Analysis Of Antibiotic Resistance And Resistance Genes Of Salmonella From Fish Pond Ecosystem

The extensive use of antibiotics in aquaculture industry leads to bacterial resistence, which brings the degree of antibiotic resistence (AG) to be more severe. And pathogens with AG can be transmitted to humans through the food chain causing health problems. Therefore it has become a major public concern around the world.In this thesis, the appearance rate and antibiotic resistance of Salmonella, antibiotic resistance genes were analyzed in a fish pond ecosystem in Nanhai, Guangdong Province. Thirty-four Salmonella strains were isolated from 6 different samples of the fish pond ecosystem, including 6 from fish intestine, fish pond soil, duck intestine and fish pond soil,5 from pond water and duck farm water respectively, were identified by biochemical and PCR tests. The microbial community of each sample was analysis using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). The result showed bacterial communitys were diverse in the animal intestine and environment, which have different dominant microorganisms. And the spectrum of sample appears the similar degree. All strains were then tested for the AG character to 15 antibiotics by Kirby-Bauer (K-B) method, including ampicillin, streptomycin, neomycin, ceftazidime, cefotaxime, nalidixic acid, trimethoprim, compound sulfadiazine, trimethoprim-sulfamethoxazole, cotrimoxazole, chloramphenicol, tetracycline, nitrofurantoin, erythromycin, penicillin, polymyxin B, tigecycline and florfenicol. Results showed that all strains were resistant to three or more antibiotics. The resistance rate to ceftazidime, cefotaxime, nitrofurantoin and polymyxin B were lower than others, and strains were sensitive to tigecycline.The multiple resistant to antibiotic was common in Salmonella isolated, and 97% strains were resistant to ampicillin, neomycin, erythromycin and penicillin at the same time.Twenty-two antibiotic resistence genes (ARG), including aadA, aac(3)-I, aphA1, aac(3)-IV, ereA, MOX, DHA, EBC, FOX, CIT, blaSHV, blaoXA, blaTEM, tetA, tetB, tetC, sulI, dhfrl, dhfrV, catI, cmlA and floR, were qualitatively detected by 4 multiplex PCR reactions.7 pairs of primers were designed to amplify the genes including sull, blaSHV, catI, dhfrV,floR, aadA and blaOXAby 7-plex PCR assays,6 pairs of primers were designed to amplify the genes including blaTEM, cmlA, CIT, ereA, dhfrI, aac(3)-I by 6-plex PCR assays, and 6 pairs of primers to amplifing the genes including aphA1, MOX, DHA, EBC, aac(3)-IV, FOXby 6-plex PCR assays. Then three multiplex PCR assays were set to amplify the resistence genes of tetracycline, including tetA,tetBand tetC. The result demonstrated that the multiplex PCR assays can effectively detect 17 out of 22 ARGs in environmental isolates, with ereA, FOX, blaSHV, catI and tetC were not found in those isolates. The prevalence of aminoglycoside resistance genes were aadA>aphA1>aac(3)-IV> aac(3)-I, and ampicillin resistant genes were CIT> DHA>EBC>MOX. The prevalence ofβ-lactam resistance genes were blaoXA> blaTEM, and that of tetracycline genes were tetA>tetB with tetC was not found in any of those isolates. The prevalence of sulfadiazine genes were 32.4%. The prevalence of trimethoprim genes were dhfrV (11.8%)> dhfrl (5.9%). The prevalence of chloramphenicol genes were cmlA (17.6%)>floR (8.8%). The result showed positive rate of drug sensitivity test was higher than 40%, and positive rate of aminoglycosides was 52.9%. The established multiplex PCR assays were simple, time-saving and could be specially to detect numbers of ARGs in only 4 PCR reactions.Salmonella isolates were further investigated for the distribution of integrons, using a 3-plex PCR assay to detect 3 integrons (intI1, intI2 and intI3) and the variable segment of intI1 gene. Results showed that 91.2% of tested strains harbour only intI1,1 strain has both intI1 and intI2, and intI3 was not found in any of these strains. The experiment indicated that integron 1 commonly present in the isolated strains, with the size of variable region fragment was 1600bp and 160bp.This study demonstrated that there were multiple antibiotic-resistant Salmonella in the fish pond ecosystem, the resistance genes and integrase gene were harboured in those strains. Integrate could carry multiple-antibiotic resistance gene horizontal transfer in the aquaculture ecosystem. It could make the resistance Salmonella strains extended, which cause human health problems and safety risks.