Isolation And Identification Of Salmonella,Construction And Biological Characteristics Of Deletion Mutants Of Genes For Biofilm Formation From Salmonella Pullorum

Avian salmonellosis is a variety of acute and chronic diseases caused by one or more than one member of the genus salmonella, which presents as septicemia and enteritis in infected animals. Salmonella can form biofilm, which can increase bacterial resistance to adverse conditions, and is of important reasons for bacterial persistent infection and recurrent infection. In this study, salmonella strains from animals were isolated and identified, and their distribution of serotypes, biofilm-forming ability and antimicrobial resistance were investigated. Further, four deletion mutants with genes related to biofilm forming ability were constructed using Red homologous recombination method, and the relationship between biofilm and salmonella pathogenicity was discussed. This study provided us an experimental basis for further study of the pathogenic role of salmonella biofilm.1. Analysis of serotype, biofilm-forming ability and antimicrobial resistance of Salmonella strains isolated from animalsSalmonella strains were isolated from diseased animals, and identified by PCR combination with slide agglutination test and sequence analysis of16S rRNA gene. Biofilm-forming ability of the isolates was detected by crystal violet assay, and antimicrobial resistance was determined by antibiotic susceptibility test. Fifty-eight strains were identified as Salmonella and belonged to seven subtypes, including pullorum, typhimurium, enteritidis, paratyphi-C, paratyphi-B, dublin and agona. The chickens were mainly infected by S. pullorum, secondly by S. enteritidis, whereas the waterfowls were mainly infected by S. typhimurium. The results of biofilm formation test showed that51.72%of the salmonella isolates could form biofilm, in which83.3%of S. typhimurium could form biofilm. The susceptibility test of19antibiotics (including Aminoglycosides, Sulfonamides, Quinolones, Lincosamides, Amphenicols, Penicillins, Tetracyclines and Cephalosporins) revealed that all of the strains were resistant to lincomycin, and51.72%of them were resistant to four and more than four antimicrobials, in which a strain of S. typhimurium displayed a high level of resistance to all test antibiotics. So, we think that the dominant serotypes of Salmonella that ate prevalent in poultry are S. pullorum, S. typhimurium and S. enteritidis. The Salmonella with both biofilm-forming ability and multiple drug resistance will bring more serious threat to the control of poultry diseases and public health.2. Construction and biological characteristics of deletion mutants with biofilm formation related genes from S. pullorumA S. Pullorum strain S6702with strong biofilm-forming ability was selected as parental strain and four mutants with deficiency of genes including csgA, csgD, adrA and bcsA were constructed using the Red recombination system. Four deletion mutants were confirmed by PCR and RT-PCR amplification, combined with gene complementation. Growth curve revealed that the growth rates of four mutants were as same as wild-type strain S6702. A quantitative microtitre assay with crystal violet staining in96-well plate showed that the△csgA and△csgD lost biofilm-forming ability completely, while the△adrA and△bcsA had similar biofilm-forming ability as wild-type strain S6702. We also found that the wild-type strain and mutants△adrA and△bcsA had stronger biofilm-forming ability in TSB culture medium than that in other culture mdium. These results indicated that the biofilm-forming ability of S. Pullorum was related with genes csgA and csgD, which controlled the expression of curli protein, but not the genes adrA and bcsA, which controlled the expression of cellulose.3. Study on pathogenicity of Salmonella Pullorum biofilmThe adherence to and invasion of Hela cells, proliferation of macrophages of the csgA, csgD, adrA and bcsA mutants were determined. The results showed that there was no significant difference in adherence ratio, invasion ratio and proliferation ration between the mutants and wild-type strain. To determine the LD50,1-day-old SPF chickens were challenged intraperitoneally with wild-type strain and mutants. The LD50of△csgD was lower than that of wild-type strain, while the LD50of△bcsA was higher than that of wild-type strain. Bacterial distribution test in vivo revealed that the number of bacteria in spleen of chickens in△csgD group was significant higher than that in other groups at6h postchallenge, while the number of bacteria in bile of chickens in△bcsA group was significant lower than that in other groups at48h postchallenge. The persistent infection experiment indicated that the S. pullorum strains were isolated in the chickens at for8-9weeks, most of them were isolated from biles. The percentage of bacterial isolation in the groups of△adrA and△bcsA which had biofilm-forming ability was higher than that in the groups△csgA and△csgD which was deficiency of biofilm-forming ability. In conclusion, there was no direct relationship between the pathogenicity of S. pullorum in acute infection and the biofilm formation. Biofilm may be related to the persistent infection of the bacteria, and Salmonella persisted in the chickens was found primarily in the bile.