| Background:Water-borne diseases post a serious threat to the health of the population. According to the publishing of year 2002 by World Health Organization,80%human diseases is water-related,1.8 million people die from diarrheal diseases a year,and 88% of dead cases is related by insanitary drinking water and insanitary water hygiene status. So determination the status of microbial contamination of water and contamination levels timely and accurately is important to monitor the water sanitary situation and ensure the population health.Under the environment pressures,intestinal pathogens can inter into a viable but nonculturable state(VBNC) except for posting of adaptation,injury,dying or death. Bacteria in the VBNC state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies,but are alive and capable of renewed metabolic activity.Many bacteria including pathogens can enter this state. VBNC state of bacteria is considered to be an adaption to adverse environmental conditions.VBNC state of bacteria expressed as no colonies formed on the routine bacteriological media through heterotrophic bacteria count,bacteria posting viable state by direct microscopy examination or cell respiration detection,and bacteria's resuscitation and regrowth in host endosomatic or under the appropriate conditions for resuscitation.Since the VBNC state theory was addressed from the 80's of the last century,The number of species described to enter the VBNC state constantly increases,with approximately 60 now reported to demonstrate this physiological response.Included are a large number of human pathogens,including Vibrio cholerae,Salmonella spp, Shigella app,Listeria app,Aeromonas app,E.coli(including EHEC strains),Legionella pneumophila.Some pathogens in the VBNC state can retain virulence and pathogenicity.If the bacteria indicators of fecal pollution was in the VBNC state and could not be detected,so the concentration of indicators in water should be underestimated,and the potential health risks of pathogens should be underestimated inevitably.So it need to study about:the induction of indicator bacteria and pathogens entering the VBNC state in water environment,virulence and pathogenicity of which in the VBNC state,and the detection methods of the bacteria in the VBNC state.Objects:Using Escherichia coli,Enterococcus faecalis,Shigella dysenteriae serotype 1 as the target bacteria,to research the induction factors of bacteria in water environment so as to Control methods about collection,preservation and detection of environmental water samples;to research the differentiation about cytotoxicity and protein expression of S.dysenteriae in the VBNC state so as to investigate the protein expression character and mechanism in this state;to research the resuscitation methods of bacteria in the VBNC state so as to heighten the positive detection rate of bacteria.Content and methods:1.To research the induction factors of bacteria in water environment entering into the VBNC state through processing bacteria by low temperature,starvation,low pH,and chlorination;2.To determine the culturable bacteria count by heterotrophic plate count(HPC),to determine the total bacteria count and the viable bacteria count by LIVE/DEAD BacLight bacterium viability kit;3.To research cytotoxicity,adhesion and invasiveness of S.dysenteriae serotype 1 through infecting Vero cell and HT-29 cell in vitro;4.To research protein expression character and molecular mechanism of bacteria in the VBNC state by 2D electrophoresis and MALDI-TOF-MS analysis of bacteria proteins through comparing bacteria in the VBNC state and in exponential phase;5.To research the resuscitation methods of bacteria in the VBNC state by temperature upshitting,adding nutrition elements,and adding chemical substances. Results:1.The induction factors of bacteria entering into the VBNC state1.1 E.coli and E.faecalis could enter the VBNC state in water environment induced by 4℃low temperature and starvation in 90days,while S.dysenteriae in 42days;1.2 Three intestinal bacteria died in 15days,15days,12days separately induced by pH2.5 acidic environment instead of entering the VBNC state;1.3 0.4 mg/L residual chlorine induced for 30 minutes made E.coli enter the injuring state,and could not induce E.coli into the VBNC state.0.2mg/L residual chlorine could not play a role in disinfection.So chlorination induced bacteria into the injuring state instead of the VBNC state;1.4 Different bacteria enter the VBNC state induced by different conditions,the application and tolerance of environmental conditions of bacteria decided them whether to enter the VBNC state of bacteria and induction time.2.The research of exotoxin expression and Pathogenicity of S.dysenteriae serotype 1 in the VBNC state2.1 S.dysenteriae serotype 1 in the VBNC state made the adherent Vero cells lost the adherent ability,while E.coli which cannot secrete Shiga Toxin and boiling sterilization S.dysenteriae serotype 1 could neither make the adherent Vero cells lost the adherent ability.So the results indicated that S.dysenteriae serotype 1 in the VBNC state could secrete cytotoxicity Shiga Toxin;2.2 S.dysenteriae serotype 1 in the VBNC state could adhere to HT-29 intestinal epithelial cells,which indicated that S.dysenteriae serotype 1 in the VBNC state still retains adherence factor and preserves the ability of the pathogenic potential;2.3 S.dysenteriae serotype 1 in the VBNC state could invade HT-29 intestinal epithelial cells,which indicated that S.dysenteriae serotype 1 in the VBNC state preserves the ability of infection;2.4 The quantity of the expressed exotoxin,adhesiveness and invasive of S.dysenteriae serotype 1 in the VBNC state were lower than in exponential phase.3.The research of protein expression pattern of S.dysenteriae serotype 1 in the VBNC state 3.1 Metabolic state of bacteria in the VBNC state changed,catabolism strengthened and anabolism reduced;3.2 Aerobic respiratory capacity bacteria in the VBNC state increased and anaerobic respiration decreased,which indicated that the viability of anaerobic bacteria reduced;3.3 The ability of decomposition and utilize of exogenous nucleotides decreased, carbohydrate and protein synthesis ability decreased,and division and multiplication ability of bacteria in the VBNC state decreased;3.4 The resistance ability to the hyperosmotic environment decreased,while to the low-temperature strengthened.4.The research of resuscitation from the VBNC state of bacteria4.1 E.coli,E.faecalis and S.dysenteriae serotype 1 could not resuscitate from the VBNC state by temperature upshitting,adding nutrition elements,and adding tween -80;4.2 Catalase and sodium pyruvate could resuscitate E.coli from the VBNC state successfully,and the optimal concentrations were 1000U and 50mg per petri plate respectively;4.3 Catalase and sodium pyruvate couldn't resuscitate E.coli from the VBNC state fully, compared to the conventional HPC,the resuscitation efficiency could increase by 1000-fold,while only one thousandth bacteria could resuscitate to culturable ones.So other resuscitation measures need to be researched.4.4 The resuscitation cultivation of bacteria in the VBNC state might greatly underestimate the level of bacterial contamination in water,so other detection methods should consider to be researched.Conclusions:1.Intestinal pathogens could enter the VBNC state in water environment induced by low temperature and starvation;2.S.dysenteriae serotype 1 in the VBNC state could express Shiga toxin,had the ability of adherence and infection;3.The catabolism ability of bacteria in the VBNC state strengthened,anabolism decreased,carbohydrate and protein synthesis decreased,and division and multiplication decreased;4.Catalase and sodium pyruvate could resuscitate E.coli from the VBNC state, compared to the conventional HPC,the resuscitation efficiency could increase by 1000-fold,while only one thousandth viable bacteria could resuscitate to culturable ones.So other resuscitation measures need to be researched.
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