| With the widely use of beta-lactam antibiotics in both human and veterinary clinical medicine,the problem about resistance of bacterial is becoming worse than before,especially about ESBLs and AmpCs-producing E.coli,which has induced larger difficulty to clinical treatment,more attention was pay to its impact of curative effect on people's clinical medicine.The production of ESBLs and AmpCs is the primary cause againstβ-lactam antibiotics in E.coli,it is more profitable for effective control drug resistance to produce and spread.Therefore,the objective of this thesis was to investigate theβ-lactam antibiotics resistance status and mechanisms in E.coli isolates from piglets,so as to provide evidences for the risk evaluation of these drugs resistance,the elimination of drug-resistance,and the rational use of drugs in clinic.1.Isolation,identification and drug-resistance analysis of piglet-associated E.coli strainsThrough investigate the drug-resistance characters of E.coli from large hoggery in Chongqing,so as to instruct the rationally application of antibiotics in clinic,and to submit evidence for further drug-resistance study.157 specimens collected from piglets with diarrhea in hoggery in Chongqing City (Rongchang,Dazu,Tongliang,Hechuan,Beibei,Changshou,Fuling,Wulong,Qianjiang,Qijiang,Jiangjin) from May 2007 to May 2008,124 strains of piglet-associated pathogenic E.coli were obtained by isolated cultivation,morphology observation,biochemical identification and pathogenicity test.124 strains of E. coli were tested with susceptibility to 34 kinds of antibacterials by Kirby-Bauer disc agar diffusion method according to Clinical and Laboratory Standards Institute.Results:124 strains of piglet-associated pathogenic E.coli had extensive resistance to 34 kinds of antibacterials.Of which,the high-level susceptible drugs were Polymyxin B,Ampicillin/Sulbactam, Aztreonam,Cefoxitin,Cefepime,Cefotamine,Ceftazidime,Ceftriaxome,Cefuroxime,Cefoperazone respectively;the high-level resistant drugs were Ampicillin,Tetracycline,Rifampin,Trimethoprim-sulfamethoxazole, Chloramphenicol and Kanamycin respectively.The percentages of all strains resistant to the cephalosporins were 4.03%to 19.35%,only Cephalothin was 39.52%.124 E.coli strains had 121 kinds of resistance spectra,and were multi-resistance mainly,the strains showed that the least resistance to 3 antibacterials,the most resistance to 27 antibacterials.Among the total,9 strains were resistance to 3～7 antibacterials(7.3%),32 strains were resistance to 8～11 antibacterials(25.8%),48 strains were resistance to 12～15 antibacterials(38.7%),19 strains were resistance to 16～19 antibacterials(15.3%),16 strains were resistance to 20～27 antibacterials(12.9%),The highest percentage of strains resistant to 12 antibacterials in tested strains,most strains had resistance to more than 10 antibacterials.Compared E.coli resistance with different areas,it was discovered that only few difference on each district and county hoggery in Chongqing.E.coli strains isolated from each district and county hoggery had high-level resistance to Rifampin,Tetracycline,Ampicillin,Trimethoprim-sulfamethoxazole,were susceptibility to Polymyxin B,Ampicillin/Sulbactam,Aztreonam,Ceftazidime and Cefoxitin.Conclusions:Piglet-associated E.coli strains had high-level resistance to most antibacterials in Chongqing,it was necessary to strengthen to monitor the drug-resistance of bacteria in hoggery,and establish reasonable principle of using drugs.2.Analysis on genotype of ESBLs and Amp Cs among E.coli strains isolated from pigletsTo investigate the genotype distribution of ESBLs and AmpCs among E.coli strains isolated from piglets in Chongqing City.E.coli-producing ESBLs and AmpCs were sieved from 124 strains of piglet-associated pathogenic E.coli by preliminary screening and phenotypic confirming respectively,and genotype analysis of ESBLs and AmpCs were analyzed by PCR and sequencing.Results:9 strains of E.coli-producing ESBLs and 5 strains of E.coli-producing AmpCs were sieved from 124 strains of piglet-associated pathogenic E.coli,the percentage of total detection was 1.3%(14/124). The genotype positive rate of TEM,CTX-M,DHA-1 and CMY-2 was 4.0%,6.5%,1.6%and 1.6%among piglet-associated E.coli in Chongqing area.Of which,ESBLs genotype contained TEM-1 type,CTX-M-14 type,CTX-M-15type and CTX-M-27type,AmpCs genotype contained CMY-2type and DHA-1type.1 strain of E.coli contained both CTX-M-15 and TEM-1genes,3 strains of E.coli contained both CTX-M-14 and TEM-1 genes.However,DHA-1 genotype was the first detected from veterinary clinic in China,GenBank accession number was obtained:FJ386455.Conclusions:There were certain proportionalβ-lactam antibiotics-resistant E.coli strains isolated from piglets in Chongqing area.TEM and CTX-M gene were the predominant genotype in this area,and the majority of ESBLs and AmpCs-producing E.coli isolates were multi-resistance to antibiotics,which should pay more attention to veterinary clinic.3.Study on the horizontal transmission mechanism of drug-resistance for E.coli againstβ-Iactam antibioticsTo investigate horizontal transmission mechanism of drug-resistance forβ-lactam antibiotics-resistant E.coli strains isolated from piglets in Chongqing area.Theβ-lactam antibiotics-resistant E.coli strains from 9 strains of E.coil-producing ESBLs and 4 strains of E.coli-producing AmpCs were adopted with drug-resistance plasmid transformed experiment,original strains and converters were tested with susceptibility to 34 kinds of antibacterials by Kirby-Bauer disc agar diffusion method as well,the plasmids of original strains and converters were extracted with kit to electrophoresis detection and analysis, converters were detected to ESBLs and AmpCs genes by PCR amplification.Results:7 converters were attained from 13 strains of drug-resistance E.coli,the successful percentage of convert was 53.8%(7/13).And converters were attained the partial or complete plasmid and drug-resistance gene from original strains,converters showed the similar resistance to original strains after converted.Conclusions:The drug-resistance of E.coli againstβ-lactam antibiotics could mediate by plasmid, this drug-resistance can horizontal transmission between bacterium by transformation. |
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