Low Temperature Storage On VBNC State Induction And Toxin Expression Of Enterohemorrhage Escherichia Coli

E coli O157, a serotype of Enterohaemorrhagic escherichia coli(EHEC), with O antigen gene rfb E, Shiga toxin gene stx1 and stx2, is an important foodborne pathogen. VBNC state of E coli O157 has been detected and can be induced at low temperature. It is a hidden source of pollution and a threat of food security because of no detection with national standard culture method. Therefore, it is urgent to establish a method to detect VBNC state of E. coli O157 and evaluate low temperature on virulence gene expression into VBNC state. Consequently, in this paper, low temperature on entry into VBNC state of E. coli O157 is discussed in order to analize the existence of VBNC state cells in low temperature storage. The method with high sensitiviy and specificity is established to detect VBNC state of E. coli O157. A comprehensive analysis is made for low temperature storage time and strain differences on three kinds of virulence gene expression.1. To evaluate the ability for entry into VBNC state of E. coli O157 standard stain ATCC43895, the fluorescence microscopy menthod is chosen.VBNC state of E. coli O157 are found at-20°C for 28 d. Morphological characteristics of VBNC cells are studied by scanning electron microscope. Results show that cells gradually change in shape from short rods to coccoids with relatively rough surface, and a reduction in size.2. The LAMP system is optimized with E. coli O157 standard stain ATCC43895. The optimal LAMP system is 150 m M magnesium ions, 50 μM calcein and 1:8 concentration ratios of manganese ions and calcein. The results of the specificity and sensitivity analysis show that the primers targeting rfb E are specific highly, and the detection limit is 100 fg/μL and 105 times higher than PCR method. O157 serotype of 66 E. coli samples is identificated by LAMP method. Results show that the positive detection rate was 36 of 66(54.55%). The specific primers targeting stx1 and stx2 are designed in order to identificate Shiga toxin of 36 E. coli O157. Identification results show that 30 of 36 is E. coli O157 targeting stx1 gene and 32 of 36 is targeting stx2 gene. The positive rate is up to 83.33% and 88.89%, respectively.3. VBNC state of E. coli O157 standard stain ATCC43895 is studied to establish the optimal PMA-LAMP method. The identification results of rfb E virulence gene show that some inhibitory on the growth of viable cells is seen with higher concentration of PMA. With the optimal 5 μg/m L PMA treatment of dead and VBNC state of E. coli O157 for stx1 and stx2 target gene amplification, the identification result is positive for stx1 and stx2, indicating that shiga toxin expression can be measured in the VBNC state of E. coli O157.4. The stains of ATCC43895, E011 and E036 are treated with low temperature storage time of 0, 6, 12, 18, 24 d. The total RNA is extracted and transcribed into c DNA reversely. The q RT-PCR method is used to detect three kinds of virulence gene rfb E, stx1 and stx2 expression with the housekeeping gene of 16 s RNA. Low temperature storage time and strain difference on the expression level of these genes is evaluated and analyzed comprehensively.The results of low temperature storage time on three kinds of virulence genes show that with the time extending, rfb E gene expression rises at first, then falls and rises up again to the value, which is higher than normal level; the expression change of stx1 and stx2 genes is same, that is, both increase and then decrease and rise ultimately for E. coli O157 strain with three virulence genes(ATCC43895 standard strain). However, for E. coli O157 strains with rfb E and stx1(stx2), the rfb E gene expression declines first and rises to a value, higher than normal levels. The expression of stx1 and stx2 is of some fluctuations.The results of strain difference on three kinds of virulence gene expression show that no regularity is suggested in rfb E and stx2 relative expression levels compared with E. coli O157 standard strain ATCC43895 for E011, E. coli O157 stain of no stx1 gene. The expression levels of rfb E and stx1 gene are significantly higher than the expression of E. coli O157 ATCC43895 in all the time points for E036, E. coli O157 stain without stx2 gene. It is suggested that the expression of rfb E and stx2 can be promoted for the strain without stx2 gene.