Study On The Resistance Mechanism Of AcrAB-TolC Efflux System In Escherichia Coli That Was Tolerdined On Oxytetracycline And Benzalkonium Bromide

As you know antimicrobial agents are very important to treat bacteriosis,but as antimicrobial agents were abused,drug-resistant bacteria has appeared.In order to avoid bacterial infection, disinfectants are widely used in hospitals, communities, and livestock farms.With the widespread irrational use of disinfectants, the sensitivity of bacteria to the disinfectants was gradual reduced.lt has been reported that bacteria could tolerate to disinfectants when it was treated by sub-bactericidal concentrations for long time.Our previous study on more than 100 clinical isolate strains suggested that bacteria that was tolerdined on disinfectants was also not sensitive to antimicrobial agents.And drug-resistant bacteria was also tolerated to disinfectants.As antimicrobial agents and disinfectants were abused, superbugs that resistant to antimicrobial agents and disinfectants have appeared in nature.And these bacterias are always not sensitivity to more than 2 kinds of antimicrobial agents.It means that many kinds of antimicrobial agents and disinfectants will do not work on them.And these bacterias may spread all over the world.When humen and animals infect with them it will be very difficult to treat.So study the cross resistance mechanisms is very important and nessary.But there is none report of the cross resistance mechanisms between the antimicrobial agents and disinfectants. In order to explore the cross resistance mechanism and provide the theory basis for rational use of antimicrobial agents and disinfectants the study was carried out.1.The cross resistant strains were getted by inducting Escherichia coli control strain ATCC25922 in vitroMethod:The MIC(minimal inhibitory concentration) values for Oxytetracycline or Benzalkonium Bromide for Escherichia coli control strain ATCC25922 were determined using standard broth microdilution method.And then Escherichia coli control strain ATCC25922 was induced on some selection steps with increasing gradient concentrations of Oxytetracycline(Oxy) or Benzalkonium Bromide(Ben).The lowest concentration is 1/2×MIC value for Oxytetracycline(Oxy) or Benzalkonium Bromide(Ben).And the highest concentration is 128×MIC value for Oxytetracycline(Oxy) or Benzalkonium Bromide(Ben).The MIC values for Oxytetracycline or Benzalkonium Bromide for the strains were determined after inducing by every concentration.Result:Resistance strains A(Oxy) and A(Ben) that were tolerdined on Oxytetracycline(Oxy) and Benzalkonium Bromide(Ben) were getted.The MIC values for Oxytetracycline for A(Oxy) and A(Ben) is 320 and 250 times higher than control strain ATCC25922, respectively.And the MIC values for Benzalkonium Bromide for the two strains is 18 times higher than control strain ATCC25922.Conclusion:It suggested that both Oxytetracycline and Benzalkonium Bromide inducing in vitro could lead to Escherichia coli control strain become resistant strain,and this strain was a cross resistant strain. 2,The study of AcrAB-ToIC function in cross resistance strainsMethod:The mutation of mar A、soxS、rob、acrA、acrB and tolC of A(Oxy) and A(Ben) were determined.The mRNA expression quantity of marA、soxS、rob、acrA、 acrB and tolC of A(Oxy) and A(Ben) were determined by using real-time quantitative reverse transcription-PCR method.To study the effect of mar A on AcrAB-TolC,A(Oxy) and A(Ben) were prepared into competent cell and named them Ac(Oxy) and Ac(Ben),and used siRNA to interfer the mRNA expression quantity of mar A. The Ac(Oxy) and Ac(Ben) were incubated in the LB medium and four different concentrations(1/4×MIC×1/2×MIC、1×MIC and 2×MIC) of Oxytetracycline or Benzalkonium Bromide was added into the LB medium,respectively.The siRNA was added into the LB medium at 6 different time points.Then determined the OD595 value of the Ac(Oxy) and Ac(Ben) by using ultra-violet and visible spectrophotometer every 2 hours during the first 8 hours.And the OD595 value was determined at the 18th and 20th hours.The mar A gene was cloned in Escherichia coli Top 10 and detected the MIC values for Oxytetracycline and Benzalkonium Bromide for the cloned strain. Then through studying the change of MIC values for Oxytetracycline and Benzalkonium Bromide to know the fuction of AcrAB-TolC in the cross resistance of Escherichia coli.Result:All the genes mar A、soxS、rob、acrA、acrB and tolC of A(Oxy) and A(Ben) occurred mutation.and the amino acid that transcribed by these genes also occurred mutation. Some mutation occurred at the same base site of the A(Oxy) and A(Ben)’ marA、soxS、rob、acrA、acrB and tolC genes.And some mutation occurred at the same amino acid site of the A(Oxy) and A(Ben)’amino acid that transcribed by these genes except rob and acrA.The expression quantity of mar A、acrA、acrB、tolC of A(Oxy) and A(Ben) were significantly higher than the control strain ATCC25922,while soxS and rob of A(Oxy) and A(Ben) were lower than the control strain ATCC25922.The OD values of experimental groups that were added Oxytetracycline that concentration was 1/2×MIC were significantly lower than negative control at the 2th、4th、6th、and 8th hours. The OD values of experimental groups that were added Oxytetracycline that concentration was 1/4×MIC were not significantly lower than negative control. The OD values of experimental groups that were added Benzalkonium Bromide that concentration was 1/4×MIC and 1/2XMIC were not significantly lower than negative control,too.There were no bacterias growth in the experimental groups that were added Oxytetracycline or Benzalkonium Bromide that concentration was 1×MIC and higher than 1×MICThe MIC values for Oxytetracycline and Benzalkonium Bromide for the strain Arctic Express-MarA overexpressing mar A is 64 and 4 times higher than the strain Arctic Express,respectively.Conclusion:It suggested that the mutation and change of expression quantity of mar A、soxS、rob、acrA、acrB and tolC may lead the two strains A(Oxy)and A(Ben) overexpress AcrAB-TolC,and because of the overexpress of AcrAB-TolC of A(Oxy) and A(Ben),the sensitivity of the two strains to Oxytetracycline and Benzalkonium Bromide were reduced then became cross resistant strains. It is difficult not to feel, AcrAB-TolC is very important for the drug-resistant of Escherichia coli and marA is an important regulon of acrA、acrB and tolC.The above results show that bacteria could become drug-resistant bacteria induced by antibacterial agents and disinfectant.And the drug-resistant bacteria also could produce cross resistance to antimicrobial agents and disinfectants and the cross resistance mechanism has high correlation to efflux pump.In order to reduce the bacterial cross resistance, we should correctly use the antimicrobial agents and disinfectants.