Rapid Detection Of Pathogens In PIF And The Stress Responses Of Pathogens

Powder infant formula(PIF),as a substitute of the breast milk,is the main dietary source for infants.Thus ensuring its quality and safety is of great significance.The microbial contamination of spoilage or pathogenic bacteria is one of the major concerns in PIF product resulting in a severe hazard in food safety.Therefore,an effective controlling and prevention method for the microbial contamination in PIF is very important.To monitor and control the microbial contamination level of PIF,two rapid detection methods aiming on Cronobacter were developed.Primary,a combination method of real-time PCR and high-resolution melting(HRM)analysis for the rapid detection and specific classification of six pathogenic Cronobacter species based on gene cgcA was developed.HRM profiles with distinct Tm(melting temperature)peaks were consistently obtained with each species represented by a single peak ranging from 86.02 to 86.80℃.The detection limit ranged from 0.1 pg to 1 pg per reaction.Combined with a 12 h pre-enrichment,desiccated Cronobacter of 2 CFU/25 g were successfully detected after four-week storage at room temperature.Then,a newly TaqMan qRT-PCR based on cgcA gene for rapid detection of C.sakazakii in PIF was developed.The detection limit of C.sakazakii was about 1,100 CFU/g in reconstituted PIF.After pre-enrichment for 6 h,it could reliably detect C.sakazakii when the inoculation level was as low as 2 CFU/25 g in PIF.These two methods greatly shorten the time-consuming required for Cronobacter detection in PIF,and simplify the detection or classification process.Pathogens in PIF developed various mechanism to protect them form adverse environment,and secreted various virulence factors at the same time.Thus they lead to a severe hazard in food safety in PIF.Research of adverse or stress response mechanism in pathogenic bacteria became one of the key scientific problems in order to pursuing effective control of pathogens in recent years.As for the stress response analysis,the stress response to desiccation in C.sakazakii and the stress response to acid(specifically,ATR induced by infant’s digestive system)in EHEC O157:H7 and S.Enteritidis were investigated.Based on the importance of the function of the Fe-S cluster in ATR mechanism in Salmonella,the function of protein NfuA in the assembly of Fe-S cluster and the maturation of proteins containing Fe-S cluster were investigated.1)According to the comprehensive proteome coverage analysis,233 DEPs were identified an altered protein expression level between experimental sets control and desiccation stress groups,and among these 233 proteins,146 proteins downregulated and 87 proteins upregulated.On one hand,C.sakazakii increase its resistance to desiccation by reducing the genes expression of unnecessary functions such as virulence,adhesion,invasion and flagella assembly.On the other hand,C.sakazakii increased the content of trehalose and betaine to hold the moisture,and activating ABC transpoter and secretion system to achieve the accumulation of trehalose and betaine in cytoplasm.2)According to the RNA-sequencing transcriptomic results,when facing the ATR environment,bacteria got different strategies to balance protons inside cytoplasm.110 DEGs were identified in E.coli.Specifically,gene cadA and cadB,mediating the lysine decarboxylation,consumed the extra protons inside and kept the neutral pH value in cytoplasm to protect E.coli from death.223 DEGs were identified in EHEC 0157:H7.In contrast,oxidation-reduction mediated by ahpC and hydrogenation mediated by mhpA consumed the protons inside and kept the neutral pH value to protect the EHEC 0157:H7 from harm.554 DEGs were identified in S.Enteritidis.By increasing the intracellular NAD(+)/NADH ratio,S.Enteritidis could also decline the protein acetylation level by promoting the consumption of acetyl-CoA via TCA,to prevent the intracellular pH from further decline under acid stress and balance extra protons.EHEC 0157:H7 increased the stability of Fe-S cluster in cells and expressed more anti-oxidation enzymes and ROS scavengers to repair damage caused by acid stress,which might enhance its resistance to acid compared to E.coli ATCC 25922.ATR S.Enteritidis underwent an oxidization damage result in an iron-lacking circumstance.Thus some DEGs related to Fe/S cluster biogenesis were responsive to acid stress to repair damage caused by acid and ROS.3)NfuA has roles in both thiamine synthesis and growth on tricarballylate as only carbon source.The results suggested that NfuA cooperated with both ISC and SUF system,while didn’t belong to any of those two main system in Salmonella.ApbC had a redundancy function over NfuA proteins in tricarballylate influx and and thiamine synthesis.△nfuA mutant results in defect at both thiC and thiH site,resulting damages in THZ and HMP synthesis step for thiamine production.The defect in the assembly of iron sulfide clusters would affect cell’s function by affecting the function of thioredoxin.Supplementation or removal of nutrients to control specific metabolism pathway would subsequently control microbial contamination in PIF.This study aimed to provide technical and theoretical support for the food safety control of PIF products,and to assist better microbial contamination control strategy in PIF.