Carbapenemase-producing Enterobacterales and their carbapenemase genes are stably recovered across the wastewater-watershed ecosystem nexus

Carbapenemase-producing Enterobacterales (CPE) have emerged as an important nosocomial threat to hospitalized patients, but CPE can also colonize the enteric microbiota of healthy individuals in the community. We hypothesized that clinically relevant CPE are frequently transported in municipal wastewater flows to treatment plants where they are reduced but not eliminated and are subsequently discharged into nearby surface waters and disseminate in the environment. We sampled untreated influent, treated effluent, and nearby surface waters weekly for a one-year period at a single large metropolitan wastewater treatment plant (WWTP) servicing Columbus, Ohio USA. In addition, we investigated the dissemination of these CPE into the environment and the downstream watershed, including sediment, fish, mammals, waterfowl, crops and soils. CPE were recovered from each (100 %) of the 44 influent samples and from 37 (84 %) effluent samples. We also isolated CPE from 50 % (22/44) of the upstream water, 82 % (36/44) of downstream, and 68 % (30/44) way downstream water. CPE were most commonly Enterobacter spp. expressing the bla_KPC-2 genotype, although a variety of other species and genotypes were observed. bla_KPC concentration was greatest in the influent (mean = 10⁶ gene copies/100 mL water) and treatment resulted in a 4-log reduction in bla_KPC concentration (P < 0.05), which was consistent with reduction in total bacteria concentration. We found 22 of 450 fish vent swabs (4.9 %) carrying CPE, but CPE were not recovered from terrestrial wildlife living in the Scioto watershed. Fish intestinal microbiome maintained approximately 1.5 × 10⁴ copies of bla_KPC per gram of feces. Our data support the hypothesized flow of CPE from healthcare settings into surface water and the downstream natural environment via municipal wastewater discharge. Our results suggest that river environments can serve as a reservoir for CPE, facilitating their broader One Health dissemination among surface water, wildlife, agriculture, and ultimately back to humans.