Reseach On The Mechanism Of Colistin Resistance By PmrB Mutations In Salmonella Enterica From Poultry

Salmonella enterica is an important zoonotic agent and also a foodborne pathogenic bacterium. Colistin is one of polycationic antimicrobial peptides and currently regarded asthe last-resort antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. The emergence and spread of Colistin-resistant Salmonella enterica is a great threat to multidrug-resistant Gram-negativebacterial infections. Studies showed that Pmr A/PmrB two-component regulatory system playan important role for the resistance of Salmonella entericato Colistin. Different MICs of S. enteritidis to colistin were sucessfully induced and Pmr A/PmrB genes were sequenced and blasted, the results showed that some mutations were observed in PmrB genesand no mutations in pmr A genes. On the basis of this result, we usedred homologous recombination and I-sce ? enzyme to construct the corresponding markerless PmrB mutant strains. Minimum inhibitory concentration(MIC) of PmrB mutant strains to colistinwas determined by broth microdilutionmethod.Real-time q PCR was performed to detect the changes of expression levels in related genes with colistin resistance. This study will be helpful to understand the regulatory mechanism of colistin resistance by Pmr A/PmrB in S. enteritidis from poultry.The 2-12(32) was induced by a serial passage experiment using colistin, and the 2-12(512) was isolated. Then the whole genome of 2-12 and 2-12(512) was sequenced by high-throughput sequencing technologies.Five specific proteins of 2-12 and nine specific proteins of 2-12(512) were found using homologous alignment under the sequence of genome. Thirty SNP sites were different between 2-12 and 2-12(512) according to the Single nucleotide polymorphism(SNP) analysis.Under the help of p KD46-rha-IS plasmid expression Red homologous recombination system and I-sce ? enzymes,Salmonella enteritidiswith PmrB partial gene deletions or point mutations was constructed using targeting chip 197-cat-sacB, 249-cat-sacB.MIC valuesof PmrB mutantstrains to 13 antimicrobial agents(including colistin) were determinated,MIC value of mutant strains to colistin was 16 ?g/m L, which iseight times higher than the susceptible strain. There was no obvious difference between2-12?PIA and susceptible strain.Compared with the susceptible strain, MIC of 2-12 D149 N to gentamicin(GM) was increased by 160 times, whilespectinomycin(SPT) reduced 64 times,no obvious difference was observed to the rest antimicrobials.Real-time q PCR was used to detect the changes inm RNA expression levels of Pmr A, PmrB, Pmr C, Pmr E, and Pmr H genesfor 2-12,2-12?PIA,2-12 D149 N,2-12(32),2-12(512), GD1-13 and GD1-14.The results showed the PmrB mutants promote the expression of relation genes Pmr A, PmrB, Pmr C, Pmr E, and Pmr Hsignificantly.