| Salmonella is one of the most important foodbome pathogens causing fever,enteritis,septicemia in humans and animals. Salmonella contamination in the food chain has a significant economic impact due to the costs of medical care and lost productivity. People usually get salmonellosis by eating contaminated foods, particularly foods of animal origin, or by direct contact with infected animals. In recent years, antimicrobial resistant Salmonella spp. are increasing due to the extensive use or inappropriate use of antimicrobial agents in both humans and food animals. Several studies have documented the increasing single and multiple antimicrobial resistant Salmonella strains originated from various sources, and antimicrobial multiresistance has been reported in various Salmonella enterica subsp. enterica serovars. These single and multiple antimicrobial resistant Salmonellae can be transmitted to humans through the food chain, which has become a significant issue in public health.Antibiotic resistance of bacteria are controled by resistance genes. Many of the currently known resistance genes are located on mobile genetic elements such as plasmids, transposons, integrons and genomic islands. Under the selective pressure imposed by the use of antimicrobial agents, bacteria can acquire resistance genes through the mobile genetic elements. And also bacteria can develop mutations that confer resistance to antimicrobial agents. Multidrug resistance is frequently linked with DNA elements such as integrons, which can be located on transposons and plasmids but also on the chromosome. They can integrate and disseminate genes encoding resistance to a number of antimicrobial drugs. Class1integrons, the most common integron type, have been detected all over the world and in a various of Salmonella serovars. They are characterized by the presence of two conserved segments, the5’conserved segment (5’CS) and3’conserved segment (3’CS). And the gene cassette of class1integrons may or not be present.Bacterial efflux pumps, where many classes of antimicrobial agents are pumped out of the cell through membrane-spanning efflux pumps, are important in resistance to multiple antibiotics. In addition to this role in antibiotic resistance, these efflux pumps seem to have been shown to contribute to the virulence of many bacterials. The AcrAB Efflux Pump is the most extensively studied efflux pump in various bacteria. Several studies have documented that the AcrAB Efflux Pump of Salmonella is involved in multidrug resistance and pathogenicity. Recent study showed that the macAB Efflux Pump of Salmonella Typhimurium contributes to the tolerance to erythromycin and deletion of the macAB genes attenuated Salmonella Typhimurium virulence more than any other efflux pumps. So it is necessary to study virulence and drug resistance roles of the macAB Efflux Pump of Salmonella Enteritidis.1Epidemiological analysis of Salmonella enterica isolates originated from various sourcesA study was conducted to describe the epidemiology of Salmonella spp. in chickens, ducks, gooses, pigs and humans in eastern China during2009-2010. A total of135Salmonella isolates were recovered from2566samples, representing5.3%of samples tested. Ten serotypes were identified among the135Salmonella isolates. The most commonly recovered serovars from different sources were Senftenberg Enteritidis, Pullorum and Typhimurium. All of the isolates were characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme Xba I.47PFGE patterns were generated among the135isolates, which grouped into six clusters (named A-F separately) with36%-100%pattern similarity. PFGE typing grouped the majority of isolates according to serotype except S.Senftenberg, S.Derby and S.Saintpaul. The PFGE typing of some S.Newport isolates recovered from different areas and sources were very similar. The results showed genetic diversity among the different areas, sources and serovars of Salmonella in eastern China.All of135Salmonella isolates were tested for the antimicrobial resistance to18antibiotics and further characterized for17risistance genes ane class1integrons. The highest percentage of resistance was found to the following antibiotics:carbenicillin (94.1%), followed by nalidixic acid (45.2%), sulfafurazole (37%), ampicillin (31.9%) and amoxicillin (28.9%). None of the isolates were resistance to ciprofloxacin. One hundred and thirty one (97%) isolates were resistant to at least one antimicrobial. We found32different patterns of resistance in135Salmonella isolates. Chicken Salmonella isolates and Salmonella Pullorum commonly displayed multidrug resistance (MDR) to>5antimicrobials.blatem-1,(89.6%), blapse-1(55.6%), sul2(28.1%) and tetA (20.7%) were the most prevalent resistance genes. We did not detect blaCMY-2, blaCMY-9, tetC, tetG and floR. The resistance phenotype is consistent with the antibiotic resistance genes except aminoglycosides resistance genes. class1integrons were detected in17(12.6%) isolates of135isolates comprising serovars Senftenberg, Derby, Pullorum and Enteritidis. All of the class1integron-positive isolates had int1and qacEΔ1-sul Ⅰ and10(7.4%) isolates harbored the gene cassettes:aadA2(amplicon sizes lkb). But the class1integrons whose amplicon sizes were600bp did not had the integron-associated gene cassettes. None of the class1integron-positive isolates harbored two different class1integrons.2Funcitional analysis of efflux pump macAB in Salmonella EnteritidisThe upstream and downstream fragements of macAB gene were amplified from genomic DNA of Salmonella Enteritidis PO4, which was isolated from human. At the same time, a chloramphenicol-resistant (CmR) gene was amplified from plasmid pKD3. And then the upstream and downstream fragments and the CmR gene were subcloned into the suicide plasmid pGMB151to construct the recombinant suicide vector pGMR151-ΔmacAB/Cm. The Salmonella Enteritidis PO4ΔmacAB was successfully constructed by homologous recombination between the mutant macAB gene in the recombinant suicide vector pGMB151-ΔmacAB/Cm and the wild-type gene in the chromosome of Salmonella Enteritidis PO4. And the mutant was confirmed by PCR method. The O and H antigens, fermentation patterns and antimicrobial susceptibility of the mutant was identical to the parent strain. However, growth rate of the mutant were differed from that of the parent strain, but not obviously. And the virulence of the mutant was3.6times lower than the parent strain. These results suggest that macAB, which are drug efflux system of Salmonella Enteritidis, contribute to the virulence of Salmonella Enteritidis. |
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