The Characteristics And Mechanism Of Polymyxin B Resistance In Salmonella Isolates

Salmonella is an important zoonotic pathogen which can seriously do harm to food safety.With the widespread use of antibiotics,the antibiotic resistance of Salmonella is increasing.Polymyxin B has been widely used as the last-line drug in treating infections caused by multi-drug resistant Gram-negative bacteria in humans and animals.In recent years,with the use of polymyxin B increasing,a variety of Gram-negative bacteria such as Salmonella have gradually increased their resistance to polymyxin B.It reduces the effectiveness of polymyxin B and brings great threat to human and animal health.Therefore,a total of 161 Salmonella strains were used and the polymyxin B resistance,serotype and mcr gene screening were analyzed in this study.SNP screening based on Whole genome sequencing,the construction and expression of ept C gene expression vector,polymyxin B induction in vitro and comparative genomics analysis were further performed to explore the mechanism of polymyxin B resistance.The major results are as follows:1.The polymyxin B resistance of 160 food-borne Salmonella strains isolated from Nanchang,Jiangxi Province was determined by microbroth dilution method,of which 92 strains were isolated in 2018 and 68 strains in 2020-2021.The isolates in2018 showed lower resistance to polymyxin B(5.4%),while 14.7% of isolates in2021 were resistant to polymyxin B,indicating the increasement of polymyxin B resistance rate.Using the Salmonella 30-species diagnostic serum kit,160 Salmonella isolates were serotyped into 24 distinct serovars,while 51 strains could not be typed.In the Salmonella strains isolated in 2018,the predominant serotypes were S.Ryssen,S.Stanleyville,S.Essen and S.Wiltevreden.In the Salmonella strains isolated in 2020-2021,the predominant serotypes were S.typhimurium,S.Vetaveriden and S.enteritidis.In total,Salmonella typhimurium showed high resistance rate to polymyxin B(20.0%).2.PCR screening for mcr-1 to mcr-9 was carried out in the 15 polymyxin Bresistant Salmonella strains.Only one S.Infantis strain(isolated in 2018)was identified to be positive for mcr-1,while the left samples did not carry mcr genes.Four mcr-negative and polymyxin B-resistant Salmonella strains were selected for Whole genome sequencing.The SNP mutations on the two-component system genes related to polymyxin resistance were screened.The four sequenced strains shared several common mutations in the same loci of ept C and mic A.Thereafter,the whole sequence of ept C of polymyxin B-resistant strain A7 was amplified by PCR.The pkk223-ept C expression vector was constructed and transferred into DH5α,polymyxin-B-sensitive strain NC28 and polymyxin-resistant strain NC141,respectively.The MIC values of polymyxin B were determined.The MICs of recombinant bacteria were not changed compared with their original strains.3.S.diarizonae is a rare serotype.The mechanism of polymyxin resistance of S.diarizonae has not been reported yet.Polymyxin B was used to induce a S.diarizonae clinical strain S499 in vitro.After 15 rounds of induction,5 mutant strains were selected.All mutant strains showed higher level of resistance to polymyxin B.The polymyxin B MIC values of mutant strains PB2,PB3 and PB4 were 267 times higher than those of wild strain S499.The polymyxin B MIC values of mutant strains PB1 and PB5 were 133 times higher than those of S499.Flow cytometry assay with PI single staining or PI/CFDA double staining was performed.The results indicated that polymyxin B could damage the integrity of cell membrane of S499 wild strain,but its destroy action on the mutant strains was significantly weakened.4.Polymyxin B-induced mutant PB5 was selected for WGS.A large fragment was repeatedly inserted into the chromosome of mutant PB5 for three times.This segment contained NADH panquinone oxidoreductase mediated by IS1 family protein Ins B,transduction protein-related gene pmr D,operon arnarn BCADTEF,synthetic heptose phosphatase gene pmr G and other polymyxin B resistance-related genes.Further analysis showed that PB5 lacked a part of flagellar assembly proteins such as Flg J,Flg I,Flg K and other sequences that need to be replicated.In summary,this study studied polymyxin B resistance of foodborne Salmonella in Nanchang City,Jiangxi Province.Polymyxin B resistance showed an increasing trend.Polymyxin resistance was mainly mediated by SNP mutation of two-component system on chromosomes.In this study,polymyxin B-resistant strains had some common SNP mutations in ept C and mic A genes.SNP mutations in ept C do not lead to polymyxin B resistance.In vitro induction experiments revealed that polymyxin B resistance in S.diarizona was also associated with pho PQ/pmr AB gene reinsertion and high expression.This study provided new data for monitoring of polymyxin resistance and provided new findings for understanding of machnism investigation.