Genomic insight into transmission mechanisms of carbapenem-producing Citrobacter spp. isolates between the WWTP and connecting rivers.

Carbapenem-resistant Enterobacteriaceae (CRE) poses major health risks worldwide. Most studies have focused on carbapenem resistance in Klebsiella pneumoniae and Escherichia coli; however, the occurrence and transmission of carbapenem-resistant Citrobacter spp. (CRCS) are poorly understood. In this study, we investigated the occurrence and potential transmission patterns of CRCS in different functional areas of an urban wastewater treatment plant (WWTP) and connecting rivers during one-year monitoring in Shandong Province, China. In total, 14 CRCS were detected in 376 environmental samples, including those from the WWTP inlet (n = 7), WWTP anaerobic tank (n = 2), and rivers (n = 5). Citrobacter braakii (n = 6) was the dominant subtype among 14 CRCS isolates, followed by Citrobacter freundii (n = 5), Citrobacter sedlakii (n = 2), and Citrobacter werkmanii (n = 1). All CRCS were resistant to imipenem, meropenem, ampicillin, amoxicillin/clavulanic acid, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, and ciprofloxacin. Plasmid analysis showed that the bla gene was located on IncN and IncFII (Yp) plasmids, whereas the bla gene was located on IncX3 and IncN2 plasmids. Clonal transmission of CRCS harboring carbapenem genes occurred between the WWTP and connecting rivers on a temporal or spatial scale. High genomic relatedness of NDM-5-producing C. sedlakii was identified between river water and WWTP aerosol, suggesting a potential exposure risk of CRCS for workers and surrounding residents near the WWTP. Furthermore, NDM-5-producing C. sedlakii isolated from rivers was related to C. sedlakii isolated from soil and well water in different regions of China, indicating that NDM-5-producing C. sedlakii may be widespread in China. These findings indicate that rare healthcare-associated pathogens such as CRCS can contribute to widespread carbapenem production in the environment; thus, CRCS should be continuously monitored.