The Research On The Quinolone-resistant Mechanism Of Escherichia. Coli O157

E.coli O157 is one of bacteria in medical science and veterinary clinically. Swif-detected method has important significance to foods safety and human health. AcrAB efflux pump plays a major role in the antibiotics resistance phenotype of Esherichia coli O157,multitiple antiobiotics resistance mutant.AcrAB-ToIC system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent, detergent and dyes.The coordinated operation of the inner membrane transporler acrB and outer channel To1C is thought to be mediatd by acrA. Here we will establish a swif-detected method, study gene mutant mechanism of E.coli 0157 resistance to quinolone, determine the level of mRNA of acrA and acrB and level of acrA protein expresion.To develop methods of multiplex PCR and real-time fluorescence PCR to detect E.coli O157:H7 rapidly, specifically and sensitively. To compare their sensitivity with conventional PCR. Four pairs of primers were designed from O antigen and H flagellar antigen and Shiga一like Toxin 1 and 2 genes. 24 strains of O157:H7 and non O157:H7 were detected by conventional PCR and multiplex PCR amplification.At the same time, we design TaqMan fluorescence probe to put up real-time fluorescence PCR .The sensitivities of PCR were estimated when the strain was diluted .O antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC 0157:H7 strains, SLTl and (or) SLT2 genes amplification generated amplicons of 210bp and(or)484bp in toxinogenic O157:H7. Other non O157:H7 failed to yield any amplicon under comparable conditions. The sensitivity of detection by conventional PCR and multiplex PCR were shown to be at least 150CFU per PCR and1500 CFU per PCR. The sensitivity of detection by real-time fluorescence PCR was 14CFU per PCR.Chromosome DNAs from 8 strains of E.coli O157 were extracted, among which 4 strains were collected from clinical samples, 3 strains were got by induction with different concentration of quinolone and 1 susceptible strain. The fragments of gyrA and parC genes of the 8 strains were amplified, cloned and sequenced by PCR. Mutaions were found at residues 83 and 87 in gyrA gene and 80 and 84 in parC gene in all the quinolone-resistant strains,while no mutations were found in the susceptible strain. Only one mutation was found in gyrA gene in 2 low resistant strains,which resulted in Ser83→Leu or Asp87→Asn, and no mutation was found in parC gene. Doublemutations were found in both gyrA and parC genes in 5 high resistant strains. Mutations in gyrA gene resulted in Ser83→Leu and Asp 87→Asn or Tyr , and in parC resulted in Ser83→Ile and Glu84→L ys.The mutations are identical with data from the published references. The results revealed that mutations of gyrA and parC genes are involved in the quinolone-resistant mechanism of E scherichia coli. And the low-resistant E.coli exist only single mutation in gyrA gene, the high resistant E.coli existmutations in both parC and gyrA genes at the same t ime. The level of mRNA of multiple-antibiotic resistance in Escherichia coli O157 gene acrA and acrB compares,the internal standard dilution of retroposon production from drug-resistant strain compares with that of E.coli O157 EDL933.The result is that the content of mRNA transcribing from acrA and acrB in the high-drug-resistant E.coli is 4or 8 times higher than in the EDL933, in the moderate-drug-resistant E.coli ,it is 1 or 2 times higher than in the EDL933, and the low- drug-resistant E.coli ,it is as same as in the EDL933.The content of mRNA describing from acrA is similar to acrB in the same bacterium. It probably because that acrA and acrB are controlled by one operon. So acrA and acrB are the multi-drug-resistant gene-marker in E.coli O157:H7.The contant of mRNA describing from acrA and acrB is direct-ratio to the drug-resistant level in E.coli O157:H7.The result is that E.coli O157:H7 can be quantitatively and qualitatively analyzed by multiplex PCR and fluorescence quantitative PCR. The mutation of E.coli O157:H7, gyrA,Ser83 and Asp87,parC gene, Ser80 and G1u84 is related with the drug-resistant mechanism of E.coli on the quinolone. There is a single mutation on the gyrA gene in low-drug-resistant strain .In high-drug-resistant strain, there also have another mutation on the parC gene. The level of mRNA describing from acrA is similar to acrB in E.coli O157:H7. The level of mRNA describing from acrA is same to the level of protein expressing from acrA. The level of mRNA and protein has relative relationship with drug-resistance in E.coli O157.