A Research On Subtypes Of Extended Spectrum β-lactamases Ctx-m-1

Objectives:The application of antibiotics and the production of drug resistance of bacteria develop in unison,and the main cause in the mechanism of bacterial drug resistance comes to the production ofβ-Lactamases.In recent years,as the combining use of the third and forth generations ofβ-Lactamases type antibiotics and enzyme inhibitors like clavulanic acid,sulbactam and tazobactam,bacterias are under double pressure in selection.β-Lactamases experience evolution and mutation,and the biological organization of enzyme change.The organization change of enzyme activity sites makes enzyme be able to hydrolyze newβ-lactam antibiotics,thus Extended-spectrumβ-lactamases come into being.ESBLs are mostly produced by Ecoli and K.Pneumoniac.Fully half of them are produced by the mutation of one or more amino acids generating by TEM-1,TEM-2 and SHV-1 on the sites of 104,164, 238 and 240.The spatial structure of zymophore changes,which broadens the range of hydrolysis substrate,therefore strengthens the affinity and hydrolysis ability of oxymineo-lactam antibiotics.At present,there are at least 387 species of ESBLs genes discovered:155 species of TEM,91 of SHV,88 of OXA,53 of CTX,and more new species are in continuous development and discover.The origin,distribution,and molecular configuration of different genes are varied,so their drug resistance abilities are different.CTX-M type is one of non-TEM/SHV ESBLs,and its features are its high hydrolysis ability to CTX and low hydrolysis ability to ceftazidime.Now more than 30 species were discovered after Bauernfeind and the others' report in 1990.They are produced mainly by Ecoli,salmonella typhimurium,protetus,enterobacter aerogenes, and etc.The nucleotide sequences in gene coding of their subtypes are varied greatly, and their homologies are between 71%-99%,pI7.5～8.9.CTX-M type of ESBLs is mainly in European countries,South America,Middle East,Far East and China.In our country,the reports about types of ESBLs are gradually increasing in recent years, and the research shows that CTX-M is the main type of ESBLs in our country.But the research on CTX-M is obviously not enough,and it's mainly focused on the conclusion of the popular subtypes and their drug resistance,thus the features of their drug resistance can not be discovered effectively,therefore,they can not be used to instruct clinical medication.This research takes the approach of PCR and intentionally amplifies the gene fragments of CTX-M,and the process of experience includes transformation,plasmid extraction,sequencing,expression protein and so on. It helps lay a solid foundation for the further research of its drug resistance and lowering its drug resistance in instructing clinical medication.Materials and methods:From July 2006 to July 2007,we collected lots of samples of clinical isolated Escherichia coli and K.Pneumoniac in the First Affiliated Hospital of Zhengzhou University.Through two-paper cooperate test and E-test,27 ESBLs positive strains were obtained.Then plasmids of positive strain mixtures were extracted.With the approach of PCR,using the primers of CTX-M-1,CTX-M-14,CTX-M-25 and CTX-M-31 to amplify some subtypes of CTX-M,observing with agarose gel electrophoresis,the targeted fragment was recovered by gel extraction.Restriction endonuclease BamHⅠand double digestion XhoⅠwere used to link the carrier pET28(a+).Through transformation of competent Escherichia coli BL-21, recombinant plasmids were extracted and sequenced,and then amino acid sequences were analyzed.After they were proved to be the targeted fragments, Isopropyl-β-D-thiogalactoside(IPTG) was used to induce expression protein.Results:The plasmids were extracted from the 27 strains of ESBLs positive samples of Escherichia coli and K.Pneumoniac mixtures.Using the four pairs of primers of CTX-M to amplify gene fragments,the result of amplifying CTX-M-1 was positive, and the others were negative.The positive fragments were between Marker 1000bp and 750bp and they were close to 1000bp.Through restriction endonuclease BamHⅠand double digestion XhoⅠ,linking the carrier pET28(a+) and the transformation of competent Escherichia coli BL-21,the plasmids of CTX-M-1-BL-21 were extracted after transformation with BamHⅠand double digestion XhoⅠ.PCR production,observed with agarose gel electrophoresis,had a clear band close to 1000bp between Marker1000bp and 750bp.Recombinant plasmids were sequenced as 907bp,which was accord with the agarose gel electrophoresis,and was also accord with the gene sequence of CTX-M-1 in Gene Bnk at 100%.Conclusion:Among the Escherichia coli and K.Pneumoniac in Henan Province,the subtype of CTX-M-1 is the main type of CTX-M of ESBLs which can be restrictively cut by BamHⅠand XhoⅠ.