Mechanisms of polymyxin resistance induced by Salmonella typhimurium in vitro

• Elevated phoP gene expression enhances polymyxin resistance in Salmonella . • The pmrAB mutation is not necessarily required for the acquisition of polymyxin resistance. • Salmonella pays a fitness cost for obtaining polymyxin resistance. • For the first time, a gene-deficient strain was constructed in Salmonella using the CRISPR/Cas9 gene-editing system. The increase incidence of multi-drug resistant (MDR) Salmonella has become a major global health concern. Polymyxin, an ancient polypeptide antibiotic, has been given renewed attention over recent years, resulting in resistance of Gram-negative bacteria to polymyxin, but its resistance mechanism is not completely clear. Thus, it is important to study its resistance mechanisms. In this study, an in vitro induced polymyxin-resistant strain of Salmonella typhimurium in the laboratory were constructed to investigate the mechanism of resistance of Salmonella to polymyxin. Gradual induction of Salmonella typhimurium ATCC13311 (AT) by concentration increment was used to screen for a highly polymyxin-resistant strain AT-P128. The broth dilution technique was used to compare the sensitivity of the two strains to different antimicrobial drugs. Single nucleotide polymorphisms (SNPs) were then identified by whole genome sequencing, and differences in gene expression between the two strains were compared by transcriptome sequencing and reverse transcription–quantitative PCR (RT-qPCR). Finally, for the first time, the CRISPR/Cas9 gene-editing system was used to construct gene deletion mutants in Salmonella to knock out the phoP gene of AT-P128. The results showed that strain AT-P128 was significantly more resistant to amoxicillin, ceftiofur, ampicillin, fluphenazine, and chloramphenicol and significantly less resistant to sulfamethoxazole than the parental strain AT. The growth curve results showed no significant change in the growth rate between AT-P128 and AT. Motility and biofilm formation assays showed a significant decrease in AT-P128. Additionally, the WGS results showed that AT-P128 had mutations in 9 genes involving 14 SNPs. RNA-seq and RT-qPCR results showed increased expression of phoPQ . The loss of the phoP gene decreased AT-P128Δ phoP resistance to polymyxin by 32-fold. These results suggested that polymyxin resistance affected the biology, genome components, and gene expression levels of Salmonella and that the PhoPQ two-component system played a key role in polymyxin resistance in Salmonella, providing insights into the diversity and complexity of polymyxin resistance in Salmonella .