Isolation And Identification Of Salmonella And Its Florfenicol Resistant Mechanism Research

Salmonella is a common zoonosis pathogens, which can infect both human and animals through contaminated food. In all parts of the world’s food poisoning cases, cases of Salmonella poisoning accounted for the first or the second. And it has taken serious threat to people’s health and the healthy development of animal husbandry. At present, many developed countries such as the United States, has established a relatively perfect Salmonella epidemiological surveillance and drug resistance monitoring system which epidemiological data is relatively abundant, and it’s related to carrying on the further research of bacterium resistant mechanism. But our country’s research lags behind in this respect. To investigate prevalent Salmonella strains from animal, their antimicrobial susceptibility, and their florfenicol resistant mechanism. In this syudy, pathogens were isolated from clinical samples with suspected Salmonellosis, and identified by mPCR. We selected floR, AcrB genes of isolation of S101022、0605E2-H by using the method of homologous recombination. This study lays a foundation for further studying the molecular mechanism of florfenicol resistance.1. Analysis of Antimicrobial Resistance of Salmonella Strains Isolated from AnimalsPathogens were isolated from clinical samples with suspected Salmonellosis, and identified by mPCR. The results showed 61 Salmonella strains were isolated, and the numbers of S.enteritidis, S.pullorum, and S.typhimurium were 10,12, and 39, respectively. In the distribution, the highest rate of separation was July to August. Antimicrobial resistance to 23 antibiotics was determined by antibiotic susceptibility test according to the K-B method. All strains were resistant to penicillin, erythromycin, vancomycin, and 90.16% of the isolates were resistant to six or more than six antimicrobials. The isolates with resistance to florfenicol were chosen to PCR amplification for floR, fexA, fexB and other genes. The floR genes were detected in 8 out of 12 florfenicol-resisitant Salmonella strains, and no other genes were detected. Nucleotide sequence analysis of floR that cloned showed that homology was 99.3%～100% and the amino acid sequence homology was 99.2%～100% with comparison of floR of animal origin which have been reported in Genbank. There were four replacements in 147、160、228、293 sites.2. Construction and Antimicrobial Resistance Analysis of Salmonella mutants with deletion of resistant genesA S. Dublin strain S101022 and a S. Typhimurium 0605E2-H were selected as parental strain, and floR,AcrB were contructed by Red recombination system. Six deletion mutants were confirmed by PCR amplification and sequence analysis. Antibiotic susceptibility test showed that the mutants lost resistance susscessfully. Growth assay indicated that growth speed of double gene deletion strains were slightly slower than the wild strains and deficiency of other mutants had no significant effect on the growth. The expression levels of floR and AcrB were determined by Q-PCR. The results revealed that the expression of AcrB increased weather it was under pression of florfenicol and the expression of floR had a related degree of dependence on CCCP. But the expression of floR was stable. And the differences of the expression of floR and AcrB were not significant.3. Determination of florfenicol gathered in the resistant and gene deletion mutants by HPLCThe determination of accumulation of wild strains (SI01022,0605E2-H) and gene deletion mutants (S101022△floR, S101022△AcrB,0605E2-H△floR,0605E2-H△AcrB) by HPLC showed that intake of florfenicol of wild strains (S101022,0605E2-H) was significantly fewer than the mutants. After ten minutes within taken into florfenicol, the accumulation of florfenicol of wild strains was half of the double genes deletion mutants. But the results showed that CCCP had significant effect on the accumulation of florfenicol of △ AcrB, and little to △floR. On the other hand, PAβN had significant effect on the accumulation of florfenicol of AfloR, and little to △AcrB. It indicated that CCCP was mainly to the pumping system floR mediated, and PAβN was about mainly to the pumping system AcrB mediated. Both of these two pumping system played a certain role in the florfenicol resistant process.