Recovery Method After Heat-injury And Knock Out Of RpoH Gene In Salmonella Enteritidis

Salmonella enteritidis, is one of the most common and important food-borne pathogens which can cause food poisoning easilly.National standards of the People's Republic of China(GB/T4789.4-2003) stipulates that processed foods Salmonella spp. testing use the buffered peptone water as a recover medium, and often use selective medium of desoxycholate hydrogen sulfide lactose agar(DHL) for the Salmonella spp. detection. There might be Salmonella enteritidis in a state of sublethal in foods which are sterilized at low-temperature such as pasteurized foods, this easily causes undetected and false negative results which can not accurately reflect the situation of food hygiene. By comparing different recover medium, temperature and time, obtaining Salmonella enteritidis revive conditions which makes the test of Salmonella enteritidis have a true and accurate reflection about the situation of food hygiene.In order to understand and elaborate the Salmonella heat shock and revive mechanisms, to provide a theoretical basis for sublethal Salmonella detection methods, the rpoH gene of Salmonella enteritidis be explored in this research, which encoding heat shock transcription factorσ32. The kanamycin-resistant cassette with 39bp short homologous sequences on both ends generated by PCR was electroporated into Salmonella enteritidis IFO3313. Recombination between linear DNA cassettes and Salmonella enteritidis chromosomes took place by Red recombinase functions. By verification of PCR, we affirmed the knock out of rpoH gene of Salmonella enteritidis. Based on this, we studied the heat shock response and recovery of wild type and mutant strains to explore how the rpoH gene deletion affects the reaction of Salmonella enteritidis to heat-shock. Using Red recombination system, we had the rpoH gene mutant of Salmonella enteritidis, it helps us to understand the heat shock response and recovery mechanisms of Salmonella enteritidis, and to make a preliminary study of the detection of food-borne pathogen in a sublethal state. The results showed that:(1) By comparing different repair media, temperature and time, we picked out the best recover conditions for Salmonella enteritidis in a sublethal state.The best recovery method is incubating the heat-injured Salmonella enteritidis in Nutrient Broth for 2 h at 37℃;(2) We obtained the strain IFO3313-ΔrpoH by successfully knocked out rpoH gene in Salmonella enteritidis usingλ-Red recombination system;(3) The rpoH gene deletion mutant strain has less tolerance to heat shock than the wild strain, and is more likely to have fatal injuries.The mutant strain recovery in the non-selective plate is worse than that of the wild strain.The deletion of rpoH gene in Salmonella enteritidis have a significant effect on the tolerance to heat and revive capacity then.