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High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Published on: August 11, 2014
Mechanisms of polymyxin resistance induced by Salmonella typhimurium in vitro
1Pharmacology and Toxicology Laboratory, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230026, PR China; Anhui Province Key Lab of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
Abstract:
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.
Insights
Multi-drug resistant Salmonella develops resistance to polymyxin antibiotics. The PhoPQ system is crucial for this resistance, impacting Salmonella
Area of Science:
- Microbiology
- Genetics
- Antimicrobial Resistance
Background:
- Multi-drug resistant (MDR) Salmonella poses a significant global health threat.
- Polymyxin antibiotics are regaining importance, yet resistance mechanisms in Gram-negative bacteria, including Salmonella, remain incompletely understood.
Purpose of the Study:
- To investigate the molecular mechanisms underlying polymyxin resistance in Salmonella typhimurium.
- To characterize phenotypic and genomic changes associated with induced polymyxin resistance.
Main Methods:
- Induced a polymyxin-resistant Salmonella typhimurium strain (AT-P128) from a susceptible parent strain (AT).
- Utilized broth dilution for drug sensitivity testing, whole genome sequencing (WGS) for SNP identification, and transcriptome sequencing (RNA-seq) with RT-qPCR for gene expression analysis.
- Employed CRISPR/Cas9 gene editing to create a phoP deletion mutant (AT-P128ΔphoP).
Main Results:
- The resistant strain AT-P128 exhibited cross-resistance to several antibiotics but reduced resistance to sulfamethoxazole, with no significant change in growth rate.
- Motility and biofilm formation were significantly decreased in AT-P128.
- WGS revealed mutations in 9 genes, while RNA-seq and RT-qPCR indicated increased expression of the phoPQ operon. Deletion of phoP reduced polymyxin resistance by 32-fold.
Conclusions:
- Polymyxin resistance in Salmonella is associated with alterations in cellular biology, genomic composition, and gene expression.
- The PhoPQ two-component system plays a critical role in mediating polymyxin resistance in Salmonella.
- This study provides valuable insights into the complex mechanisms of polymyxin resistance in Salmonella.
