Contribution of novel substitutions in PmrAB to the development of resistance to colistin in mcr-negative Salmonella isolates

The emergence of colistin-resistant Gram-negative bacteria poses a serious challenge for healthcare, and the two-component system PmrAB is closely associated with colistin resistance. This study aimed to characterize the colistin-resistant Salmonella isolates collected from 211 retail chicken meat samples between December 2020 and January 2022 in Shanghai, China. Overall, 90 Salmonella isolates (42.7%, 90/211) were identified, which were identified as 13 serotypes. Antimicrobial resistance profiling showed that 15.6% (14/90) of the isolates exhibited resistance to colistin. Among these, 5 isolates were found to carry the mcr-1 gene, and the mcr-1-positive IncHI2 plasmid pSJTUF15550 from Salmonella Agona could be horizontally transferred to other hosts. The mechanism of resistance in the remaining 9 mcr-negative colistin-resistant isolates was further studied, and it was found that there were 7 amino acid substitutions in PmrAB. Site-directed mutagenesis was used to construct mutants, demonstrating that 3 novel substitutions (L105P in PmrA, P94L, and L331R in PmrB) contributed to colistin resistance in Salmonella (MIC = 8 or 16 μg/mL). Quantitative PCR and lipid A analysis were then employed to explore the resistance regulatory pathway. It was found that these substitutions resulted in the production of L-Ara4N by upregulating the expression of the genes udg and pmrK, which in turn modified lipid A. Finally, the genes udg and pmrK in the mutants were knocked out to investigate whether these substitutions conferred colistin resistance through these genes. The colistin MIC of the udg or pmrK deletion in mutants was similar to that of the parental strains (MIC = 0.25 μg/mL), indicating that these substitutions might confer colistin resistance through the udg and pmrK pathways. These findings demonstrate that amino acid substitutions in PmrAB contribute to the development of colistin resistance in Salmonella by modifying lipid A through the genes udg and pmrK to produce L-Ara4N, providing insight into the mechanisms of colistin resistance in Salmonella.