Studies On Multidrug Resistance Of E. Coli And Integron Gene Cassettes System

Escherichia coli (E.Coli) is one of the most common pathogens in the medicine and veterinarysurgeon clinically, which endangers the poultry industry and humans. Antimicrobial agents play avery important role in controlling diseases caused by Escherichia coli. Along with the widely useof antibiotics, antibiotic resistance to bacteria is getting worse. There is a significantly increasingtendency of multi-resistance to bacteria. The resistance of Escherichiacoli has raised severalconcerns related to human health and the development of poultry industry. Many researchers havepaid attention to the mechanism of integron gene cassette system in multidrug resistant,and havegot a lot of evolution. Integrons are genetic elements being able to capture foreign genes and makethem to functional genes, it is mobile DNA. It is more frequently existed in gram-negative bacteria,such as enterobacteria and P.aeruginosa.It is the important reason of rapid development aboutmultiple resistance in gram negative bacteria. It is composed of two conserved segments, beingseparated by a segment of variable length and sequence which includes inserted antibioticresistance genes,being called insertion cassette, also being called gene cassette. The integron ofbacterial is DNA system which carry gene cassette encoding antibiotic resistance, integrase has animportant molecular foundation of bacteria to capture the resistance gene, the upstream promoterhelps integron express the resistance gene,which causes the dissemination of bacteria muitidrugresistant. Examining the bacterial sensitivity to common antimicrobials and detecting the integrongene by PCR and investing its types was suitable for the research of bacteria multidrugresistant.This research aims at getting the message of integron gene cassette system distributiondissemination in Escherichia coli and its multidrug resistant mechanism, researching a method todetect integron and its type in clinic.In this experiment, E.coli drug resistance of different resource in Harbin nursery wasinvestigated, as well as the integron gene cassette system was analyzed firstly, determining thedevelopment of multidrug resistant E.coli under the selection pressure of antibacterials,which hadsignificant meaning in multidrug resistant E.coli disseminated and evaluated its chanciness.lntegrase degenerate primer amplified Int product. Int, Intâ… and Intâ…¡digoxin labeled nucleic acidprobes were utilized to detect if the integron in E.coli existed. By comparision, in situ colonyhybridization detect integrase then sure if the E.coli possessed integron is more suitable, thismethod had not be found in internal report. The possessed integron E.coli strain's genotypes weredivided by ERIC-PCR firstly, revealing epidemiology information in strain level.1. 301strains of human swine and fowl source E.coli were isolated from two nurseries bygeneral method and biochemistry characteristic in Harbin. All isolates were examined to better understand the prevalence of antibiotic resistance using K-B method suggested by WHO.Antimicrobial susceptibility testing of 16 antimicrobial agents was performed on the isolatedstrains. Based on the NCCLS results, streptomyci, gentamicin, cotrimoxazole andtrimethoprim-sulfamethoxazole's drug fast rates were always in fore four position. No one swineor fowl source E.coli strains,was found the sensitivity to sulfonamides. There were 247 strainsmultidrug resistant E.coli, which was 82ï¼…of the total isolated strains. In aminoglycosidesantimicrobial, if the multidrug resistant E.coli was not sensitive to streptomycin, it must have ahigh drug fast rates to gentamicin, nyacyne and kanamycin,for example, human source E.coli crossdrug fast rates was 100ï¼…, swine source E.coli cross drug fast rates was higher than 80ï¼…, fowlsource E.coli cross drug fast rates was higher than 74ï¼…. Three source E.coli cross drug fast ratesto cotrimoxazole and trimethoprim-sulfamethoxazole were 97ï¼…or so.2. The integron in multidrug resistant E.coli was detected by PCR method. Polymerase chainreaction amplifiing 1009bp, 1319bp, 1664bp, 199 lbp, 2449bp two types and five different sizesintegrons. Among them 2449bp PCR product was classâ…¡integron, 1319bp PCR product wasclassâ… integron, they were only found in fowl source E.coli. The 1009bp PCR product wasclassâ… integron only found in swine source E.coli.The 1991bp PCR product was classâ… integronfound both in swine and fowl source E.coli. Three sources of E.coli were all found 1664bpclassâ… integron. Analysis cleavage map of Hinfâ… incision enzyme we could find, except for1009bp single band and 1009bp of double bands had different gene cassette, the band size close toeach other had the same gene cassette.3. All the integron of PCR-generated DNA fragment were sequenced in Shanghai sangoncompany. DNA sequences were analyzed by searching the Gen-Bank database of the NationalCenter for Biotechnology Information via the BLAST network service. The results showed that1009bp integrons possessed aadA23b gene cassette or aadA2 gene cassette, 1319bp integronspossessed arr-3, dfrl6 gene cassette, 1664bp integrons possessed dfrl7,aadA5 gene cassette;1991bp integrons possessed dhfrâ…«, aadA2 gene cassette and orfF open reading fram; 2449bpintegrons possessed dfrA1, sat1, aadA1 gene cassette and orfX open reading fram. All the integronhad six specieses of different structure, two kinds of different gene cassettes, and two kinds ofacataleptic function open reading frams.4. The Int,Intâ… and Intâ…¡digoxin labeled nucleic acid probes' color development was 2pg.These probe did not reacted with DNAs from Staphylococcus aureus ATCC25923, pseudomonasaeruginosa ATCC₂₇₈₅₃,and lung tissues of normal rats. The specificity of the three probes was verywell. The southern hybridization proved the three probes detected integrase as well as PCR method.The three probes could efficiently detect integrase gene species of multidrug resistant E.coli.Compared with the dot blot hybridization, in situ colony hybridization detected integrase then sureif the E.coli possessed integron was more suitable for multiplicity strains integron types detection.5. The genotypes of isolates were determined by ERIC-PCR. 171 possessed integron E.colistrains were divided into seven kinds of genotype, each kind of genotype were similar inERIC-PCR band.In a word, in this study, PCR method was used to investigate the kind of integrons in Harbin, examining epidemiology genotypes of E.coli strains, and preparing nucleic acid probe to detect thetype of integrons. The situ colony hybridization being used to integrase then monitors integron wasa better method to investigate multidrug resistant in E.coli, which mediated by integron. This studyhad provided a base for the research of multidrug resistant mechanism of E.coli. Integrons not onlyrelated to multidrug resistant of E.coli, but also plaied important roles in resistant gene convey anddissemination. The study would inturduce a new way and direction for bacterium multidrugresistant mechanism.