 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.