Study On Molecular Detection And Some Resistant Gene Tags In Escherichia Coli O157:H7

E.coli 0157 is one of bacterial in medical science and veterinary clinically. Swif-detected method have important significance to foods safety and human health. AcrAB efflux pump plays a major role in the antibiotics resistance phenotype of Esherichia coli 0157 mμLitiple antiobiotics resistance mutant.AcrAB-TolC system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent, detergent and dyes.The coordinated operation of the inner membrane transporter AcrB and outer channel TolC is thought to be mediatd by AcrA. Here we will establish a swif-detected method, study gene mutant mechanism of E.coli O157 resistance to quinolone, determine the level of mRNA of AcrA and AcrB and level of AcrA protein exprssion.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 (only one sets of primers) Four pairs of primers were designed from O antigen, H flagellar antigen and Shiga - like Toxin (SLT) 1 and 2 genes. 24 strains of O157:H7 and non O157:H7 were detected by conventional PCR and mμLtiplex 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 from 101 - 106 .0 antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC O157:H7 strains, SLT1 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 was shown to be at least 150CFU per PCR tube and > 1 500CFU bymμLtiplex PCR. Thesensitivity of detection by real-time fluorescence PCR was 14CFU per PCR.Chromosome DNA s from 8 strains of Escherichia coli were extracted, among which 4 strains were collected from clinical samples, 2 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 by PCR, cloned and sequenced. 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 resμLted in Ser83→Leu or Asp 87Asn, and no mutation was found in parC gene. Double mutations were found in both gyrA and parC genes in 5 high resistant strains. Mutations in gyrA gene resμLted in Ser83→Leu (n= 5) and Asp 87→Asn (n= 4) or Tyr(n= 1), and in parC resμLted in Ser83→Ile(n= 4)and Glu84→Lys (n= D.The mutations are identical with data from the published references. The resμLts revealed that mutations of gyrA and parC genes are involved in the quinolone-resistant mechanism of Escherichia coli. And the low-resistant Escherichia coli exist only single mutation in gyrA gene, the high resistant Escherichia coli exist mutations in both parC and gyrA genes at the same time.Exogenous 6-actin DNA was inserted in cloned AcrA and AcrB by PCR, constructing a internal standard DNA respectively.Total RNA of E.coli was extracted.A sensitive reverse transcription-ploymerase chain reaction (RT-PCR) was performed , it was found that the expression of mRNAs were detected in all selected E.coli.PCR was performed using identical volum of product of reverse transcription and internal standard of different concen- tration .Concentration of internal standard while brightness of two special band in a lane was same was compared between resistant and sensitive E.coli, showed that mRNA level of 11 m^Litiple resistant clinical isolates of E.coli (MIC of enrofloxacin=32/ug-mL"1 ) was higher than that of EDL 933. mRNA level of 2 m^Litiple resistant clinical isolates of E.coli (MIC of enrofloxacin=16^g-mL'! ) was no significant higher than that of EDL 933.Contrasting to EDL 933, mRNA relative level of AcrA and AcrB of were similar. Its show that...