Formation Mechanism Of Viable But Nonculturable Escherichia Coli O157:H7Induced By High Pressure CO₂

Induction of Escherichia coli O157:H7, a foodborne pathogen, to enter into the viable but nonculturable (VBNC) state by high pressure CO2(HPCD) was investigated, and the formation mechanism of VBNC E. coli O157:H7induced by HPCD was also studied. Results obtained by this research will provide theoretical foundation for further optimizaing HPCD processing strategy and ensureing food safty produced by HPCD, so this research has a significant value for theory and practice. The main research contents and results were summarized as follows:(1) Determine the induction conditions of HPCD for inducing E. coli O157:H7to enter into VBNC state. With temperature as a parameter, the VBNC state induction for E. coli O157:H7by HPCD at5MPa was investigated. It was shown that E. coli O157:H7could be induced into VBNC state by HPCD at5MPa and four temperatures (25℃,31℃,34℃,37℃), and the time required for E. coli O157:H7to enter into VBNC state shortened with the treatment temperature increasing. When incubated in TSB at37℃, except for the VBNC cells induced at5MPa and37℃, VBNC cells treated by other three HPCD treatments all achieved resuscitation, while the resuscitation capability decreased with the treatment temperature increasing. This research demonstrated for the first time that HPCD could induce E. coli O157:H7to form the VBNC state.(2) Analysis of biological characteristics of HPCD-induced VBNC E. coli O157:H7cells. As measured by API ZYM kit, it was shown that intracellular enzymatic activities in VBNC cells were reduced with the treatment temperature increasing. When observed by scanning electronic microscopy and transmission electronic microscopy, it was found that the morphology and interior structure of VBNC cells and resuscitated cells were significantly changed from those of the exponential-phase cells. In addition, membrane fluidity of HPCD-induced VBNC cells did not change when measured by fluorospectrophotometer, and the resistance of VBNC cells to sonication was significantly enhanced. These results indicated that VBNC state was a survival strategy adopted by E. coli O157:H7when treated by HPCD.(3) Using transcriptomics and proteomics to analyze the formation mechanism of VBNC E. coli O157:H7induced by HPCD. According to the results of transcriptome and proteome, the metabolic situation in HPCD-induced VBNC cells was summarized below, the transport ability for carbonhydrate, amino acid and other substances was reduced in HPCD-induced VBNC cells, leading to the substance exchange between VBNC cells and environment decrease, and thus causing the metabolic activity decreasing in VBNC cells; the ATP output was enhanced by metabolisms of carbonhydrate, amino acid, nucleotide and respiration chain, which will provide energy for metabolisms proceeding in VBNC cells, thus it is benifit for maintaining cell viability; in VBNC cells, activity of DNA replication and recombination decreased, and cell division was also repressed, which will cause the VBNC cells cannot divide and grow; the weakening of defense capabilities in periplasmic space will increase the sensitivities of VBNC cells to chemical stimuli; and the pathogenicity of VBNC cells decreased. In conclusion, through decreasing or repressing the activity of substance transports and metabolisms, the cell division capability, as well as the resistance to chemical stimuli in E. coli O157:H7, HPCD treatment cause the cells cannot grow and division although remain the low metabolic activity, that is induce the E. coli O157:H7cells to enter into the VBNC state.(4) Verify the results of transcriptomics and proteomics by the resistance of VBNC cells to acid and oxidative stresses and the pathogenicity of VBNC cells to host cells. HPCD-induced VBNC E. coli O157:H7cells were more sensitive to acid and oxidative stresses than the exponential-phase cells, but the VBNC cells had a certain tolerance to oxidative stress. Compared with the exponential-phase cells, the adherence ability of VBNC cells to HeLa cells and HT-29cells decreased, and the A/E lesion produced by VBNC cells to these two host cells was also reduced, which indicated that the pathogenicity of VBNC cells decreased. These results were accordant with the results of transcriptome and proteome, which partly demonstrated that the results of transcriptome and proteome were correct.