Effect Of Bacteriophages With Different Genetictypes On Inactivation And Mutation Of Shiga Toxin Producing Escherichia Coli O157:H7

Antibiotics have been widely used as feed additives or medicine in livestock for the promotion of animal health and products. However, the emerge of antimicrobial-resistant bacteria and residual problem of antibiotics in animal products and environment have been raised as public health issues with global attention. In the past few years, series of measures have been developed for preventing of the food-borne pathogen Escherichia coli O157:H7(E.coli O157:H7) from entering into food chain, but outbreaks of E.coli O157:H7 infection associated with beef products consumptions still occur. Therefore, effective interventions in mitigating contamination of E.coli O157:H7 of beef are desirable. The objectives of this research were to study the efficacy of four phages with different genetictypes as a biocontrol strategy in preventing contamination of E.coli O157:H7 on beef, then study the effect of phages on mutation of E.coli O157:H7 and thereby construct an effective biological control stage to ensure food safety. Meanwhile, the usefulness of an optimized PMA-qPCR method to quantify survival of E.coli O157:H7 cells infected by four types of phages was evaluated.O-antigen flippase, wzx was amplified by PMA-qPCR for detection of efficacy of phages T5-like (AKFV33), T1-like (AHP24), T4-like (V7) and rV5-like (AHP24S) on inactivation of E.coli O157:H7 R5O8N as well as the degradation of host DNA by phages. DNA (< 71ogioCFU) from heat lysised E.coli O157:H7 was completely combined by 100μM PMA but without affecting the amplification of DNA from survival cells. After infection by 4 phages, the survival cells detected by PMA-qPCR was siginificantly higher than tranditional plate count method, and the degradation of host DNA by 4 phages was different. This is the first successful application of PMA-qPCR in dection of phage efficacy on pathogen, which shortened the detection time from 48h to 5h.Efficacy of four phages (AKFV33, AHP24, V7 and AHP24S) for biocontrol of five nalidixic acid resistant E.coli O157:H7 strains (E318N, E32511, H4420N, R5O8N, CO281-31N) was assessed on beef samples stored at 4,22 and 37℃, respectively. A virucidal solution of lOmM FeSO4+15% Tea was included for accurate detection. As a result, efficacy of phages on mitigating of E.coli O157:H7 was different (P<0.001):AKFV33 was the most effective phage, the highest sterilization rate was 99%, followed by AHP24 and V7, then AHP24S. The efficacy of phages on biocontrol of E.coli O157:H7 was significantltly effected (P<0.01) by temperature (negative correlation). The highest inactivation of E.coli O157:H7 was obtained at 37℃ (average inactivation of 3.2log10CFU/cm2), followed by 22℃, then 4℃ (average inactivation of 3.0log10CFU/cm2). Efficacy of phages on inactivation of E.coli O157:H7 was also effected (P<0.001) by reaction time (positive correlation).The genovariation and genetic relationship among 120 E. coli O157:H7 survival strains from beef study above were determined by pulsed field gel electrophoresis (PFGE). Susceptibility of these strains (5 mutant and 115 wild types) to the four phages was determined by mircoplate virulence assay. As a result, four phages exhibited highest activity against E318N, followed by E32511, but showed lowest activity against H4420N. Five mutant E.coli 0157 strains were isolated from phages AFKV33(4/5) and V7(1/5) treatment, but none from AHP24 and AHP24S treatment. Consequently, phages with higher lytic capability were more likely to make the host bacterium mutations. However, the microplate virulent assay showed that the mutant strains still sensitive to the four phages.In conclusion, this study demonstrated the effect of bacteriophages with different genetictypes on inactivation and mutation of E.coli O157:H7. Meanwhile, it also realized the accurate detection of phages efficacy on biocontrol of E.coli O157:H7, as well as demonstrated that phages as biocontrol agents have great potential to effectively mitigate E.coli O157:H7 on beef contamination and thus pioneered commercial strategy to ensure food safety.