Experimental Studies On Disinfectant Resistance To Vibrio Vulnificus And Immunity Of O-antigen, Cytotoxin Antigen

Vibrio vulnificus(V.vulnificus,Vv) is a species of grarrmegative,motile,curved bacterium that is part of the Vibrio genus and the Vibrionaceae family.Based on phenotypic characteristics and host criteria,V.vulnificus isolates have been grouped into three different biotypes.We studied strains from biotype 1 which have been associated with pathogenicity in humans,having a positive indole reaction,and are serologically heterogeneous.This gram-negative bacterium is an opportunistic human pathogen.This virulent,bacterium causes two distinct syndromes.The first is an overwhelming primary septicemia caused by consuming raw or undercooked seafood, particularly raw oysters.The second is a necrotizing wound infection acquired when an open wound is exposed to warm seawater with high concentrations of V.vulnificus. Those with primary infection,develop sepsis and severe cellulitis with rapid development to ecchymoses and bullae.In severe cases,necrotizing fasciitis can develop.Case-fatality rates are greater than 50 percent for primary septicemia and about 20-30 percent for wound infections.It is often found in patients with a high plasma iron level.In immunocompromised individuals,V.vulnificus is often fatal. Physician awareness of risk factors for V.vulnificus infection combined with prompt diagnosis and treatment can significantly improve patient outcomes.Since these diseases share the characteristics that the bacteria multiply extremely rapidly in host tissues resulting in extensive tissue damage and high fatality rate.How can we reduce disease incidence and high fatality rate due to V.vulnificus? Sterilization and activity immunity maybe the effective approach to prevent infecting V.vulnificus.METHODS1.biochemistry characters and resistance of Vibrio vulnificus(1) Cultivate fresh inoculation of Vibrio vulnificus in the biochemistry assessor of Vibrio,read the results after 24h.(2) The diluted suspension and flat plate of V.vulnificus ATCC27562 and Escherichia coli ATCC25922 were directly irradiated for different time to the UV with 90μW/cm~2 intensity,Finally,the viable organism were enumerated both before and after irradiation,the average kill rate were calculated and the curve of inactivation(t-△log) were drawed then.(3) DGHM test was in progres on Glutaraldehyde,peracetic acid and ethanol to Vibrio vulnificus for minimum inhibitory concentration(MIC) and minimal bactericidal concentration(MBC),the qualitative suspension test of V.vulnificus in the fastest effective bactericidal timing.2.Extraction,purification and identification of the rVVCWe obtain the positive clone pET-32a-vvhA/E.coliBL21(DE3) by Blue spot screening,then choose a white plaque for PCR amplification and agarose gel electrophoresis to verify the existence of the target gene.Then the recombination proteins were expressed under the optimized inducing condition.The induced E.coli BL21(DE3) were mechanical lysised by sonication.Then the recombination proteins of V.vulnificus cytolysin(rVVC) with His-tag in the cell lysis were purified by binding to Ni-IMAC resin.The purification of recombination proteins were detected by sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE) and Western-blot using antibody against His-tagged.The concentration Purify rVVC were calculated using BCA protein quantitative method.3.The animal experimental study on the active immunization of V.vulnificusThere were three groups in experiment:Vv inactivated O-antigen group(OG), rVVC protein antigen group(PG) and control group(CG),18 BALB/c mice per group. Three groups mice were hypodermic injection Vv inactivated O-antigen 1×10~8 cell, rVVC protein antigen 50μg or NS 0.2ml per mice respectively,totall 6 time.Then we assayed the subsets of the lymphocytes(CD4-T cell,CD8-T cell and CD19-B cell) in peripheral blood of the 3 mice per groups with flow cytometry.We assayed the transformation efficiency of the splenocytes of the three groups of(6) mice with MTT chromatometry.We assayed antibody IgG in the sura of the mice by ELISA.We also detect the specific antibody by serum agglutination test and western-blot using antibody against V.vulnificus.Finally,the mice(12mice/group) were attacked by the live V.v about 10~7-10~8 live cell.Live V.v were intraperitoneal injection into all the mice,then we observe general state of health of the mice,three groups mice case fatality rate and survival rate were valued.The autopsy were carried out when the mice died or been infected with V.vulnificus in 48h or 10d,and hemoculture for the pathogenic bacterium were undergone in the same time.The tissues were made to paraffin blocks then were sliced and HE stained.RESULTS:1.Biochemistry characters and resistance of V.vulnificus(1) The biochemistry characters of Vv-ATCC27562 was coincidence with the V.vulnificus perfectly,%id=99.9 T=0.74(2) V.vulnificus exposured to the UV with 90μW/cm~2 intensity,qualitative cultivation were negative for 15min;after exposure to it for 1 min,the average log inactivation value of Vibrio vulnificus viable organism in suspension and flat plate were4.37±0.13,5.10±0.19 and the average kill rate were(99.99±0.01)%, (99.99±0.05)%respectively.None of V.vulnificus were survival for 15min. Escherichia coli exposured to the UV after 1min,the disinfection level(LIV≥4.00 lg) had been achieved,None of Escherichia coli were survival for 10min.(3) To V.v,glutaraldehyde on the MIC for the 0.31g/L,MBC 10.00g/L;peracetic acid on the MIC was 0.04g/L,MBC is 0.31g/L;ethanol 4.69%for the MIC,MBC was 37.50%;V.v were be the effective killing for 20g/L glutaraldehyde,5g/L peracetic acid and 75%ethanol in the 10s,10s and 60s respectively.2.Extraction,purification and identification of the rVVCThe fragment of vvhA gene in the positive clone E.coliBL21(DE3)were amplified by PCR,we obtain the 1356bp gene fragment on agarose gel.The rVVC protein could be highly expressed in E.coli BL21(DE3) under the optimized inducing conditions.The rVVC protein were certified purified by SDS-PAGE.A 70-kD band was observed in gels,matching the predicted molecular weight.The recombination protein rVVC expressed with His-tag was confirmed by Western-blot.The concentration of the purified protein with His-tag was 2.542mg/ml.3.The animal experimental study on the active immunization of Vibrio vulnificus(1) The spleen weight of PG were higher than that of control group,and there is significant difference(P=0.004),but there is no significant difference between OG and CG.There is no significant difference in efficiency of the thymic weight among the three groups of mice.(2) The efficiency of the splenocytes of OG in ConA and LPS were higher than that of control group,and there were significant difference(P=0.018,P=0.001);but there is no significant difference in Vv.There is no significant difference between PG and CG in efficiency of the splenocytes.(3) There is significant difference in CD19~+B cell subsets of the lymphocytes in peripheral blood among the three groups(P=0.03),the percentage of CD19~+B cell subset of OG and PG were higher than that of control group.There is no significant difference in the CD4~+T cell and CD8~+T cell subsets of the lymphocytes in peripheral blood.(4) The results of serum agglutination test of the mice's antisera:the OG and the PG were positive,the CG were negative.(5) The valence of anti-O IgG in antisera of OG were 1:25600,was higher than that of control group,and there were significant difference.The valence of anti-rVVC IgG in antisera of PG were 1:25600,was higher than that of control group,and there were significant difference.(6) The specific antibody detected by western-blot:The rVVC can be specific binding with antisera on PVDF tissue,confirm that mice can produce specific antibody against rVVC.(7) The general state of health of mice since been intraperitoneal injection with live V.vulnificus:there were 10 mice died in 36h,2 mice were survival,the case fatality rate and survival rate were 83.33%and 16.67%.One survival appear cellulitis and ulcer on the left posterior limb.All the mice of OG and PG were survival,survival rate were 100%,higher than that of control group and there were significant difference (P=0.0004).The result of hemoculture on control group were positive,and V.vulnificus was testified to be the pathogenic bacterium by biochemistry characters tests.The result of hemoculture on OG and PG were positive in 48h but were negative in 10 day.Pathological changes:The control group died with internal organs tissues serious bleeding and congestion.Partial tissue was liquefied necrosis,on which we can observe neutrophilic granulocyte.All pathological changes were most serious in heart, lung,liver,kidney.The surviving mice suffer cellulitis in musculature.After Vv intraperitoneal injection,multiple organ congestion were observed on the OG and PG, the damage were lighter than that in the CG.And inflammatory cells infiltration can been observe in partial tissue.Partial tissue was necrotic in liver of PG mice in 10d.CONCLUSION1.V.vulnificus ATCC27562 was sensitive to ultraviolet,belongs to the UV-resistant low-grade bacteria;It can be rapidly inactivated effectively by 20g/L glutaraldehyde,5g/L peracetic acid and 75%ethanol.2.V.vulnificus inactivated O-antigen,V.vulnificus cytolysin protein antigen may be the potentiality vaccine of the V.vulnificus,since they can effectively stimulate and enhance BALB/c mouse specific humoral immune function,and support mice to obtain effective immune protection.3.Lethal dose of V.vulnificus violates BALB/c mice,V.vulnificus anti-O and rVVC antibody can not completely kill the pathogenic bacteria,but can reduce its toxicity,improve the survival rate of mice and reduce disability significantly.