Effect Of Integron And OmpF On The Phenotype Of Cefoxitin-Resistance Escherichia Coli

Objective The distribution, characterization and effect on antibiotic resistant gene disseminateion of the integron among 59 Cefoxitin-resistance Escherichia coli clinical isolated strains were investigated. Ompf gene and porin is detected using PCR and SDS - PAGE respectively. The effect of integron and ompF deficiency on drug-resistance phenotypic was explored to guide selection of antibiotic in clinic and surveillance of bacteria. Methods Degenerate primers of class 1, 2 and 3 integrase were designed to screen int I among 59 Cefoxitin-resistance strains of Escherichia coli from clinic. The amplicons of int I were classified by restriction endonucleases. Integron was determined by PCR with integron specific primer. PCR mapping and DNA sequencing were used to analyze resistant gene in integron. gene cassettes were examined by using specific primers of varied region according to intl type by PCR. And then they were sequenced after amplification of bacterium, the results were BLAST on GeneBank (http: //www.ncbi.nlm.nih.gov/ BLAST/) . The 36 strians puritied amplicons of 1664bp were digested with restriction endonucleases-Dral and EcoRV to indentify them. Plasmid and chromosome DNA of the integrase- positive strains were extracted by their kits. The integrase were located by PCR. OmpF gene was amplicated by PCR and porin was dected by SDS-PAGE. The susceptibility of Fifty-nine E. coli strains was test by Microdilution. Results Forty-five of 59 isolates were positive and showed that the forty-five isolates all carried intI 1 by RFLP. Six sort of different integron were detected in 59 isolates. An band of 1664bp were found in 36 strains and DNA Sequencing revealed that the amplicon carried aadA5 and dfr17 on a class 1 integron, conferring resistant to spectimomycin, streptomycin and trimethoprim respectively. GenbankAY748452, AY828551. An band of 1913bp were detected in 2 isolate. The amplicon harbored gene cassette of aadA2 conferring resistant to spectinomycin and streptomycin, and gene cassette dhfrXII conferring resistant to trimethoprim. Genbank AY748453.There were an band of 2360bp in 2 isolate, Which contained aacA4 and cmlA1, conferring resistant to spectimomycin, streptomycin andchloramphenicol respectively. Genbank AY912492. Sequencing revealed that ECO16 has amplicon of 1417 bp and contained gene cassette of blaTEM-lb conferring resistant to to beta-lactams ,and gene cassette Tn2* tnpR, producting TnpR resolvase. Genbank DQ406736. DNA Sequencing revealed that ECO24 carried amplicon of 3000bp and contained gene cassette of blaTEM-lb conferring resistant to to beta-lactams, and Int I, producting integrase. DNA Sequencing between them could be performed, thirty-six of 59 isolates digested with restriction endonucleases -Dral and EcoRV showed that all 36 strains was digested into 200 bp,300 bp and 1500 bp with EcoRV, altogether 234 bp and 1430 bp with Dral. The int I were amplicated by template extracted from Plasmid and chromosome DNA of the 45 integrase-positive strains by PCR. The results were that there are 26 positive-strains (58%) not only in Plasmid but also chromosome. There are also 13 positive strains (29%) only in Plasmid, and 6 positive-strains (13%) in only chromosome. An amplicon of OmpF gene, 1 lOObp was detected in 29 isolate. The other 30 isolates were OmpF gene depletion and Ompf deficient. Six of the 30 isolates were OmpC deficient simultaneously. Five of 29 positive isolate were OmpF deficient yet. ATCC25922, standard strain carried OmpA, OmpF, and OmpC at the same time. Resistant rate of 59 isolates against 22 antimicrobial agents were to nalidixic acid 100%, ciprofloxacin98%, Ofloxacin95%, Cefoxitin 95%,Ticarcillin93%, ticarcillin/Clavulanic acid 83%, Rifampicin 88%, Tetracycline 81%, Cefoperazone 75%, Cefoperazone/sulbactam 73%, gatifloxacin 73%,Sulfamethoxazole 73%,gentamycin 69% respectively. 58 tested strains were susceptible to Imipenem. There were significant differences of the MIC of the Escherichia coli strains only in Cefoxitin (P < 0.05) between producing AmpC (3-lactamase groups and the other groups;No significant differences were found for the other 21 antimicrobial agents in the two groups. So was the MIC of the ompF deficient Escherichia coli strains. This difference of the MIC of Escherichia coli strains was statistically significant between producing integron groups among sulfanilamide, kanamycin , amikacin, Netilmicin, tobramycin, gentamycin, tetracycline, chloramphenicol. Compared with the other antimicrobial agents used in this study, there was no significant difference between resistance rates for the two groups. Conclusion PCR assay to screen class 1 , 2 and 3 integron at one time was confirmed to be practicable by application, and it is feasibiity to analysintrograse with restriction endonucleases. Class 1 integron resided in 59 strains of Cefoxitin- resistance Escherichia coli widely. aadA5 and dfrl7 were the most common resistance gene insert, beta-lactams gene cassette does not play important role in integron-mediated drug-resistance. Integron is located not only Plasmid but also chromosome in most strains. Producing AmpC strains is seriously resistant to Cefoxitin than the other group. Multiresistance gene cassettes could play an important role in causing the antibiotic resistance on aminoglycosides and trimethoprim.The deficienc of porin chiefly affect on Cefoxitin. Thought the 59 strains Escherichia coli is hightly resistant to these antimicrobial agents such as quinolones, cephamycin , penicillins, third generation cephalosporins, beta-lactams combined with beta-lactamase inhibitor, the forth generation cephalosporins et al, they were susceptible to carbopenems and also kept highter susceptible to aminoglycosides such as Amikacin, and Netilmicin. Aminoglycosides may be choiced except carbopenems, when infected by Escherichia coli strains which producing both high-level AmpC P -lactamase and ESBLs, But infected by Escherichia coli strains which carrying integron, Aminoglycosides might be inefficient.