Study On The Biological Characteristics Of Desiccation-adapted Salmonella Typhimurium

In recent years,Salmonella has been linked to outbreaks associated with low water activity(a_w)foods,especially powdered infant formula(PIF).The capability of biofilm-forming and entering the Viable but Non-Culturable(VBNC)state may be closely related to the long-term survival of the bacteria in dry stress.Meanwhile,Salmonella prior dehydration may provide cross-protection to improve its survival rate in food and its processing environment and bring risks and hidden dangers to human health and economic development.The purpose of this study was to examine the effect of the physiological state(biofilm versus planktonic)on the survival of Salmonella at low a_wand the act of dehydration-adapted Salmonella Typhimurium ATCC 14028 storage in PIF to enter the state of VBNC state,as well as,the effect of the physiological state and different a_w to provide cross-protection against the harsh conditions of heat,sanitizer and antibiotic treatment.Furthermore,the effect of physiological state,dry storage and heat treatment time on select stress(rpo S,rpo E),virulence(inv A),chaperone protein(dna K),heat shock protein(grp E)gene expressions of S.Typhimurium were investigated.The findings are as follows:(1)PIF(a_w 0.30)was inoculated with biofilm or planktonic state S.Typhimurium stored at 25?for up to 270 days,but Salmonella with a a_w of0.85 was lower than the detection line on the seventh day,indicating that dehydration-adapted S.Typhimurium ATCC 14028 can survive in PIF for a long time at room temperature.(2)CTC-FCM was used for rapidly variable cell enumeration.Culturable cells were counted based on colonies from proliferation of single cell on plates.The number of VBNC Salmonella was considered as the difference between the number of viable cells(CTC-FCM)and culturable cells(xylose lysine desoxycholate agar,XLD).The results demonstrated that the number of the biofilm state Salmonella entering the VBNC state increased after 180-day storage significantly,while the Salmonella in the planktonic state increased significantly after 270-day storage.The results showed that low a_w could induce S.Typhimurium to enter the VBNC state,and biofilm-forming Salmonella was more likely to enter the VBNC state(p<0.05).(3)By comparing desiccation-adapted Salmonella with non-adapted Salmonella after heat treatment and disinfectant treatment to reduce the number of Salmonella,it was found that for the planktonic cells,desiccation treatment can induce cross-protection to heat treatment and hydrogen peroxide easily.By comparing the sensitivity between desiccation-adapted Salmonella and non-adapted Salmonella after antibiotic treatment,it was found that planktonic desiccation-adapted Salmonella more easily to produce cross resistance to cephalexin,levofloxacin,and gentamicin.While the biofilm state desiccation-adapted Salmonella was only cross-resistant to heat treatment and levofloxacin treatment.The above results show that low water activity treatment can induce Salmonella cross resistance to a variety of stress environments.However,the cross resistance of Salmonella in different physiological states was different under different stress conditions(4)The stress response genes and virulence genes of Salmonella were detected by quantitative reverse transcription polymerase chain reaction(RT-q PCR).It revealed that for the biofilm state,the upregulation of two desiccation stress-related genes,rpo S and dna K,following the seven-day PIF incubation,while for the planktonic state,merely rpo S gene upregulated.Invasion gene inv A,grp E,and alternative sigma factor rpo E were downregulated for both the state of the biofilm and planktonic state,but there were significant differences in gene expression in the two states(p<0.05).When studying the effects of heat treatment,it was found that only the expression of rpo S gene was up-regulated.For the planktonic state,the expression reach highest after 1 h of treatment and for the biofilm state,the expression reach highest after 2 h of treatment.Therefore,the physiological state,storage time,and heat treatment of Salmonella affect the expression of genes.The expression of rpo S gene was up-regulated,which indicated that rpo S played an important role in the process of low water stress and heat treatment.