| Bacteria in a viable but nonculturable state(VBNC state for short)cannot grow and reproduce on ordinary medium to form colonies,but can still carry out metabolism.Viable but nonculturable(VBNC)state of microorganisms has attracted much attention due to its characteristics,such as difficult detection by the cultured methods,virulence retention,high resistance,and so on.As a foodborne pathogen,Staphylococcus aureus is widely distributed,and has been found to enter VBNC state under some environmental stresses,posing a potential threat to human health.In view of the fact that freezing is a common condition for food storage,in this study,the ability of common food additive citric acid to induce S.aureus into VBNC state at-20? was studied.S.aureus was induced by citric acid buffer(p H 4.0),0.85%Na Cl(p H 7.0)and peptone water(p H 7.0)at-20?.The number of culturable bacteria was determined by plate counting method.The results showed that after 72 days of induction,S.aureus entered the nonculturable state in citric acid buffer at-20?,but did not enter the nonculturable state in 0.85% Na Cl and peptone water.The number of viable bacteria was detected by Live/Dead Baclight bacterial viability kit combined with flow cytometry.It was found that there were still viable bacteria in the non culturable cells,which proved that S.aureus could be induced into VBNC state in citric acid buffer at-20?.The above induced VBNC cells successfully resuscitated at 37? in trypsin soybean medium(TSB),and TSB supplemented with sodium pyruvate,Tween 80 and yeast extract,and after heat shock treatment(72?,10 s),VBNC cells were also resuscitated in TSB.However,the resuscitation ability of VBNC cells under these conditions was different,and heat shock treatment was the best,the resuscitation ability of adding sodium pyruvate and Tween 80 was better than that directly in TSB,but not as good as that of heat shock treatment;the resuscitation ability of adding yeast extract was the same as that in TSB.Transmission electron microscopy(TEM)was used to observe the internal structure of S.aureus in exponential phase and VBNC state.The results showed that the internal structure of cells in VBNC state changed significantly(cytoplasmic density increased,nuclear matter loose,membrane structure deformed).API ZYM kit was used to measure the intracellular enzyme activity in logarithmic growth phase and VBNC state.The results showed that the enzyme activity decreased after entering VBNC state.it was found that the resistance of cells to simulated gastrointestinal fluid increased after entering VBNC state through Live/Dead Bac Light kit combined with flow cytometry.Quantitative reverse-transcription polymerase chain reaction was used to detect pbp1,adhesion and biofilm formation 10 related genes(ebp S,ica A,ica B,ica C,ica D,fnb A,fnb B,ebh,agr A,and sar A)of cells in exponential phase and VBNC state.The results showed that the down-regulation of pbp1 in VBNC cells inhibited the division of S.aureus cells,which may be related to the morphological changes of VBNC cells.The ability of adhesion and biofilm formation of VBNC cells might be decreased due to the down-regulation of related genes,thus reducing the pathogenicity of VBNC cells.But it should not be ignored the recovery potential of biofilm-forming ability of VBNC cells caused by the high expression of sar A.Sar A encoded by sar A is a global regulatory factor.Stable expression of sar A in VBNC cells will help to increase the expression of ica A,ica B,ica C,ica D,fnb A,and fnb B under certain conditions,so as to improve the ability of biofilm formation.In conclusion,it should not be ignored the recovery potential of biofilm-forming ability of VBNC cells.It is necessary for food industry to pay attention to the hazard caused by VBNC S.aureus under frozen conditions. |
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