The Effects Of Salmonella Typhi Conjugative Plasmid On Biofilm Formation In Vivo And In Vitro

Objective:To investigate the effects of plasmid pRST98, which was originally isolated fromSalmonella enterica serovar typhi (S.typhi), on biofilm (BF) formation, theexperiments were performed both in vitro and in vivo. It provides the theoretical andexperimental basis for controlling BF related diseases and discovering new drugs.Methods:Ⅰ. Experiments in vitro1. The effects of plasmid pRST98originally isolated from S.typhi on biofilmformation in different strainsSalmonella enterica serovar typhimurium (S.typhimurium) wild-type strainχ3306、virulence plasmid-deleted S.typhimurium strain χ3337, Escherichia coli (E.coli)K12W1485and pRST98-transconjugant S.typhimurium χ3337/pRST98, E.coliK12W1485/pRST98were used in this section. All the strains were cultured at30℃for3dto compare the formed BF by using crystal violet staining method, semi-quantitativemeans, and scanning electron microscopy (SEM) was used to observe BF structure andconfocal laser scanning microscopy (CLSM) was used to compare BF thickness.2. The effects of different concentrations of IL-10on biofilm formationOur previous studies discovered that the conjugative plasmid pRST98can promoteIL-10expression in vitro and in vivo.1ng/ml,10ng/ml and100ng/ml IL-10wereadded and0ng/ml IL-10was set as a control. Crystal violet staining method,semi-quantitative means, CLSM and SEM were used to determine the effects of IL-10.Ⅱ. Experiments in vivo S.typhimurium is used as surrogate for S.typhi, since S.typhi only leads to humaninfections, and no suitable model has been setup for investigation of S.typhipathogenesis. S.typhimurium, was employed to set up the mouse infection model for itsclose relative to S.typhi. To tract the route of infected bacteria in mice, thebioluminescent strains of S.typhimurium were constructed by electroporation ofplasmid pBEN276containing a constitutive lux expression cassette, and the followingrecombination of lux expression cassette within bacterial chromosome. Six-toseven-week-old female BALB/c mice were subcutaneously inoculated with CT26cellsat the pre-abdomen site, followed by injecting intravenously with S.typhimuriumχ3337lux or χ3337lux/pRST98. Normal mice injected with χ3337lux/pRST98were set as acontrol. In-Vivo Imaging was performed on1d,2d and3d post infections (p.i.) usingIn-Vivo Imaging System FX Pro (IVIS) to observe the route of injected bacteria inmice. Mice at3d p.i. were sacrificed, and tumor, livers and spleens were collected forSEM and colony forming unit (CFU) analysis. Secreted IL-10in serum were assayedby ELISAat desired points.Results:Ⅰ. Experiments in vitro1. The effects of Salmonella typhi plasmid pRST98on BF formation: Bacteriaharboring pRST98developed slimy and viscous pellicles, while pRST98-free bacteriaformed loose and less coherent BF. Semi-quantatitive analysis showed that BF formedby S.typhimurium carrying pRST98was significantly robust, compared to those withoutpRST98in S. typhimurium (P <0.05). E.coli K12W1485/pRST98had stronger ability to formBF than E.coli K12W1485(P <0.05). There was no difference between χ3306and χ3337(P>0.05).2. The ability of BF formation in different strains harboring pRST98: Salmonelladeveloped thicker BF than E. coli did, and the difference was even more significantwhen both Salmonella and E. coli harbor pRST98(P <0.05).3. The effects of IL-10on biofilm formation: Compared with control group, bacteria treated with1ng/ml and10ng/ml of IL-10were more gathered and the abilityof BF formation was improved. The bacteria were coated with denser matrix especiallyin the presence of10ng/ml IL-10(P <0.05). BF was significantly promoted asindicated by SEM, which showed bacteria formed three-dimensional BF structure, andsurrounded by mass matrix. IL-10in100ng/ml inhibited bacterial BF formationcompared with control group (P <0.05). The effects of different concentrations ofIL-10on χ3337/pRST98BF formation were greater than χ3306and χ3337.Ⅱ. Experiments in vivo1. The effects of lux on bacterial growth and biofilm formation: lux gene showedno effects on bacterial growth; genelux had no effects on BF formation as indicated bycrystal violet staining method and semi-quantitative means (P>0.05).2. The dissemination of bacteria in tumor bearing mice: Once intravenouslyinfecting mice, S.typhimurium quickly circulated with the blood in the body. Tumorcolonizing χ3337lux and χ3337lux/pRST98were detected by IVIS at3d p.i., andχ3337lux/pRST98in tumor emitted stronger bioluminescence signals than χ3337lux did.3. Biofilm formation in tumor: SEM showed tumor colonizing χ3337lux/pRST98developed BF, while χ3337lux only scattered in tumor.4. Changes of organs and viable bacteria count: Livers and spleens in bothinfected groups were showed enlargement, and more severe pathological changes werediscovered in χ3337lux/pRST98; the number of viable χ3337lux/pRST98in tumor wasalso higher, and livers and spleens from mice infected with χ3337lux/pRST98wereloaded with more bacteria as well (P <0.05).5. IL-10assay by ELISA: The expression of IL-10was gradually increased in therange of1-5d. IL-10level was significantly higher in χ3337lux/pRST98than χ3337lux(P <0.05).Conclusions:1. The S.typhi plasmid pRST98can promote BF formation in S.typhimurium and E.coli. It showed that E.coli K12W1485/pRST98had weak ability in BF formation, compared with S. typhimurium harboring pRST98.2. The S.typhi plasmid pRST98can promote bacteria to colonize tumor and developBF in animal.3. The effects of S.typhi plasmid pRST98on BF formation were related to IL-10level in some degree.