Co-occurrence of mcr-1.1, mcr-3.40, and tmexCD1-toprJ1 on a hybrid IncFIB/IncHI1B plasmid in Klebsiella pneumoniae from pork in Hainan, China.

The spread of antimicrobial resistance genes in the food production chain poses a major threat to food safety and public health. In particular, the emergence of tmexCD1-toprJ1 RND efflux pump gene cluster, which confers tigecycline resistance in foodborne Klebsiella pneumoniae heightens this risk. This study aimed to investigate the prevalence and genetic features of tmexCD1-toprJ1-positive isolates from retail food in Hainan, China, and to characterize the co-occurrence of mcr-1.1, mcr-3.40, and tmexCD1-toprJ1 on a hybrid IncFIB/IncHI1B plasmid. A total of 663 retail food samples were collected in 18 administrative regions of Hainan Island. Nine tmexCD1-toprJ1-positive Klebsiella spp. isolates were recovered, including eight K. pneumoniae and one K. quasipneumoniae subsp. quasipneumoniae. All isolates exhibited resistance to tigecycline (MIC = 4-32 mg/L), cefotaxime and ciprofloxacin, while remaining susceptible to meropenem and ceftazidime/avibactam. Conjugation assays showed that six isolates successfully transferred tigecycline resistance to recipients, and two of these isolates also transferred colistin resistance. These two isolates carried both tmexCD1-toprJ1 and mcr genes. Notably, K. pneumoniae HN2023DA127 simultaneously harbored tmexCD1-toprJ1, mcr-1.1, and mcr-3.40, all located on a hybrid IncFIB/IncHI1B plasmid designated pHN2023DA127-1. After 15 days of passaging without selective pressure, 75.7 % of cells in K. pneumoniae HN2023DA127 retained all three determinants, with mcr-3.40 most frequently lost. Further genomic analysis revealed that the mobilization of tmexCD1-toprJ1, mcr-1.1, and mcr-3.40 was mediated by IS26-, ISApl1-, and ISKpn40-related insertion events, respectively. This is the first report of the co-occurrence of mcr-1.1, mcr-3.40, and tmexCD1-toprJ1 on a hybrid IncFIB/IncHI1B plasmid in K. pneumoniae. The convergence of last-resort antibiotic resistance highlights the urgent need for integrated One Health surveillance to limit the dissemination of clinically important pathogens in the food chain.