Relation of acrAB-tolC Efflux Pump and marOR Regulatory Gene Mutation with Antimicrobial Resistance in Shigella.spp
|Course||Epidemiology and Biostatistics,|
|Keywords||Shigella MDR- mar0R gene the acrAB a tolC initiative outside pump genes|
With the application of antibiotics, drug resistance is increasingly common in Shigella bring some difficulties to the prevention and treatment of bacterial dysentery. Studies have shown that bacterial resistance mechanisms involve a number of aspects, including active efflux pumps may play a more important role. Our previous studies have demonstrated that the efflux pump AcrAB-Tolc its regulatory genes marOR of Shigella. Study of the acrAB-tolc pump gene, multidrug-resistant strains of acrA mRNA levels were significantly higher than the sensitive strains, but no statistically significant differences in mRNA levels acrAB-tolC gene mutant and non-mutant; in 4 bp deletion in 1376-1379 of the gene loci exist and not exist Yu Zhi Shigella multidrug-resistant strains in the sensitive strains and reference strains of the regulatory genes marOR the study, which speculated that the gene mutation may cause increased efflux pump expression levels, thereby affecting the resistance of Shigella. Purposes on the basis of the preliminary studies, this project aims to research the the Shigella marOR, acrA, acrB and tolC gene mutations, regulation the gene marOR the mutation on the AcrAB-Tolc active efflux pump expression and bacterial resistance relationship. Method 1 strain was identified: LB plates crossed complex Su Zhi He strains and re serological and biochemical identification. Drug sensitivity test: Kinby-Bauer disc agar diffusion method, quality control bacteria as ATCC 25922. Organic solvent tolerance test: the proportion of n-hexane and cyclohexane strains grown in the mixed solution of 3:1 considered organic solvent tolerance strains. 4. Shigella marOR, acrA, acrB and tolC gene single-strand conformation polymorphism (PCR-SSCP) analysis: PCR amplification marOR, acrA, acrB and tolC genes, and polyacrylamide gel electrophoresis, stained with silver nitrate observed single strand conformation polymorphism and photographed. The nucleic acid sequence analysis: According to the results of SSCP analysis, 11 mutants and a sensitive strain, a reference strain marOR gene sequencing. 6. Shigella acrAB-tolC gene transcription level analysis: using TRIzol extracted total RNA Shigella, in accordance with the instructions of the reverse transcription kit first strand synthesis, PCR amplified fragment was gel imaging analysis the system detects marOR mutation group with unmutated group acrAB-tolC relative content. 7. Of Shigella strains 4 base deletion marOR cloning four bases deletion the joint three point mutations marOR cloning: using overlap extension the PCR amplification 4 base deletion marOR gene, and the T vector clones directly into DH5a, four base deletion clones; 4 base deletion mutation United three points with the mutant strain marOR gene was amplified the DH5a proceeds into T vector. Finally, the nucleic acid sequence analysis confirmed. Gene mutations of marOR on bacterial resistance: detection DH5a, into T vector DH5a, DH5a clones complete marOR genes, four base deletion DH5a clones and four base deletion United 3 DH5a clones point mutations resistant to many antibiotics and compared. Results 1. Shigella strains resistant detection: results of susceptibility testing of of 159 Shigella clinical isolates of the TE, C, AM, GM, NOR and SMZ-TMP six drugs, sensitive strains of four resistance single drug strains 18 strains of MDR-137, MDR-rate of 86.2%. 2. Organic solvent tolerance test: 122 pairs of organic solvent tolerance, tolerance was 76.7% of the 159 Shigella. Single drug-resistant strains of the organic solvent tolerance was 83.3%, and the multi-drug-resistant strains of the organic solvent tolerance was 75.9%. 3. Shigella marOR, acrA, acrB and tolC gene single-strand conformation polymorphism (PCR-SSCP) analysis: 159 Shigella clinical isolates, four sensitive strains did not find the marOR, acrA, acrB and tolC genetic mutation; 155 resistant strains, marOR gene mutation rate was 17.4%; the acrA gene mutation rate was 5.8%; the acrB gene mutation rate was 3.9%; the tolC gene mutation was 2.6%. The nucleic acid sequence analysis: the reference strains, sensitive strains (Z10) and of 11 marOR Mutant sequencing showed that, compared with sensitive strains and reference strains, there are four bases 1376-1379 loci eight mutants (CATT) missing and three point mutations; 3 resistant strains, strains 200011 exist the 1381-1384 sites four bases (ATTT), deletions and multiple point mutations, including more than three point mutations (with sensitive the matching degree of only 86% of the strains), strains Z23 exist five point mutations, strains Z24 exist two point mutations. AcrAB-tolC, Shigella analysis of the level of gene transcription: gel imaging analysis showed that the the mutation group acrA, acrB tolC expression relative content was significantly higher than the unmutated group acrA acrB tolC the relative content is, P lt; 0.05 The difference was statistically significant. The 6. MarOR mutation cloning: After PCR amplification, cloning and nucleic acid sequencing confirmed, has been successfully cloned empty vector, no mutations marOR gene 4 bp deletion mutation marOR gene and four base deletion joint 3 point mutations Cloning marOR, denoted as a in DH5a (T), DH5a (marOR,), of DH5a (marOR-ACTT), DH5a (marOR-ACTT 3m). 7. MarOR mutant bacterial resistance: a difference of more than 5mm difference for antibiotic resistance zone diameters: DH5a (T), DH5a (marOR-ACTT) compare streptomycin, tobramycin Pioneer V, cephalexin increased resistance DH5a (marOR ACTT 3m) of streptomycin and tetracycline resistance increased; DH5a (marOR) comparison, of DH5a (marOR-ACTT) and DH5a (marOR-ACTT 3m) of streptomycin, tetracycline, chloramphenicol, Pioneer V, levofloxacin, ciprofloxacin and norfloxacin increased resistance; DH5a (marOR-CATT) and DH5aT (marOR-CATT 3m) phase zone diameters with antibiotics than experimental trimethoprim no significant difference. Conclusion 1. Shigella resistant strains higher rate of marOR gene mutations. 4 bp deletion, and three point mutations in the detected 11 Shigella multi-drug resistant strains, 8 marOR genes at 1376-1379, while the deletion mutants and point mutation is not present in the sensitive strain and the reference strains. 2. MarOR gene mutations in Shigella acrAB-tolC gene transcription level was significantly higher than the non-mutated strain. Gene at 1376-1379 marOR four base deletion, Shigella increased resistance to some antibiotics. Smaller. The gene at 1411,1417,1435 marOR point mutations in the impact resistance of Shigella.