Tigecycline-resistance mechanisms and biological characteristics of drug-resistant Salmonella Typhimurium strains in vitro.

Increased drug resistance of Gram-negative bacteria to tetracycline caused by the unreasonable overuse of tigecycline has attracted extensive attention to reveal potential mechanisms. Here, we identified a tigecycline-resistant strain called TR16, derived from Salmonella Typhimurium ATCC13311 (AT), and examined its biological characteristics. Compared with AT, the TR16 strain showed significantly higher resistance to amoxicillin but lower resistance to gentamicin. Although the growth curves of TR16 and AT were similar, TR16 showed a significantly increased capacity for biofilm formation and a notably decreased motility compared to AT. Furthermore, transcriptome sequencing and reverse transcription-quantitative PCR (RT-qPCR) were implemented to evaluate the genetic difference between AT and TR16. Whole genome sequencing (WGS) analysis was also conducted to identify single nucleotide polymorphism (SNPs) and screened out two genetic mutations (lptD and rpsJ). The acrB gene of TR16 was knocked out through CRISPR/Cas9 system to further elucidate underlying mechanisms of tigecycline resistance in Salmonella Typhimurium. The up-regulation of acrB in TR16 was verified by RNA-seq and RT-qPCR, and the lack of acrB resulted in a 16-fold reduction in tigecycline resistance in TR16. Collectively, these results implied that AcrB efflux pump plays a key role in the tigecycline resistance of Salmonella, shedding light on the potential of AcrB efflux pump as a novel target for the discovery and development of new antibiotics.

BaeR overexpression enhances the susceptibility of acrB deleted Salmonella enterica serovar Typhimurium to polymyxin.

The mechanism of polymyxin resistance is complex, and the modification of lipopolysaccharide mediated by two-component system is one of the main cause of polymyxin resistance. To date, no studies have reported the contribution of the BaeSR two-component system to the polymyxin resistance of Salmonella. In this study, baeR, acrB single and double gene deletion strains of Salmonella typhimurium (AT-P128) which induced polymyxin resistance in vitro were constructed by using CRISPR/Cas9 gene editing technology, and the baeR gene was overexpressed in the acrB single gene deletion strain by the pUC19 plasmid. The susceptibility of different strains to polymyxin was determined by broth dilution method. Time-kill assay was carried out with different concentrations of polymyxin. The difference of gene expression among strains was compared by transcriptome sequencing (RNA-seq) and RT-qPCR. As a result, the MIC of the BaeR overexpression strain (AT-P128ΔacrB/pbaeR) to polymyxin was significantly reduced by 8-fold compared with the other tested strains. The growth curve results showed no significant change in the growth rate of the strain before and after gene deletion and overexpression. The time-kill assay showed that AT-P128ΔacrB/pbaeR was more susceptible under different concentrations of polymyxin. RNA-seq and RT-qPCR results showed that the expression levels of several polymyxin resistance-related genes including phoPQ, pmrD, pmrAB, arnT, eptB, lpxD, pagC and pagL changed significantly. These results indicate that overexpression of baeR in the context of the acrB gene deletion increases the polymyxin susceptibility of the strain and affects the expression level of polymyxin resistance genes, providing insight into the polymyxin resistance mechanism of S. typhimurium.

Quantitative phosphoproteomics reveals the effect of baeSR and acrB genes on protein phosphorylation in Salmonella enterica serovar typhimurium.

The BaeSR two-component system and the AcrB efflux pump are closely associated with Salmonella resistance to antibiotics. However, the relationship between the two-component system, efflux pumps and protein phosphorylation of Salmonella is poorly understood. In this study, Salmonella typhimurium ciprofloxacin-resistant strain CR, baeSR gene deletion strain CRΔbaeSR, acrB gene deletion strain CRΔacrB, and double gene deletion strain CRΔbaeSRΔacrB were used to explore phosphorylated proteins with significant difference, based on non-marker, quantitative phosphorylation modified proteomics technique. Consequently, 363 phosphosites of 213 phosphoproteins were identified in the four strains. More than 70% of the phosphosites were serine phosphorylation. In the CRΔbaeSR/CR, CRΔacrB/CR and CRΔbaeSRΔacrB/CR comparison groups, 36, 37 and 49 phosphosites were significantly altered, respectively. Bioinformatic analysis revealed that the main enrichment pathways of these differentially phosphorylated proteins were metabolic pathways, biosynthesis of antibiotics, phosphotransferase system (PTS), ABC transporters, and lipopolysaccharide biosynthesis. Furthermore, 21 differentially phosphorylated proteins were identified to be associated with antibiotic resistance. These results suggest that the BaeSR two-component system and the AcrB efflux pump affect the phosphorylation of proteins in S. typhimurium and may influence the drug resistance and virulence of S. typhimurium by affecting protein phosphorylation, providing a new idea to explore the mechanism of drug resistance in Salmonella.

Mechanisms of polymyxin resistance induced by Salmonella typhimurium in vitro.

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-P128ΔphoP 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.