Municipal wastewater monitoring revealed the predominance of blaGES genes with diverse variants among carbapenemase-producing organisms: high occurrence and persistence of Aeromonas caviae harboring the new blaGES variant blaGES-48.

Abstract

We characterized carbapenemase-producing organism (CPO) detected in municipal wastewater to better understand the epidemiology of CPOs in the community. In total, 36 samples were collected at six sampling sites every other month from December 2020 to October 2021. CPOs were not recovered from influent taken from inlet A receiving separated sewer line, treated effluents, and river water samples upstream and downstream of the effluent outlet. By contrast, 75 CPOs were detected in all influent samples taken from inlets B and C receiving combined sewer lines collecting both domestic/industrial wastewater and rainwater runoff. Aeromonas caviae was the dominant species (25/75, 33.3%), and the other 11 Aeromonas spp. together accounted for 48% of CPOs. The remaining 39 Enterobacterales strains mainly comprised 17 Klebsiella spp. and 10 Raoultella spp. CPOs carrying blaGES carbapenemase genes were overwhelmingly dominant, accounting for 72 of 75 isolates, including two isolates harboring both blaGES-24 and blaIMP-1 (96%), followed by three blaIMPs-positive isolates, where those carbapenemase genes were mainly carried in diverse class 1 integrons. Among blaGES variants, including six new variants (blaGES-47, blaGES-48, blaGES-49, blaGES-50, blaGES-51, and blaGES-54), blaGES-5 was detected in 28 CPOs, with Aeromonas spp. accounting for 53.6% of these organisms. Quantitative analysis revealed that the repeated detection of blaGES-48-positive A. caviae ST1056 from both inlets B and C ranked the total number of this bacterial clone highest in the wastewater influent. In summary, our study revealed the high prevalence and persistence of diverse blaGES carbapenemase genes among CPOs isolated from influent inlets connected to combined sewer systems.IMPORTANCEThe emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of blaGES genes, including six new blaGES variants, and less frequent blaIMP genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were Aeromonas spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.