The Effect Of Salmonella Typhi Plasmid On Bacteria Virulence In Different States And E. Coli Biofilm Formation In Vivo

Objective:Biofilm formation of S. typhimurium and E. coli are available to all kinds materialsurface, which is important for pathogenicity of bacteria. The study researched thebiofilm formation of S. typhimurium and E. coli influences bacterial inherence andinvasion capability and virulence, as well as S. typhi plasmid pRST98impact on hostbacteria virulence in the BF state. And a model of mouse urinary catheter was created tostudy the impact of S. typhi plasmid pRST98for E. coli BF formation in vivo.Methods:1. The effects of biofilm formation on bacterial phenotypesThe tested bacteria STM, STM/pRST98，E. coli K12W1485and K12W1485/pRST98werecultured under suitable conditions for3d to form mature BF, then dispersion bacteria inBF using ultrasonic. The planktonic bacterial was prepared in the routine method. BFbacteria and planktonic bacteria were adjusted to the same concentration then carried onadhesion and invasion experiments with CT26, anti-serum killing experiments andanti-macrophage experiments to compare bacterial inhesion and invasion capacity andvirulence in different state.(1)Adhesion and invasion experimentsThe tested bacteria STM，STM/pRST98，E. coliK12W1485and K12W1485/pRST98wereco-culture with CT26with MOI100:1for1hour, discard the broth and washed withPBS for3times, then broke the cell wall with0.2%Triton-X100and carried on theCFU test to compare the inhesion capacity. The invasive test basically was carried outin the same way of inhesion experiments, except after discarding the broth and washingcells with PBS for3times, adding RPMI1640containing AMK to culture3hours thencarry on CFU assays.(2) Anti-serum killing assays.The tested bacteria in different state were diluted to1×104CFU/ml,20ul bacteriawere respectively added the tubes containing0.2ml serum and the tubes containing0.2ml broth as the CFU control. According to the results of pre-experiment, we have chosen37℃and2hours as the killing time. Then CFU assay was carried on to countthe number of survival bacteria.(3) Macrophage survival testsAs an intracellular bacterial, S. typhimurium can live in the intracellular ofmacrophages. Thus anti-macrophage experiment was used to compare the virulence ofbacteria in planktonic state and the state of BF. The concentration of tested bacteria wasadjusted to107CFU/ml. The J774A.1was adjusted to5×105cell/ml, and added to cellculture plates, culture1hour in the conditions of37℃，5%CO2, then washed J774A.1with RPMI1640containing100μg/ml AMK for3times to discard the extracellularbacteria, suspended with RPMI1640containing100μg/ml AMK, at last J774A.1wascleavaged to carried on CFU assays at different point.2. pRST98promotes E. coli BF formation in vivo.(1) Building mouse urinary catheter model.PE10catheters were co-cultured with the same concentration of bacteria E. coliK12W1485and E. coli K12W1485/pRST98for24h after disinfection treatment, so that thetested bacteria adhere to the surface of the catheter.6-8weeks mice were anesthetizedand fixed, around the urethra were disinfected with75%ethanol. And then PE10catheter prepared in advance is slowly inserted the mouse urinary tact and pushed to themouse urinary bladder.(2) Using SEM, CLSM and CFU methods detect the differences of BF formed byE. coli K12W1485and E. coli K12W1485/pRST98on the catheter.Observation the BF using CLSM: PE10catheter removed from the mouse at the5d after infection, washed three times with PBS and then dyed with acridine orange at aconcentration of0.01%in the dark for10min, washed with PBS buffer3times, and thecatheter is placed on a slide, observed under the CLSM.Observation the BF with SEM: on the5th day after infection, the mice weresacrificed and the PE10catheter in the bladder were removed, gently washed threetimes with PBS, and then fixed for2h in2.5%glutaraldehyde solution,4℃, thenrinsed three times with0.1mol/L of phosphate buffer, after fixed with1%osmiumtetroxide for1h, with a continuous gradient tbutanol solution was dried, the catheter iscarried out after the critical point drying and gold was observed under SEM.Comparison the number of living bacteria in BF by CFU: the catheters were obtained according to the above method, after gently rinsed with PBS, then placed in1.0ml PBS, worked20min in an ultrasonic vibrating apparatus, so that the biofilmbacteria scattered to the PBS solution, at last CFU experiments was carried on to detectthe number of viable cells in the biofilm.(3) Comparison the pathological changes of these two groups of infected mice.On the8th d of inserting the catheter, each group mice were anesthetized andsacrificed. The liver and kidneys were taken, fixed in10%formalin. After ethanoldehydration, xylene, embedded in paraffin and other steps before slicing patch, at last,after HE staining was observed under the microscope after.Results:1. The effects of biofilm formation on bacterial phenotypes(1) BF influences bacterial inherence and invasion capacity.BF promotes all tested bacteria inherence to CT26except for E. coli K12W1485;Also found in the state BF and the planktonic, S. typhi plasmid pRST98has no effect onadhesion of bacteria to the CT26. Adherence capacity of Salmonella to CT26is strongerthan E. coli. Invasion assay showed that BF formation of S. typhimurium and E. colidecreased invasiveness to CT26cells. In addition, in BF and planktonic S. typhi plasmidpRST98has no significantly effects on bacterial invasion to the CT26cell.(2) BF promotes bacterial resistance to serum killingThe survival rates of STM，STM pRST98in BF state were higher than the testedbacteria in planktonic state in rabbit serum and guinea pig serum. In BF state theanti-rabbit serum and guinea pig serum bactericidal capacity of STM pRST98weresignificantly higher than STM. We can see the same trend in E. coli group.(3) BF promotes bacterial survive in macrophage.Three strains of S. typhimurium survival within macrophages in BF state issignificantly higher than under planktonic bacteria, indicating that BF enhances thebacterial anti-macrophage phagocytic activity, the difference was statistically significant,(p <0.05). In addition, you can find under the BF and planktonic state STM pRST98survival within macrophages were higher than STM.2. S. typhi plasmid pRST98promotes E. coli biofilm formation in vivo.(1) Successfully establish intubation mouse model. After intubation, Mice normalactivities, the mice were sacrificed under anesthesia, PE10catheter can be obtained from the mouse bladder.(2) pRST98promotes E. coli biofilm formation in vivo.At the5d of infection, E. coli K12W1485and E. coli K12W1485/pRST98BF may beformed on the catheter, can be observed by CLSM. E. coli K12W1485/pRST98formedmature BF on catheter surface, issued strong fluorescent signal, BF signal emission by E.coli K12W1485formed in the catheter is relatively weak.PE10catheter will be removed from the mice after intubation for5d, scanningelectron microscopy was used to detected E. coli biofilm formed on the surface of thecatheter, and found E. coli K12W1485/pRST98clump each other and secrete extracellularmatrix to form BF, and E. coli K12W1485just scattered distribution of the catheter surfacecan not form mature BF.CFU quantitative experiments show that the living bacteria in E. coliK12W1485/pRST98BF is more than in E. coli K12W1485BF.(3) BF of E. coli K12W1485/pRST98formation in mice results in significantinflammatory response, apathetic, sluggish activity in mice. At the8th day of infection,the mice were sacrificed, HE staining showed that in mouse liver inflammatory cellinfiltration, lobular structural damage, a large number of lymphocytic infiltrating in thekidneys of mice and renal ball structural were damage. E. coli K12W1485has a sparse BFformation in mice, causing minor response to infection.Conclusion:1. BF formation promotes bacterial adhesion to cells while inhibiting bacterial cellinvasion force.2. BF formation enhances bacterial anti-serum killing and anti-phagocytic ofmacrophage, which enhances bacterial virulence.3. pRST98increases the virulence of bacteria in the state of BF.4. pRST98can promote BF formation of Escherichia coli in vivo, BF can enhancethe virulence of E. coli.