Study On Viable But Non-culturable Escherichia Coli And Lactobacillus Lactis

Viable but non-culturable state in bacterial cells has become the fundamental research issues in medical microbiology, epidemiology, general microbial ecology and sanitation quarantine since it was put forward in 1982. When in this state, bacterium are no longer able to grow and form colonies on conventional culture media, but maintain their patheogenicity, may become latent infection escaping detection to threaten enviroment and human security. Now, VBNC research range is relatively constrictive and VBNC bacteria variety is limited, only including some gram-negative pathogenic bacterium in the field. At least 40 species contained in 20 genera have been demonstrated to enter the VBNC state except prebiotics bacteria. To supplement variety and quantity of VBNC bacteria, differential source of Escherichia coli and Lactobacillus lactis were studied in experiment. First, induction condition of entering VBNC was decided. Second, VBNC state was detected by plate count, acridine orange and LIVE/DEAD kit staining, flow cytometry detection. Third, resuscitation experiments were demonstrated in VBNC population on returning to suitable conditions. At last, the main biology characteristics were compared between normal L.lactis and VBNC L.lactis.Conclusions as follow:1. E1 and E2 were cultured in LB(2% glacial acetic acid), aerobic culture at 4℃, after 12 days and 22 days,respectively,may enter VBNC. But VBNC in E1 and E2 disapeared after 72 days and 60 days,respectively.2. R1,R2 and R3 were cultured in MRS(pH2.0-3.0), anaerobic culture at 4℃, after 41 days, 35days and 46 days, respectively, may enter VBNC. But VBNC disapeared in R1 and R3 after 65 days, and VBNC R2 existed during 100 days.3. Different bacteria, even different strain, the time of induction and maintain is aslo different, maybe be correlat with its biology characteristics and resistance to harmful environment.4. VBNC E. coli and L. lactis could resuscitate with Tween-80. Generally, it is easy to resuscitate for just entering VBNC, but difficultly for lossed VBNC.5. Normal R2 could resist strong acid, 0.2% bile salt, and 1% pepsin. Contrastly, VBNC R2 could only resist strong acid and 1% pepsin except for bile salt.In addition, in order to approach VBNC development mechanisms, the DDRT-PCR primers were designed according to highly iterated sequence in E.coli genome. Three VBNC correlated genes were obtained by mRNA differential display PCR. Conclusions as follow:1. Three gene fragments which obtained by DDRT-PCR were expressed by VBNC E. coli, rather than normal E. coli.2. There was more nucleotide homology betwwen three differential genes and E. coli genes, the most homology C1,99%, A1 and A2 aslo above 98%.3. There was more amino acids homology betwwen three differential fragments and E. coli 23srRNA, all above 97%. Maybe three genes participated transcription or ribosome construction.4. Three differential fragments were housekeeping genes, extensively existing in Shigella and Salmonella.5. Three differential genes of 23srDNA which obtained by DDRT-PCR maybe not be expressed or lowly expressed in normal E.coli. When in VBNC, they could be highly express to participated transcription or protein synthesis.