Role Of VirSR-VirAB In Antimicrobial Resistance And Biofilm Formation Of Listeria Monocytogenes

Two-component system(TCS)which consists of histidine kinase and response regulator is an important mechanism to sense and adapt to the external environment for bacteria.Listeria monocytogenes,a food-borne pathogen,contains 15 complete TCSs and an orphan response regulator.Our previous study found that the ATP-binding cassette(ABC)transporter VirAB is required for VirS to sense due to the lack of the extracellular sensing domain in VirS,which constitutes a complex TCS/ABC transporter system.The aims of this study were to investigate the role of VirSR-VirAB system in antimicrobial resistance and biofilm formation of L.monocytogenes.The virSR-virAB gene deletion mutant strains were constructed and minimum inhibitory concentration(MIC)of antimicrobial agents for mutants and wild type was detected;biofilm formation ability of wild strain EGD-e and virSR-virAB gene deletion mutant was measured by microtiter plate,confocal laser scanning microscopy and scanning electron microscopy;the role of VirSR-VirAB in resistance to phenyllaetie acid(PLA)and the effect of PLA on the formation of L.monocytogenes biofilm were also investigated.Results are shown as follows:(1)Under cefotaxime stress,the gene transcription level of VirSR-VirAB in the wild strain EGD-e increased significantly,indicating that VirSR-VirAB may mediate L.monocytogenes resistance to cephalosporins.We used homologous recombination technology to construct gene-deficient strains AvirS,?virR,?virSR,?virA,?virB and ?virAB.Compared with the wild strains,the MIC values of these deleted strains to cephalosporin antibacterial drugs were significantly reduced,which proved the VirSR-VirAB system is involved in the resistance of L.monocytogenes to cephalosporins.Compared with the wild strains,the MICs of all deleted strains to nisin and ethidium bromide also decreased,indicating that VirSR-VirAB is also involved in the resistance of L.monocytogenes to nisin and ethidium bromide.Under the action of BC,the gene-deficient strains ?virS,?virR,?virSR,?virA,?virB and ?virAB showed growth defects,indicating that the VirSR-VirAB system is involved in the resistance of L.monocytogenes to benzalkonium chloride.(2)The results of microplate quantitative detection,laser confocal microscopy and scanning electron microscopy showed that virS and virAB are involved in the formation of L.monocytogenes biofilm,while virR has nothing to do with the formation of Lm biofilm.The adhesion of the mutant ?virS and AvirAB decreased significantly,while the adhesion of ?virR did not change significantly.Compared with the wild strain,the motility of the mutant strains ?virR,?virS and ?virAB has no significant difference,and the transcription levels of the motility-related genes motA and motB in the mutant strains have no significant changes.(3)Under PLA stress,the gene expression levels of virSR and virAB did not change significantly;the wild strain EGD-e and gene deletion mutant strains ?virS,?virR,?virSR,?virA,?virB and ?virAB all had MIC values for PLA of 6 mg/ml;Under PLA stress,the wild strain EGD-e has increased MIC values for ampicillin,cefotaxime,cephalothin,ceftazidime,cefepime,kanamycin,and Ethidium bromide.The MIC value of ciprofloxacin,tetracycline,chloramphenicol and nisin was unchanged.PLA stress had an uncertain effect on Lm resistance.PLA with sub-inhibitory concentration has no effect on the formation of L.monocytogenes biofilm,and PLA with inhibitory concentration can effectively remove the formed biofilm.In addition,the presence of PLA does not affect L.monocytogenes motility.