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

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 bla(GES) carbapenemase genes were overwhelmingly dominant, accounting for 72 of 75 isolates, including two isolates harboring both bla(GES-24) and bla(IMP-1) (96%), followed by three bla(IMPs)-positive isolates, where those carbapenemase genes were mainly carried in diverse class 1 integrons. Among bla(GES) variants, including six new variants (bla(GES-47), bla(GES-48), bla(GES-49), bla(GES-50), bla(GES-51), and bla(GES-54)), bla(GES-5) was detected in 28 CPOs, with Aeromonas spp. accounting for 53.6% of these organisms. Quantitative analysis revealed that the repeated detection of bla(GES-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 bla(GES) carbapenemase genes among CPOs isolated from influent inlets connected to combined sewer systems.