| E coli 0157: H7 is the main strain of many serum types of enterohemorrhagic Escherichia coli (EHEC). Since 1982 when the first case of food induced gastroenteritis caused by beef contaminated with O157:H7 appeared in the USA, many cases of food poisoning related to the bacteria have been reported in Occident one after the other. An epidemic outbreak caused by O157:H7 occurred in Japan in 1996, in which accumulatively near ten thousands of people were involved in and several cases died. This drew extensive attention of the world. The condition is listed in the notifiabe diseases in many countries.Sporadic cases have been reported in China since 1987 and O157:H7 was detected in more than 10 provinces or municipalities. In 1999, an outbreak caused by O157-.H7 happened in some areas of Jiangsu and Anhui Provinces, which resulted in the death of more than 100 people. This was likely to be the maximal prevalence in the world so far and indicated that EHEC has become a prominent problem in China and it is essential to attach great importance to it.0157 strains retain in the intestines of domestic animals such as cattle, sheep, pigs and chicken and induce diarrhea of the infected animals and therefore result in the contamination of meat and dairy products and eggs, which bring about a great loss in farm and stock breeding and a serious threatening to human beings. The infection caused by 0157 strains has become a global problem in public health.The application of antibiotics in high dose is common in order to improve theyield of animal foods, which provide a favorable condition for the formation of drug resistance of many animal disease inducing bacteria and their spread in the body, which is especially true in the case of using antibiotics to prevent diseases of food animals and human beings. Therefore the buildup of drug resistance of food origin pathogenic bacteria is inevitable.The drug resistant strain of O157:H7 was reported frequently overseas in recently years and the drug resistance mechanism has also been investigated. However the accumulated data was not comprehensive and systemic. The drug resistant strain of O157:H7 has been isolated in clinic from both human beings and animals in China but there were no report about the drug resistance mechanism. It is necessary to make a deep probe into the exact mechanism of drug resistance of 0157 strain.In this experiment E coli 0157 resistant strains were isolated from animals in clinic and the genes related to drug resistance to β -lactam and aminoglycoside antibiotics were detected. The expression level of mRNA and proteins of some related genes were determined aiming at the search for the relationship between the gene expression and the drug resistance of E coli 0157. The experiment consisted of four parts.l.The isolation and identification of E coli 0157 from animals.E coli O157 was isolated from different animals in clinic. Drug resistance of E coli 0157 to 20 kinds of antibiotics were examined with drug sensitivity test papers after the identification of the strains with microbiological test, biochemical reactivity and PCR. The results showed that over 50% of the strains were resistant to one or more medications. The minimal bacterial inhibition concentrations of drug resistant strains to six a-lactam and five aminoglycoside antibiotics were determined with broth double dilution. The results indicated that drug resistance of some strains was surprisingly serious.2.1solation of drug resistant E coli 0157 in clinic and detection of genes related to β -lactam and aminoglycoside antibiotic resistance of 0157 strains and gene cloning and sequence analysis.Amplification of the genes: Primers were designed according to the sequences of ￡ coli blaAmpC, WaTEM-121, aphA, aac(&) -Ib, aac(3)-IV, aph(T)-Tb, aadA and￡ coli O157:H7 EDL933 pbpA, ompF, ompC, rpsL, rrsA released in GeneBank. The gene fragments of interest were amplified with the genome DNA and plasmid DNA of strains isolated in clinic as templates.Cloning of gene fragments: the gene fragments of interest were ligated to pGEM-T-Easy vector and then transformed into E coli DH5 a strains. The positive clones were screened with a complementary assay.Sequencing and analysis of recombinant plasmid nucleotide sequences: the nucleotide sequences of the correct clones confirmed were determined and analyzed.The results showed: the plasmids isolated from drug resistant strains in clinic were about 1.9KDa and blaAmpC gene of ESBLs, and aphA gene of APH were amplified from the plasmids and the identity in nucleotide level to the sequences reported was 99.6% and 100% respectively. Comparing with the nucleotide sequences of O157.H7 EDL933 released in GeneBank, 1,7,4,0 and 3 mutations occurred in the amino acids encoded respectively by pbpA, ompF, ompC, rpsL and rrsA genes amplified from genomes of drug resistant strains isolated in clinic.3.Quantitative detection of mRNA expression of blaAmpC^ aphA genes of ￡ coli 0157 drug resistant strains isolated from clinic.Design of primers and probes: two pairs of primers were designed according to the nucleotide sequences of 6/aAmpC and aphA genes. In the middle of the amplified sequences were two probes labeled with fluorescence group FAM in the 5 end and fluorescence diminishing group TAMRA in the 3 end.Amplification of each chain of cDNA: Total RNA was extracted from each strains and reverse transcription were performed. The products of RT-PCR were observed with agarose electrophoresis.Quantitative PCR: the cDNA amplified were used as templates and the primers and probes were added into the reaction solution. Fluorescence quantitative PCR amplifier was used to detect the fluorescent optical density. Standard curve wasestablished with plasmid DNA.Comparing of mRNA level of WaAmpC and aphA genes of different Ecoli 0157 drug resistant strains: The results showed that WaAmpC mRNA expression of the strains with high level (MIC 256/ig mL"1) of drug resistance to P -lactam antibiotics were higher than that of the strains with relatively low resistance (MIC 3^ug mL'1) . aphA mRNA expression of the strains with high level (MIC 256/zg mL"1) of drug resitance to aminoglycoside antibiotics were higher than that of the strains with relatively low resistance (MIC 32,ug mL"1) , indicating that the expression level of WaAmpC and aphA mRNA is positively related to its drug resistance4.The expression of proteins encoded by WaAmpC and aphA genes of E coli 0157 strains isolated in clinic.Cloning and prokaryotic expression of WaAmpC and aphA gene fragments WaAmpC and aphA gene fragments with restriction sites were amplified from E coli 0157 and then cloned into pGEM-T Easy vector. The positive clones confirmed by PCR and double digests were sequenced. The results showed that identities of WaAmpC and aphA gene amplified to the reported sequences were 88.6% and 100% respectively. One of the amino acids encoded by WaAmpC altered and no changes occurred in amino acids encoded by aphA gene. The constructed expression plasmids of WaAmpC-pET28a (+) and aphA- pET28a (+) were transformed into host strain BL21 (DE3). The expression level of WaAmpC and aphA genes was determined after induction by IPTG.Preparation of antibodies against WaAmpC and aphA proteins. The purified expression products were used to immunize rabbits. The antibodies against WaAmpC and aphA were obtained for the first time in China as confirmed by ELASA and Western-Blotting.Western-Blotting of WaAmpC and aphA The level of WaAmpC and aphA of E coli 0157 strains from animal sources were determined with specific antibodies raised against WaAmpC and aphA. The results of Western-Blot showed that there was a positive relativity between the level of WaAmpC and aphA proteins and their drug... |
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