| Salmonella is an important pathogen, which has a widely hosts range. It is easy to lead a variety of animal salmonellosis. So Salmonella pathogen became an important source of human salmonellosis and played a significant role in the veterinary science and medicine. Currently because of antibiotics abuse for preventing and treating human or animal diseases, there had make a rapid growth of Salmonella resistant to antimicrobial drugs, especially the emerging of multi-drug resistance phenomenon. It was difficult to control Salmonella illness effectively. In recent years, the mechanism of bacterial resistance studies have shown that drug-resistance not only relevant to selective pressures under the antibiotics environment and wide dissemination of drug-resistant genes movable element(resistance plasmids.transposons,etc), but also relevant to virulence. This experimental research focused on the biological characteristics, drug-resistance, resistance genes distribution, pathogenicity and virulence gene distribution of Salmonella isolates from chicken and expect to provide a theoretical basis for colecting clinical salmonellosis medication, drug resistance mechanisms, pathogenesis and epidemiology.Conventional methods were used for collecting70isolates from organs of chicks, and dead chickens around of Harbin farms. Isolates by culture characteristics, gram staining, biochemical identification, serotype cation and PCR skills. Isolates were isolated. Gram-negative bacteria, and biochemical characteristics were in line with Salmonella spp characteristics. Isolates belonged to four serogroups:A group was eight (18%), B group was five (12%), C group was1(2%), D group was30(68%). Group D was the dominant serogroups.According to NCCLS recommendation, measuring sensitivity isolates to10kinds of antibiotics by K-B method. Resistance genes (blatem-1, blaCMY-2, tetA, tetB, tetG, sul I, sul2, aadAl, catl, qnr) were detected by PCR skills. Animal pathogenicity test was used to determine the isolates pathogenic strength. PCR methods were used to test distribution of Salmonella virulence genes (stn, finiA, invJ, virK, sipA, sopA, ssaB. misL, orf319. pipC, spvC).Membrane bonding method was used for conjugation test, and this test was based on isolates which carried virulence plasmid gene spvC regard as donor bacteria and E.coli NK5449with rifampicin resistant regard as acceptor bacteria. Variable temperature-SDS method was used to eliminate plasmid of isolates with virulence plasmid gene spvC. Detecting virulence plasmid gene spvC by PCR skill aimed to determine whether or not to eliminate the plasmid. Then detection animal pathogenic strength of sub-strains eliminating virulence plasmid gene spvC. which was compared with the isolates did not eliminate virulence plasmid gene spvC.Isolates to ampicillin resistance rate was95.5%. tetracycline. sulfamethoxazole, gentamicin resistance rates were relatively high, reaching to68.2%,50%.50%,respectively; while sensitive to ofloxacin.and the resistance rate only was4.5%.2-resistance strains were10(22.7%),3-resistance strains were12(27.3percent),4-resistance strains were4(9.1%),6-resistant strains were12(27.3%).7-resistance strains were4(9.1%) and8-resistant were2(4.5%). There were26strains with three or more antimicrobial drug resistance, the multi-drug resistance was59.1%. Isolates were detected9kinds of drug-resistance genes, and the rates of Blatem-1, aadA1,tetA,tetB,sull and sul2were over50%, while detection rate of qnr was45%and tetG was40.9%. Sequencing results showed that the resistance genes were highly homologous corresponding to GenBank sequence. The results showed that22isolates with strong pathogenicity,4isolates with moderate pathogenicity,18isolates with weak pathogenicity. Pathogenic Salmonella isolates were detected carrying enterotoxin gene stn, virulence island virulence genes invJ, virK, sipA, ssaB. misL. orf319. pipC, and pili fimA gene detection rate was82%,sopA gene detection rate was95.5%,spvC virulence plasmid gene detection rate was68.2%. Sequence analysis showed that11kinds of virulence genes sequences were highly homologous homology corresponding to GenBank virulence genes and the reference sequences rate reached98.5%. Plasmid conjugation test results showed that24strains were conjugated successful, and conjugated isolates can detect the presence of virulence plasmid gene spvC.Plasmid eliminatin test results showed that all of samonella isolates were eliminated virulence plasmid successefully and isolates without plasmid had a weak pathogenicity.The studyings suggested that there was a widely drug-resistance, and showed a serious multi-drug resistance.44isolates resistant phenotype consistent with drug-resistant genotypes and drug resistance and resistance genes are directly related. All isolates had strong pathogenicity. And there was a positively correlation between pathogenicity and pathogenic virulence genes. But there was a negative correlation between resistance and pathogenicity. The plasmid as a movable member, which can transfer between different strains with virulence genes.There was an attenuated pathogenicity trend of strains after the plasmid elimination. Furthermore, experimental study gene expression of virulence strains after combining the existence of pending changes. |
PDF Full Text Request