Phenotypic And Genotypic Characterization Of Antimicrobial Resistance In Porcine Pathogenic Escherichia Coli And Salmonella

Since recent decade ago,many antimicrobials have been extensively used in pig production to prevent infectious diseases and improve growth. The wide and irrational application of the antimicrobials resulted in heavy antibiotic selection pressure, and increasingly severe bacterial resistance and multi-resistance to pathogenic Escherichia coli and Salmonella. Some clinical isolates could survive even in several new drugs which even not yet introduced in clinic.Therefore,it's wise and favorable to monitor the drug resistance, characterize antimicrobial resistance both phenotype and genotype, so that better contain the development and dissemination of pathogenic E. coli and Salmonella, and more rationally administer regimens of antimicrobial agents.Samples from sick piglets in intensive pig farms of Zhejiang Province were collected for isolates of pathogenic E. coli and Salmonella. Through biochemical tests, PCR and virulence tests in mice, total of 79 pathogenic E. coli isolates and 35 Salmonella isolates were identified. The susceptibility testing was carried out by Kirby-Bauer(K-B) method according to CLSI(Clinical and Laboratory Standards Institute) against 21 commonly used antimicrobials, respectively. The results indicated that, apart from great sensitivity to cefotaxime/clavulanate, cefotaxime and polymyxin B,the E. coli isolates exhibited severe resistance to the majority of antimicrobials.The resistance frequencies of oxacillin, rifampicin, sulphamethoxazole and trimethoprim, doxycycline, carbenicillin, ampicillin, amoxicillin, streptomycin and chloromycetin were all above 90%, and the resistance frequencies of gentamycin, ciprofloxacin, neomycin, enrofloxacin, norfloxacin, amoxicillin/clavulanate and florfenicol were also over 50%.The resistance patterns analysis demonstrated that all the E. coli isolates were resistant to 1 and more antimicrobials, and the majority of the isolates are resistant to 16-18 antimicrobials(60.8%,48/79).Compared with E. coli, the Salmonella isolates are sensitive to most antimicrobials except for oxacillin and rifampicin. They showed totally sensitive to cefotaxime/clavulanate, cefotaxime, amikacin, norfloxacin and polymyxin B.The sensitivity frequencies of gentamycin, ciprofloxacin, amoxicillin/clavulanate, spectinomycin, florfenicol,ampicillin, neomycin and sulphamethoxazole and trimethoprim were over 80%,and the sensitivity frequencies of enrofloxacin and chloromycetin were also over 60%.Though the 35 Salmonella isolates were also resistant to 1 and more antimicrobials, the multidrug resistance was less severe than E. coli isolates, and the majority of the isolates were resistant to 2-4 antimicrobials.The ESBLs(extended-spectrum beta-lactamases) genes(TEM, SHV, OXA), aminoglycoside-resistant genes(aadA1,aadA2, aadB, aadD, aph(3')-Ⅱa, aacC2, aacC4, aac(3)-Ⅰa, aac(3)-Ⅱa), sulfonamide-resistant genes (sulⅠ, sulⅡ, sulⅢ), chloramphenicol-resistant genes(Cat1, Cat2, CmlA, CmlB, Flor), tetracycline-resistant genes(tetA, tetB, tetC, tetD, tetE, tetG) were amplified by Touch-down PCR and detected by agarose gel electrophoresis, respectively. The results showed that the following antimicrobial resistance genes were more prevalent among the 26 E. coli isolates randomly selected:sulⅡ(96.2%), TEM(88.5%), aph(3')-Ⅱa(80.8%), aacC2(69.2%), Cat1(69.2%), Cat2(69.2%), aac(3)-Ⅱa(65.4%), tetB(65.4%), tetE(61.5%), aadA2(57.1%), sulⅠ(50%). The positive rate of the gene Flor, CmlA, aadA1, tetA, sulⅢand aadB was 34.6%,26.9%,19.2%,19.2%,11.5% and 3.9%, respectively. However, SHV, OXA, aadD, aacC4, aac(3)-Ⅰa, tetC, tetD, tetG and CmlB genes were not detected. There were not so many resistance genes in the 35 Salmonella isolates. The positive rate of the gene was lower than that of E. coli isolates:tetA(28.6%), TEM(20%), sulⅡ(20%), aacC2(17.1%), Car1(17.1%), Cat2(17.1%), tetB(11.4%), aph(3')-Ⅱa(8.6%), sulⅠ(8.6%), aadA2(2.9%), aac(3)-Ⅱa(2.9%), tetG(2.9%). However, SHV, OXA, aadA1, aadB, aadD, aacC4, aac(3)-Ⅰa, sulⅢ, tetC, tetD, tetE, CmlA, CmlB and Flor genes were not detected.The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of enrofloxacin and ciprofloxacin hydrochloride against sensitive Salmonella isolates from piglets, which were confirmed by K-B method, were determined by microdilution and agar dilution methods,respectively.The MICs of enrofloxacin and ciprofloxacin hydrochlorid were 0.031-0.5μg/mL and 0.031-0.13μg/mL, and the sensitivity rates were both 100%, which were consistent with the sensitivity testing by K-B method. The MPCs of enrofloxacin and ciprofloxacin hydrochlorid to the sensitive isolates were 1-4μg/mL and 0.25-1μg/mL, and the selection index (SI) values of ciprofloxacin hydrochlorid (8-16) were lower than those of enrofloxacin (16-32). Therefore, the efficacy of ciprofloxacin hydrochlorid for containing the selection of Salmonella resistant mutants was greater than that of enrofloxacin.The results in the study indicated that the pathogenic E. coli isolates from piglet in Zhejiang province had severe resistance to antimicrobials, and they harbored more varieties of the resistance genes with high positive rates. However, the Salmonella isolates were sensitive to the majority of antimicrobials tested. Enrofloxacin and ciprofloxacin hydrochlorid presented different capacity to restrict the selection of strains resistant mutants. In order to control the development of antimicrobial resistance, so not only the MIC should be considered in clinical application, but the MPC and MSW (mutant selection window, MSW) values of isolates should also be taken into account to contain the development and dissemination of bacterial resistance.