Persistence and Transmission Dynamics of mcr-1 and mcr-9 in Enterobacteriaceae from Pig Farms in Fujian, China after the Colistin Ban

Polymyxins are last-resort antibiotics for multidrug-resistant Gram-negative bacteria infections. However, their efficacy is threatened by the emergence of plasmid-mediated colistin resistance genes ( mcr-1 to mcr-10 ). Antibiotic use in agriculture has been recognized as a major driver of antimicrobial resistance, prompting China to ban colistin as a feed additive in 2017. This study investigated the persistence and transmission mechanisms of mcr genes in Enterobacteriaceae isolated from pigs, farm workers, and the surrounding environment on four pig farms in Fujian Province, China. Totally, 930 samples were collected, including pig rectal swabs, farm worker fecal samples, as well as environmental samples from both inside and outside the farms. From these, a total of 263 Klebsiella pneumoniae and 521 Escherichia coli isolates wererecovered, with isolation rate of 28.3% and 56.0%, respectively. PCR screening revealed that mcr-1 and mcr-9 were the only detected variants. Among K. pneumoniae isolates , 39 (14.8%) carried mcr-1 and 21(8.0%) carried mcr-9 . In E. coli , mcr-1 and mcr-9 were detected in 8 (1.5%) and 6 (1.1%) isolates, respectively. In addition, three Raoultella ornithinolytica and one Enterobacter roggenkampii isolates carried mcr-9 genes were identified . Besides animal sources, mcr -positive strains were also identified in environmental samples, particularly from inside the farms, and in farm workers, indicating potential zoonotic and environmental transmission.Antimicrobial susceptibility testing revealed that all mcr -positive isolates exhibited multi-antibiotic resistance, with mcr-1 -positive strains displaying broader resistance profiles than mcr-9 -positive strains. The minimum inhibitory concentrations (MICs)of colistin ranged from 2-32 μg/mL for mcr-1 -positive isolates and 1-8 μg/mL for mcr-9 -positive isolates. Whole-genome sequencing and conjugation experiments showed that mcr-1 was primarily located on IncHI2 (n=5), IncX4 (n=14), and IncI2 (n=15) plasmids, while mcr-9 was predominantly carried by IncHI2 plasmids (n=4) and IncF(n=2) plasmids. Notably, mcr-9 -positive plasmids showed higher conjugation efficiency, lower fitness cost, and greater persistence within bacterial hosts compared to mcr-1 -positive IncHI2 plasmids. These finding suggest that the increasing prevalence of mcr-9 in Enterobacteria may be driven by its enhanced transferability and stability, even in the absence of antibiotic selection pressure. This highlights the urgent need for continued environmental surveillance and targeted interventions to solve the dissemination of plasmid-mediated antibiotic resistance in livestock production.