Characterization of novel mutations involved in the development of resistance to colistin in Salmonella isolates from retail pork in Shanghai, China.

Salmonella is an important foodborne pathogen that poses a significant threat to food safety. This study aims to assess the prevalence, genomic features, and colistin-resistant mechanisms of Salmonella isolates collected from 118 retail pork samples from January 2021 to January 2022 in Shanghai, China. Overall, 46 (39.0 %, 46/118) Salmonella isolates were collected, which were identified as 12 serotypes by genomic analysis, including Salmonella Typhimurium (n = 17) and Salmonella London (n = 6). Antimicrobial resistance profiling revealed that the resistance rate of these isolates to colistin was 13.0 % (6/46), while 60.9 % (28/46) exhibited multidrug-resistant. It was found that there were 51 distinct antimicrobial resistance genes in these 46 isolates, which were predominantly associated with resistance to aminoglycosides, fluoroquinolones, and β-lactams. More importantly, among six colistin-resistant isolates, two isolates (Salmonella Schwarzengrund and Salmonella Indiana) were found to carry the mcr-1 gene. The mechanism of resistance in the remaining four colistin-resistant isolates was further studied, and it was found that there were nine amino acid substitutions in PmrAB. It was demonstrated by site-directed mutagenesis that novel substitutions G53W in PmrA and I83V in PmrB led to colistin resistance in Salmonella (MIC = 2 or 4 μg/mL). Analysis results by real-time quantitative PCR and mass spectrometry indicated that the mutants PmrA and PmrB displayed higher expression levels of the gene pmrE than in the parental strain. This upregulation resulted in an increase in the production of 4-amino-4-deoxy-l-arabinose (L-Ara4N) that modified lipid A, thereby conferring resistance to colistin. These findings demonstrated that there was a high prevalence of MDR Salmonella isolates in retail pork in Shanghai, and the substitution G53W in PmrA and I83V in PmrB were independent factors contributing to the development of resistance to colistin in Salmonella via modification of lipid A with L-Ara4N.