Study On The Influencing Factors And Mechanism Of Salmonella Biofilm Formation

Part Ⅰ The effect of Salmonella typhimurium plasmid on Salmonella biofilm formationObjective:The ability of Salmonella typhimurium biofilm formation on different surfaces contributes greatly to its pathogenicity. Our study was to investigate the effect of Salmonella typhimurium (S. typhimurium)100kb plasmid on the S. typhimurium biofilm formation on polystyrene and glass surfaces, which can provide the theoretical and experimental basis for further studying on the pathogenic mechanism of the plasmid and discovering new antibacterial agents.Methods:(1) The optimum medium for Salmonella typhimurium biofilm formationDiluted logarithmic phase bacterial suspension of wild type S. typhimurium strain carrying100kb plasmid was cultured in96wells and24wells with glass discs placed at the bottom at30℃using TSB and LB respectively for biofilm formation on polystyrene and glass surfaces for48h. Semiquantitative crystal violet stain method was used determine the optimum medium for biofilm formation on polystyrene and glass surfaces at the indicated intervals.(2) Construction of GFP-labeled strain and determine the growth curve of wild type strain and mutant strainThe strains labeled with green fluorescent protein (GFP) were constructed used the method described by Ma et al. Transformants were selected by plating on LB supplemented with Amp at the concentration of100μg/ml, the expression and uniformity of GFP label cells were determined with an fluorescence microscope. The single colonies of wild type and mutant strains were cultured in LB and the growth curve of the strains were determined.(3) Effect of plasmid on S. typhimurium biofilm formation on different materialsWild type S. typhimurium strain carrying100kb plasmid and mutant strain were used to build biofilm formation model according to the condition acquired by above experiments. Semiquantitative crystal violet stain method was used to evaluate the biofim formation ability of wild type strain and mutant strain on polystyrene and glass discs at the indicated intervals. Confocal laser scanning microscopy was used to observe the biofilm formation ability of wild type strain and mutant strain on round glass discs.Results:(1) The optimum medium for Salmonella typhimurium biofilm formationThe OD590of wild type strain cultured in LB was higher compared with TSB. Therefore, LB was used as the optimum medium to allow bacteria biofilm formation in the following experiment procedures.(2) Construction of GFP-labeled strain and determine the growth curve of wild type strain and mutant strainThe results of fluorescence microscope showed that both the wild type strain and mutant strain were able to expressing GFP protein. Furthermore, no difference of growth curve was observed between the GFP-labeled strains and their corresponding parental strains.(3) Effect of plasmid on S. typhimurium biofilm formation on different materialsThe result of semiquantitative crystal violet stain method showed that the OD590of wild type strain carrying100kb plasmid was higher than mutant strain on glass discs surfaces at6h,12h,24h and48h, when cultured in LB medium (P<0.01). However, no difference of OD590was observed on polystyrene surface (P>0.05). When observed in CLSM, wild type strain tended to form more bigger colonies on round glass discs and the average thickness and maximum thickness was25μm and30μm. For the mutant strain, only some small colonies were observed and the average thickness and maximum thickness was15μm and18μm. Conclusions:The carriage of100kb plasmid leads to increased Salmonella typhimurium biofilm formation on glass discs, however, no significant increased biofilm formation on polystyrene was observed. Here, we demonstrate a new link between plasmid carriage and biofilm formation on hydrophilic material, which indicated BF formation is correlated with surface structure of materials besides bacterial adhensive structures. Part Ⅱ Effect of extracellular DNA on the biofilm development of Salmonella and its releasing mechanismObjective:To detect and characterize extracellular DNA (eDNA) in Salmonella typhimurium SR-11and Salmonella typhi ST6biofiom matrix and study the role of eDNA in Salmonella biofilm formation, its effect on biofilm integrity and explore the the releasing mechanism of extracellular DNA, which can provide the theoretical and experimental basis for controlling Salmonella infection, discovering new drug target and inventing disinfectants.Methods:1. Detection of extracellular DNA in Salmonella biofilm matrix(1) Detection of the molecular weight of eDNA and quantitative analysis of eDNA in biofilm matrixDiluted logarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were cultured in6wells at30℃under static condition over48h. Biofilm samples were collected by scraping the biomass from the well surface with a cell scraper at the indicated intervals. The collected biofilm samples were then treated by proteinase K and cellulase to determine the molecular weight by agarose gel electrophoresis and the concentration of eDNA by ultraviolet spectrometry in Salmonella biofilm matrix.(2) CLSM analysis the location of eDNA in Salmonella biofilm matrixDiluted logarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were inoculated in24well with round glass discs placed at the bottom of the wells at30℃for48h and then the biofilms formed on the round glass discs were stained by DDAO to determine the location of eDNA in Salmonella biofilms.2. Role of eDNA in Salmonella biofilm formation and on biofilm integrity(1) Microtitre plate assayDiluted logarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were inoculated in96wells and DNase I (50μg/ml) was added at the inoculation at30℃for48h. Semiquantitative crystal violet stain method was used to evaluate the role of DNase I in Salmonella biofilm formation at different amounts of time during48h.Diluted logarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were inoculated in96wells and eDNA (5μg/ml) was added at the inoculation at30℃for48h. Semiquantitative crystal violet stain method was used to evaluate the role of DNase I in Salmonella biofilm formation at different amounts of time during48h.Diluted logarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were inoculated in96wells at30℃to allowed biofilm formation for48h. Then the biofilms were treated by DNase I (50μg/ml), nontreated as a control. Semiquantitative crystal violet stain method was used to evaluate the effect of DNase I on biofilm integrity.(2) CLSM analysisDiluted logarithmic phase bacterial suspension of GFP-labeled wild type Salmonella typhimurium SR-11and wild type Salmonella typhi ST6were inoculated in the24wells with round glass discs placed at the bottom of the wells. DNase I (50μg/ml) was added at the inoculation and allowed to incubate for48h under static conditions to performe CLSM analysis. For experiments using DNase Ⅰ(50μg/ml) on established biofilms, diluted logarithmic phase bacterial suspension of GFP-labeled wild type Salmonella typhimurium SR-11and wild type Salmonella typhi ST6were inoculated in the24wells with round glass discs placed at the bottom of the wells for48h. Then two days biofilms formed by SR-11and ST6were treated with/without DNase (3) Effect of DNase I on viable count in the biofilmsLogarithmic phase bacterial suspension of wild type Salmonella typhimurium SR-11and Salmonella typhi ST6were diluted to OD600of0.05and inoculated into6well,3ml per well. DNase I (50μg/ml) was added at the inoculation at30℃and allowed to incubate for48h under static conditions. Colony forming unit enumeration was performed to evaluate bacterial cell viability of the sessile phase on the well surface and planktonic phases during biofilm formation.3. Analysis the source of extracellular DNA through RAPDGenomic DNA extracted by phenol chloroform extracting method and eDNA was performed RAPD analysis using two different primers (P1254,5’-CCGCAGCCAA-3’;23L,5’-CCGCAGCCAA-3’) to determine the sourse of eDNA.Results:1. Detection of extracellular DNA in Salmonella biofilm matrix(1) Wild type Salmonella typhimurium SR-11and Salmonella typhi ST6biofilm matrix contain extracellular DNA and both of their molecular weight were higher than10.0kb. Furthermore, the concentration of extracellular DNA in the wild type strain and mutant strain were increasing as time goes on and remarkable more eDNA was observed in SR-11biofilm matrix than in ST6biofilm matrix (P<0.01).(2) After48h of incubation, the biofilms were stained by DDAO which binds to extracellular DNA or DNA inside the cells that have compromised membranes. The results showed that biofilms formed by wild type strain Salmonella typhimurium SR-11were mainly composed of cells which were not stained by DDAO, and thus have impermeable membranes. In addition to the green-staining live cells, bright red-staining dead cells were observed along with a more diffuse weaker red stain which is indicative of eDNA. Similar to SR-11, ST6biofilms exhibited both bright red-stain interspersed among the GFP-expressing live cells and comparatively less red eDNA was observed in ST6biofilm matrix.2. The role of extracellular DNA in Salmonella biofilm formation and on biofilm structure integrityThe results of Semiquantitative crystal violet stain method showed that the presence of DNase I in the culture medium at the time of inoculation resulted in a time-dependent effect on the ability of both the species to form biofilms. At the early time-point of6h, the addition of DNase I had no significant impact in enhancing biofilm growth for the strain ST6. In contrast, at12h or later of biofilm growth, DNase I significantly enhanced the ability of both SR-11and ST6to form biofilms. Furthermore, the addition of extracellular DNA (5μg/ml) inhibited Salmonella biofilm formation compared to the control group (P<0.01). However, for the established biofilms, no difference was observed with/without DNase Ⅰ treatment (P>0.05). For both SR-11and ST6strains, after incubation for48h in the presence of DNase I, the glass discs was extensively colonized resulting in the visualization of a thicker layer of cells compared to the non-treated controls. However, no difference was observed between the DNase Ⅰ (50μg/ml) treated and non-treated two day old biofilms.The analysis of CFU assay performed on sessile phases both on DNase Ⅰ-treated biofilms and controls of each examined strain showed great differences (P<0.01). However, no difference was observed for the planktonic phases for both strains (P>0.05).3. RAPD analysisThe RAPD analysis performed on the two Salmonella strains displayed great similarity between the eDNAs and their corresponding genomic DNA fingerprintings, which indicated that genomic DNA is the source of eDNA.Conclutions:Both the Salmonella strains biofilms contain eDNA, their molecular weight is higher than10.0kb. Extracellular DNA inhibits Salmonella biofilm formation, however no effect of eDNA on the biofilom integrity was observed. The main source of eDNA is from genomic DNA.