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

Salmonellosis is a large group of acute and chronic diseases caused by Salmonella, which presents as septicemia and enteritis in infected animals. In China there are pullorum disease、fowl typhoid and paratyphoid fever coexist in clinic, which are not only economic problem in all the period of poultry industry but the barrier for the trade of the poultry. A number of studies have demonstrated that Salmonella are capable of forming biofilms on a wide variety of contact surfaces, and the formation of biofilms by bacteria may improve the ability of these organisms to resist disadvantages such as desiccation, extreme temperatures, antibiotics, and antiseptics. Salmonella enterica serovar Enteritidis (Salmonella Enteritidis, S. enteritidis) has emerged as one of the most important food-borne pathogens for humans and is mainly associated with consumption of contaminated poultry meat and eggs. The biofilm allows Salmonellae to survive long-term in the poultry farm environment and contaminate poultry meat and eggs, which remain the leading vehicles of food-borne salmonellosis outbreaks. In this study, we identified some genes for biofilm formation of S. enteritidis biofilm, explored the relationship between biofilm and pathogenicity preliminarily. It will provide some useful information for further research on pathogenicity of S. enteritidis biofilm and development of potential vaccine candidate against S. enteritidis.1. Identification of genes for biofilm formation in a Salmonella Enteritidis strain by transposon mutagenesisSeventy-four strains of S. enteritidis, S. pullorum and S. gallinarum isolated from chickens were determined for biofilm using crystal violet staining, eighty-four percent of these Salmonella strains produced biofilm. The strain CMCC50041with well biofilm formation was chosen to construct a mutant library using transposon mutagenesis. We screened1924mutants with transposon insertion, and15inserted genes were identified by growth curve determination of mutants, sequence analysis of the chromosomal DNA, and further confirmed by southern blot. These genes included metE, ompR, rpoS, rfaG, rfaJ, rfaK, rfaP, rfbH, rhlE, spiA, steB, tpx, ybdN and other two genes with unknown function. Among them,12genes associated with Salmonella biofilm formation were reported firstly, these findings may help to understand the regulation mechanism of biofilm formation and develop an attenuated Salmonella vaccine.2. Construction and biological characteristics of the ompR gene deletion mutant from Salmonella EnteritidisA ompR mutant of S. enteritidis was constructed by suicide plasmid pGMB151, and the ompR mutant was confirmed by RT-PCR and pattern of outer membrane protein. Biofilm forming ability of the mutant was detected by crystal violet assay and scanning electron micrography, cellulose was detected by bacterial culuture on plates supplemented with Calcofluor, and curli was determined by expression of curli protein.The results showed that the mutant did not produce biofilm as well as cellulose and curli. These data indicates that the ompR gene was involved in biofilm formation of S. enteritidis by down regulation of cellulose and curli expression.3. Construction and biological characteristics of the spiA gene deletion mutant from Salmonella EnteritidisIn this study we constructed a spiA gene mutant with a suicide plasmid pGMB151. Phenotypic and biological analysis revealed that the spiA mutant was similar to the wild type strain in growth rate, morphology. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and under scanning electron microscopy, significantly decreased curli production. These data indicates that the spiA gene is involved in biofilm formation of S. enteritidis by down regulation of curli expression.4. Construction and biological characteristics of the ompR-spiA gene deletion mutant from Salmonella EnteritidisA ompR/spiA mutant of S. enteritidis was constructed by suicide plasmid pGMB151. The ompR-spiA mutant was confirmed by RT-PCR and pattern of protein of ompR, spiA gene.The growth curve showed that the growth rates of the ompR/spiA mutant was similar as the spiA mutant. Biofilm forming ability of the mutant was detected by crystal violet assay and scanning electron micrography. The mutant did not produce cellulose, curli, and biofilm. The ompR/spiA mutant was suitable for the future study of S. enteritidis virulence.5. Virulence-determination of different genes deletion mutants from a Salmonella Enteritidis strainVirulence of the ompR、spiA、ompR/spiA mutants were determined by assay of adherence to and invasion of epithelial cells, proliferation of macrophages and mouse challenge experiments. The mutants showed similar adherence percentage to and invasion percentage of epithelial cells as wild type strain, but significantly decreased intracellular proliferation of macrophages during the biofilm phase.The result of intraperitoneal challenge of bacteria in BALB/c mice revealed that LD50s of the spiA mutant with both phenotypes of logarithmic growth and biofilm phases were107.47and107.13CFU, while those of the wild-type strain were10"003and100.13, respectively. The wild-type strain in biofilm phase was more virulence than that in logarithmic growth phase, howere, the spiA mutant in biofilm phase was less virulence than that in logarithmic growth phase. In addition, LD50s of the ompR、ompR-spiA mutant in logarithmic growth phases were106.67CFU,107.13CFU, respectively. Therefore, the ompR、 spiA、ompR/spiA mutants were attenuated. These data suggest that ompR and spiA gene are involved in biofilm formation and virulence of Salmonella Enteritidis.6. Immunoprotection against Salmonella Enteritidis C50041in miceThe mutants and wild-type strain were inoculated in BALB/c mice from oral adminstration, and LD50s were determinated. Consequently, the LD50s of ompR、spiA、rpoS、ompR/spiA、ompR/rpoS mutants were all more than109CFU, and that of the wild strain was105CFU. To futher investigate the distribution of bacteria in vivo, the mice were inoculated108CFU by oral route, the numbers of the wild-type strain present in livers, spleens, and peyer’s patches were significantly higher than that of the mutants and the infected BALB/c mice died in a week (p<0.05). The double deletion mutants in livers were eliminated, but they were dectable in spleens and peyer’s patches by the end of a month. The single deletion mutants were detected in livers, spleens, and peyer’s patches all the time. The observation of pathological section showed that the wild-type strain caused serious pathological lesions in livers and spleens while the mutants caused mild pathological lesions. All the mutants induced the high IgG antibody in sera, which reached the peak at3weeks postimmunization.ln addition, the spiA mutant induced IgA antibody in sera, ompR/rpoS mutant induced igA antibody in mucosa. When the immunized mice were infected100times LD50of the wild-type strain, at4weeks postimmunization, the protection rates of the o spiA、rpoS、ompR/rpoS and ompR/spiA mutants groups were62.5%、50%50%、37.5%and50%, respectively. Due to deficiency of biofilm forming ability and higher protection rate, the ompR mutant may be a potential vaccine candidate against S. enteritidis.