Relation Of AcrAB-tolC Efflux Pump And MarOR Regulatory Gene Mutation With Antimicrobial Resistance In Shigella.spp

With the application of antibiotics, the antibiotic resistance in shigella.spp is increasingly widespread, followed by the difficultion of prevention and cure in bacillary dysentery. It has been certified that the antibiotic resistance mechanism involves many aspects including the initiative excretion pumps. Our study on acrAB-tolC gene showed that the acrA gene mRNA levels in antibiotic resistance strains were significantly higher than those in the sensitive strains, but there was no significant difference between the mutant and unmutant strains. Our study on marOR gene showed that there were four basic loss of the gene in multi-antibiotic resistant strains, suggesting that this mutation maybe cause the increase of the pumps, further lead to the antibiotic resistance.ObjectiveAccording to the results above, this study researched the influence of marOR gene mutation to the transcription of AcrAB-TolC gene and its relation with multi-antibiotic resistance in shigella.Methods1. Identification of the bacteria strains:clinical strains were resuscitated. Biochemistry and serology methods were carried out to identify the bacteria strains.2. Antibiotic susceptibility test:Susceptibility tests used Kinby-Bauer paper agar diffusion method. ATCC25922 was used as control.3. Organic solvent tolerance:If the spots were scored for confluent growth in a misture of hexane and xylene(vol/vol=3:1), it demonstrated toleranceto the solvents.4. PCR-SSCP analysis of marOR, acrA, acrB and tolC genes:Amplified the four genes by PCR, then digested the products with restriction endonuclease. SSCP analysis was performed. The strains which appeared different single strand conformation or number can be called as mutative strains.5. Sequence analysis:According to the results of SSCP, we selected 11 mutative strains, one reference strain and sensitive strains to sequence.6. The transcriptional level analysis of the acrA, acrB and tolC genes:Total RNA was abstracted with TRIzol. Synthesize the first strand in accord to the reverse transcription kit instruction, then amplified the target genes by PCR. Analyze the transcriptional levels of 16sRNA, acrA, acrB and tolC genes with gel image analytical system.7. Preparation of the clone of four-basic deletion and the clone of four-basic deletion and three point mutations:The marOR gene having four-basic deletion was amplified by overlap PCR, then connected and transformed into DH5a. The clone of four-basic deletion and three point mutations were prepared from the strain having these mutations. Sequencing was preformed to certify.8. Compare of the antibiotic resistance:Susceptibility tests were preformed in these clones. Compare the results.Results1. Antibiotic susceptibility test of Shigella:According to the results, we got 4 sensitive strains,18 single-antibiotic resistant strains and 137 multi-antibiotic resistant strains. The multi antibiotic resistant rate is 86.2%.2. Organic solvent tolerance:122 of 159 strains tolerated to organic solvents. And the organic solvent tolerance rate is 76.7%.3.. PCR-SSCP analysis of marOR, acrA, acrB and tolC genes:SSCP showed that the mutation rates of the marOR, acrA, acrB and tolC genes were 17.4%,5.8%,3.9%, 2.6%, respectively. And the four sensitive strains had not mutation.4. Sequence analysis:Compared with sensitive strain Z10 and reference strain S51522,8 mutation strains all had the 1376-1379 four base deletions and three point mutations in marO gene. Strain 200011 had the 1381-1384 four base deletions and many point mutations including the three point mutations described above, and it was 86% similar to the sensitive strain. Strain Z23 had five point mutations and strain Z24 had two point mutations. 5. The transcriptional level analysis of the acrA, acrB and tolC genes:The transcriptional levels of the acrA, acrB and tolC genes in mutation strains were higher than those in the non-mutation strains in according to the results of gel image analytical system. P<0.05 and there was statistical significance.6. Prepare the clone of matOR genes with mutations:The sequence results certified we had gotten these clones successfully.7. The antibiotic resistance of the clone of four-basic deletion and the clone of four-basic deletion and three point mutations:According to the rule formulated in this study that it was different in resistance, if the scope of different diameters exceeded five millimeters, the antibiotic resistance of the clone of four-basic deletion to streptomycin, tobramycin, cefazolin and cefalexin was different with that of DH5a (T), and the antibiotic resistance of the clone of four-basic deletion and three point mutations to streptomycin and to tetracycline was different with that of DH5a (T). The diameters of streptomycin, tetracycline, chloramphenicol, cefazolin, levofloxacin, ciprofloxacin and norfloxacin to (marOR-CATT) and to DH5a (marOR-CATT+3m) were shorter than that to DH5a (marOR). The diameters of the antibiotics except the trimethoprim between DH5a (marOR-CATT) and DH5a (marOR-CATT+3m) had not significant disparity.Conclusions1. The mutation rate of marOR gene in resistant Shigella was high. In 11 antibiotic resistent Shigella strains sequenced, there are 8 strains whose marOR genes had the 1376-1379 four base deletions and three point mutations which were absence in the sensitive strains and the reference strain.2. The trancriptive level of the acrA, acrB and tolC genes in the strains which had the mutations in the marOR gene were significantly higher than non-mutation strains.3. The four-basic deletion in 1376-1379 sites of the marOR gene increased the resistance of Shigella spp to some antibiotics.4. The point mutations in 1411,1417,1435 sites of the marOR gene have little influence on the antibiotic resistance of Shigella spp.