Gene Regulation Study On The Active Efflux Pump AcrAB-TolC In Shigella Spp.

Shigella(called as dysenteriae bacillary) is the main pathogen of shigellosis. The bacillary dysentery shigellosis is still one of the acute intestines contagions. since 1960s Shigella bacteria have got universal resistance to traditional antibiotics such as tetracycline.Since 1980s,quinolones,in particular the fluoroquinolone drugs, for their broad antibacterial spectrum,unique mechanism,excellent pharmacokinetic characteristics and good antibacterial ability,became the preferred drugs in treatments to Shigellosis.However,with the use and abuse of these medicines,there was a rapid increase in bacterial resistance,which directly reduced the clinical effect, increased treatment costs,reduced the application cycle of the new drugs and increased research and development costs.Therefore,it's urgent to research the antibiotic resistance mechanisms and develop new antibacterial drugs to stop or slow the drug resistance.Recent studies showed that some bacteria have the ability to pump a variety of antibacterial drugs of different structures out of the cells initiatively to get resistance. More research is about Escherichia coli AcrAB-TolC multiple drug efflux pump.It has been confirmed that the pump also exists in the Shigella bacteria and plays an important role in the Shigella multi-drug resistance.The mechanism is mediated by the chromosome in a regulatory loci under the control of multiple genes collaboration phenotype,and its related loci on chromosome mutation may lead to the increase of multi-drug resistant pump protein expression,which will pump different structure drugs out of the membrane,induced multiple drug resistance.The AcrAB-TolC system is regulated by a variety of regulatory factors,mainly by AcrR inhibitor and MarRAB operon.It is reported that AcrR inhibit the transcription of acrAB.The structure of MarRAB operon including:①marO;②marR;③marA;④marB.It has been found that mutations in marO or marR,may activated ACrAB-TolC system,and then induced multi-drug resistance.The study also has found that mar mutation concentrated in the region of marOR,but has not found a mutation hotspot in this region.It seems that any mutation in this region may reduce the sensitivity to antibiotics,and what is similar to other resistance mutations is that the accumulation of point mutations and greater loss at gene segments would lead to a high degree of bacterial resistance.Our study on the distribution and expression of active effiux pump acrAB in clinical isolates of Shigella found that the acrA gene mRNA levels in Mar strains were significantly higher than those in the sensitive strains,but there was no significant difference between the mutant and unmutant strains,suggesting that the acrAB-tolC gene mutation does not affect its own expression,and may be regulated by the MarRAB operon and AcrR inhibitor.In view of this,we studied the gene regulation of active effiux pump AcrAB-TolC in Shigella spp.,to understand the role of the upstream regulatory genes and hope to find common mutations related to the multi-drug resistance.We collected 54 clinical Shigella strains;,susceptibility tests of antibiotic and organic solvent tolerance were performed,acrR,marOR genes were amplified by polymerase chain reaction(PCR) respectively,the PCR products were digested by restriction endonuclease TaqI,and were analyzed by single strand conformation polymorphism(SSCP) and finally were sequenced.Methods1.Identification of the bacteria strains:There are 54 Shigella spp.strains preserved in semi-solid agar at 4℃refrigeratory.We inoculated them into LB solid culture medium and identified them with biochemical and serological methods over again to make sure the reliability of the strains.2.Filtration the Mar strains and senstive strains:Susceptibility tests used Kinby-Bauer agar disk diffusion method,According to the guidelines formulated by the United States National Committee for Clinical Laboratory Standards(NCCLS). Five antibiotics were tetracycline,chloramphenicol,ampicillin,ciprofloxacin and norfloxacin.The strains which were sensitive to all the five kinds of antibiotics can be called senstive strains,the ones which were resistant to the two or more different types of antibiotics can be called multi-drug resistant strains(Mar),the rest were the single-drug-resistant strains.3.Organic solvent tolerance(OST):Mid-logarithmic-phase cultures(A530,0.4 to 0.5) grown in LB broth were diluted by phosphate-buffered saline(PBS) to approximately 10~6 to 10~7 cells/ml.Five microliters was spotted onto LB agar and allowed to dry.The surface of the agar was covered with either hexane,cyclohexane or a mixture of hexane and cyclohexane(3:1,1:1,or 1:3[vol/vol]) to a depth of 2 to 3 mm.The plates were incubated at 30℃in a closed container to prevent evaporation of the solvent.After 24 to 36 h,the spots were scored for confluent growth,which demonstrated tolerance to the solvents.Tests were repeated three times.4.PCR-SSCP analysis of acrR,marOR genes:The two genes were amplified by PCR,the products of which were digested with restriction endonuclease TaqI.Then SSCP analysis was performed.It can detect a single nucleotide change and the loss or insertion of oligonucleotide by electrophoresis.5.Sequence analysis of the PCR products of AcrR,marOR gene mutant strains were performed.Results1.Identification of the bacteria strains:A total of 54 clinical isolates were resuscitated.There were 41 Shigella flexneri strains,10 Shigella sonnei strains,and 3 Shigella dysenteriae strains.2.Filtration the Mar strains and sensitive strains:According to the susceptibility test results,we got 35 Mar strains,11 sensitive strains and 8 single-drug-resistant strains.The MDR rate is 64.8%.3.Organic solvent tolerance(OST):38 of 54 strains tolerated to organic solvents,the total rate of OST is 70.37%,in which 33 were Mar strains,3 were single-drug-resistant strains,and only 2 were sensitive strains.The OST rate in Mar Group is 94.29%,significantly higher than that in the non-multiple-drug-resistant strains group(26.32%),the difference was statistically significant(P＜0.05).4.PCR of acrR and marOR:Every strain had been amplified two segments of 564 bp and 604bp,which were the products of acrR and marOR.The lack of the two genes was not found.5.SSCP analysis of acrR and marOR:(1) acrR gene:The same single-strand conformation in all Mar strains and sensitive strains was found,mutant strains were not found.(2) marOR gene:There were five in all Mar strains mutant in marOR gene.The mutation rate was14.29%.6.DNA sequence analysis of PCR products of acrR and marOR gene:(1) acrR gene:Mutation in the acrR gene in 3 Mar and sensitive strains were not found.(2) marOR gene:Compared with sensitive strain Z10 and reference strain S51520,5 mutant Mar strains(H6,H12,N10,N13,198) all had one deletion mutation which was the 1376-1379(CATT) four base deletions in marO gene,and there was also found two points mutations in 3 Mar strains.Conclusions1.The study found that the rate of the multi-drug resistance was high,and the tolerance to organic solvents in Mar strains was much higher than that in other strains.2.acrR,marOR genes were amplified by PCR in the Shigella spp.,the lack of the two genes was not found,which confirmed that the regelatory genes acrR,marOR of AcrAB-TolC system were widely found in the Shigella spp..3.The acrR gene sequence in Shigella Mar strains were found consistent with that in the reference and sensitive strains,which showed that the gene may be not related to Shigella multi-drug resistance. 4.The 1376-1379 base deletion in marOR gene was found only in Shigella Mar strains and wasn't found in reference and sensitive strains for the first time,may be it is related to multi-drug resistant in Shigella spp..