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Detection of the Viable But Nonculturable in Escherichia Coli O157:H7

Author: LuoZhongJie
Tutor: HuangZheng
School: Huazhong University of Science and Technology
Course: Occupational and Environmental Health
Keywords: E.coli O157:H7 VBNC ELISA IF
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 73
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Abstract

Escherichia coli O157:H7(E.coli O157:H7) is a most important serotype of Enterohemorrhagic Escherichia coli(EHEC) which could express Shiga-like toxins, erythrolysin, adhesion, and could cause hemorrhagic enteritis, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura. Several large outbreaks of E. coli O157:H7 were reported all over the world over the past 30 years. Therefore, there is an urgent need to develop a sensitive and reliable method for detection of E.coli O157:H7.Traditional study of environmental microbes relies on conventional culture which is time-consuming with low detection limits. Xu Huaishu first proposed that many of the cells have been denmonstrated to enter a viable but nonculturable (VBNC) state under certain environment conditions in 1982. Through a series of genetic differentiation, the VBNC state has been reported to be a survival mechanism adopted by many bacteria when exposed to environmental stresses. Bacteria in the VBNC state still keep metabolic activity and virulence but could not be resuscitated in conventional growth medium. They could be capable of regaining culturability when the environment is suitable for bacteria. Enzyme-linked immunosorbent assay (ELISA) is a common microbiological detection method which has been widely used in environmental monitoring, in addition to being highly specific, sensitive and rapid. this procedure even can be directly observed by eyes. Indirect immunofluorescence (IF) is an intuitive and specific method which is based on the specific conjugation between antigen and antibody, it could not only identify the types of bacteria, but also observe the bacteria directly by the fluorescence microscope.At present, there is little comparative analysis and experimental evidences for ELISA and IF on the detection of VBNC E.coli O157:H7. Therefore, ELISA and IF were applied and compared in detection of VBNC E.coli O157:H7 in this research.Objectives: To monitor the survival of E.coli O157:H7 in aquatic microcosm of Han River water and drinking water; to explore the feasibility of ELISA and IF in detection of VBNC E.coli O157:H7.Methods: E.coli O157:H7 were added into aquatic microcosm of Han River water and drinking water with low temperature and olig-nutrition and E.coli O157:H7 survival was detected by acridine orange direct count(AODC), direct viable count-nucleic acid(DVC-NA), direct viable count-CTC(DVC-CTC)and heterotrophic plate count (HPC). The ELISA and IF were combined to detect the real time changes on E.coli O157:H7 in aquatic microcosm of Han River water and drinking water, and characteristics of the two methods were illustrated.Results: 1. 3.6×10~6 CFU/mL of E.coli O157:H7 became 7.2×10~4CFU/mL of VBNC E.coli O157:H7 in drinking water after 10 days at 4℃with olig-nutrition. 3.1×10~6CFU/mL of E.coli O157:H7 became 1.2×10~5CFU/mL of VBNC E.coli O157:H7 in Han River water at the same condition. 2. As to the ability of detecting viable bacteria, DVC-CTC is better than DVC-NA. 3. The best parameters of ELISA were as follows: coating antibody concentration on multiwall plate was 10μg/mL, 3% bovine serum albumin (10% calf serum) was used to block the wells at 37℃for 1h, optimal dilution of mouse anti-E.coli O157:H7 monoclonal antibody connecting with HRP was 1:1000, and the detection limit was104 CFU/mL for E.coli O157:H7. The best parameters of IF were as follows: coating antibody concentration on multiwall plate was 1:100, optimal dilution of goat anti-mouse antibody connecting with FITC was 1:60, and the detection limit was 103CFU/mL for E.coli O157:H7. ELISA and IF are the methods for detection of the total number of E.coli O157:H7, either of them combined with fluorescence staining and HPC could be used in detection of the VBNC E.coli O157:H7.Conclusions: The viable E.coli O157:H7 could survive longer in Han River water and enter VBNC state much later than in drinking water. DVC-CTC was more suitable for viable count and observation than DVC-NA. Either ELISA or IF combined with fluorescence staining and HPC could be used in detection of the VBNC E.coli O157:H7.

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