| Objective:Benzalkonium bromide is widely used in many fields like medical industry,animal husbandry and veterinary industry,food processing industry and other industrial environments.However,resistant bacterial to Benzalkonium bromide have been observed in many studies.In this study,water-borne Enterococcus from some rivers and pig farms in fujian province were isolated to identified the resistant character to Benzalkonium bromide and other antibiotics;the expression of some virulence factors related genes of Enterococcus were detected aim at understanding the role of those genes in the forming of biofilm resistant character to Benzalkonium bromide;meanwhile,model biological systems were established to determine the effect of Benzalkonium bromide on the resistant character of Enterococcus to different antibiotics,the role of quorum-sensing system in the form of resistant Enterococcus was studied as well.Method:(1)Enterococcus selective medium and color medium were used to clinical isolation and identification of the water-borne Enterococcus.Broth micro-dilution method was used to detect MIC value of the isolated strains.The PCR method was used to detect drug resistance genes vanA,vanB,vanC,VanC2/3,gryA,tetM,tetL,tetS,mefA,ermB and msrC and quaterna-salt disinfection resistance genes qacA/B,qacC,qacG,qacE?1 and qacJ.Multiple PCR techniques were used to identify the species of Enterococcus.(2)There were five sets of benzalkonium bromide concentration group,which were 1000 ng/mL,500 ng/mL,100 ng/mL,10 ng/mL and 0 ng/mL.The cells were adhered to Enterococcus with the growth logarithmic metaphase(3.5 h),late logarithmic phase(5 h)and stable period(7 h).A fluorescence quantitative PCR method was used to determine the relative expression of the associated virulence factors in each period.(3)Model ecosystems have been established.5 benzalkonium bromide concentrations were set.Water samples were collected regularly for drug susceptibility testing and resistance genes detection.Fluorescence quantitative PCR was used to detect the relative expression of mRNA in the fsr quorum sensing system and cyl quorum sensing system in different concentrations in model ecosystems.Result:(1)There were 75 strains of Enterococcus isolated from water samples collected from some rivers and pig farms in Fujian Province,and the isolation rate was 71.43%.Including Enterococcus faecalis,31 strains(41.33%)and Enterococcus faecium 11(14.67%).Drug sensitivity test showed that the resistance rate of 75 Enterococci to erythromycin was the highest,which was 81.33%(56/75),followed by tetracycline,74.67%(56/75).It is most sensitive to ampicillin and vancomycin,and the resistance rate is only 4%(3/75)and 2.67%(2/75).The MIC value of 22.67%(17/75)isolates to benzalkonium bromide was higher than that of standard bacteria.The highest value is 8 times that of the MIC standard bacteria.The multidrug resistance rate of clinical isolates was 74.67%.Most of them showed resistance to 4 or 5 antibiotics,and the highest reached 6 kinds of drug resistance.9 kinds of resistance genes were detected by PCR resultsThe highest detection rate of gyrA gene was 93.33%,followed by tetL,and the detection rate was 80%.The resistant phenotype is higher than that of the susceptible strain.The identification results of different Enterococci showed that Enterococcus faecalis was superior to Enterococcus faecium,accounting for 41.33%.(2)Cell adhesion experimental results show that the adhesion of Enterococci to host cells was different at different growth stages.The adhesion ability of the logarithmic phase is higher than that in the stable period.The adhesion of the low concentration group was higher than that in the high concentration group.The up regulation effect of low concentration(10 ng/mL)of benzalkonium bromide on pili related genes ebpA,ebpB,ebpC and strA was mainly in the middle growth stage of Enterococcus.The stability period of the higher concentration group has greater influence on the ebpR.Benzalkonium bromide significantly inhibited the expression of atn genes in the middle stage of logarithmic growth.The expression of gelE gene is mainly in the late stage,and the lower benzalkonium bromide concentration can significantly improve its expression.(100?1000 ng/mL),Benzalkonium bromide can inhibit the expression of cylA gene at logarithmic growth stage.In the middle stage of logarithmic growth,the expression of mRNA in ace increased correspondingly with the increase of benzalkonium bromide concentration(100?1000 ng/mL).The opposite of the late logarithmic period.(3)In microecosystem,when the benzalkonium bromide treated for seventh days,the MIC value of Enterococci to 8 drugs increased significantly.Resistance to VAN,CHL,BB,CIP,ERY and TET were also observed,but resistance genes such as tetL and ermB were detected on the first dayfsrA,fsrB andfsrC genes were hardly expressed,but the expression ofgelE and sprE in the downstream was significantly promoted,especially in the seventh and twenty-eighth days.But the expression of gelE and sprE increased significantly,and the expression peak appeared on the seventh day and the twenty-eighth day.The expression level of cylLL and cylLs is much higher than that of other cyl related genes.The expression level of cylR1 and cylR2 on the first 21 days was similar,and the latter was significantly higher than that of the former on the 28 day.The expression peaks of other cyl genes all appeared at 28 days,and the high concentration of benzalkonium bromide(>200 ng/mL)promoted and low concentration(less than 20 ng/mL)showed significant inhibition in the later period.Conclusion:(1)In some areas of Fujian,the isolation rate of Enterococcus was high and multiple drug resistance was serious.Most of the isolates carry multiple resistance gene's to antibiotics.On the whole,the isolated strains were sensitive to benzalkonium bromide and there were only low tolerance level.Enterococcus faecalis was the predominant bacterium which have higher resistance rate than that of Enterococcus faecium.(2)Benzalkonium bromide significantly increased the adhesion ability of Enterococci to host cell IPEC-J2 cells in the middle of logarithmic growth.Low-concentration benzalkonium bromide's ability to adhere to host cells is related to fimbriae production.High concentration of benzalkonium bromide mainly stimulates the high expression of adhesion gene ace thus affecting the adherence of host cells.Benzalkonium bromide can inhibit the selective expression of virulence factors such as gelE and rnjB genes to some extent.(3)The subinhibitory concentration of benzalkonium bromide can increase the MIC value of Enterococci to antibiotics and produce drug resistance;Benzalkonium bromide inhibited fsr quorum sensing related genes fsrA,fsrB and fsrC completely.High concentration of benzalkonium bromide M(?200 ng/mL)significantly promoted the expression of gelE,sprE and cyl related genes in quorum sensing system.Low concentrations(?20 ng/mL)have inhibitory effects after 21 days.There are some links between antibiotic resistance caused by benzalkonium bromide and the quorum sensing system. |
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