| Abstract:Active efflux system (efflux pump) is a major mechanism that causes multipleresistance in Escherichia coli. AcrAB-TolC is the most important one. AcrAB-TolC comprises medicine proton transportor AcrB and periplasm fusion protein AcrA and outer membrane passage protein TolC. It has many substrates, its high standard expression shows tolerance against many kinds of compounds, even high tolerance. Escherichia coli is a major pathogenic bacterium in fowls and livestocks, it exists in many places and animals. Multiple resistance is produced and spreaded easily.Multidrug resistance mechanism mediated by two efflux pumps i.e. AcrA and AcrB in Escherichia coli was studied by detecting the changes on the resistance level in control strains induced by single drug, comparing with clinical isolated strains in three experiments. The specific resisitance mechanism of ESBLs is mediated by plasmid,it is relevant to the plasmid with more resistant genes,ESBLs genes and the inherent drug-resisitent mechanism.we detected ESBLS by CLSI commended methods to 45and 12 inducted E. coli.from chicken We detected MIC by duplation dilution and Ten antibacterial drugs activities were detected by 2-fold agar dilution method with or without efflux pump inhibitors,carbonylcyanidem chlorophenylh-dra—zone(CCCP)and reserpine in Escherichia coli.12 ESBLs-producing clinical strains could amplification 861 bp destination-segment of Specificity by PCR with TEM-type-primer,SHV-type-primer 905bp destination-segment of specificity by PCR with CTX-M-type-primer, and 831bp destination-segment of specificity by PCR with OXA-1-type-primer.The results show that 35 strains producing ESBLs among 35 the positive efflux pup strains Chicken E. coli join in reserpine.; 39strains producing ESBLs among 42 the positive efflux pup strains Chicken E. coli join in CCCP. E. coli plasmid the fragment TEM-phore, However, the subtypes of TEM genes is TEM-1, classified as common P-lactamase.9 strains found to carry fragment CTX,9 strains E.coli were also found to fragment TEM-phore and CTX-phore; PCR products were sequenced after cloning, the results of sequence analysis showed that the sequence homology of various strains producing TEM-type enzyme is 98.4 % to 100%, there are two mutations compared to EU979560 in Gene Bank. The sequence homology of various strains producing CTX-type enzyme is from 96% to 100%, the genotypes of strains ESBLs-producing are CTX-M-14,CTX-M-24,CTX-M-65. The inducted E.coli O78-B and published on Gne Bank sequence FJ812110 (CTX-M-65) is 100%, the inducted E.coli G30-A,G30-C,G31-A,G31-C,O78-A,O78-C and on Gne Bank sequence AJ972956(CTX-M-24) is 98% to 100%; the inducted E.coli G30-B,G31-B and on Gne Bank sequence AJ972953 (CTX-M-24) is 98% to 100%; so the ESBLs produced by the strains inducted in this test is CTX-M-65-type,CTX-M-14-type,CTX-M-24-type.Using real time RT-PCR to detection of mRNA expression of acrA genes in E. coli drug resistant strains induced and isolated from clinic, the results of test showed that:compare with the negative ESBLs,the mRNA expression of the positive strains is significantly twenty-thress times higher in the positive efflux pump;the positive efflux pump is seven times higner than the negative strains in the positive ESBLs;the positive efflux pump and ESBLs is hundred times higher than the negative strains.We could concluted that Extended-spectrumβ-lactamase and efflux pump have collaborative relationship. |
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