| Staphylococcus aureus is one of the most easily detected foodborne pathogens in cold food,which can easily cause serious health problems and severe economic losses.In this experiment,wild-type S.aureus isolated from low temperature food was used as the research object,and the low temperature tolerance ability of S.aureus was distinguished,and a strain that could form Small Colony Variants(SCVs)was found.The differences in cell membrane integrity of different low temperaturetolerant S.aureus were indirectly compared by measuring the leakage of cell contents,the conductivity of bacterial suspension and crystal violet staining,and the changes in cell morphology were observed by transmission electron microscopy.In addition,the contents of trace glutaraldehyde,superoxide dismutase and reduced glutathione of S.aureus in low temperature treatment were determined to evaluate the difference of oxidative stress ability of S.aureus at low temperature.Then,transcriptome sequencing technology(RNA-seq)was used to analyze the difference in transcriptional expression ability of two S.aureus strains with different survival strategies at low temperature,which provided a theoretical basis for the control of S.aureus in low temperature food.The main findings are as follows:(1)178 strains of S.aureus isolated from low temperature food were treated with low temperature for 7 days to evaluate the difference of their low temperature tolerance types.As a result,38 low temperature-sensitive strains and 26 low temperature tolerant strains were found,and one BB-1 strain that could form microcolony variants was found.After 7 days of low temperature stress,the number decreased by only 1.00 log CFU/m L,and the survival rate was 89.9%.Subsequently,three strains of S.aureus(BK-1,BH-25 and FA-3)and BB-1 with different low temperature tolerance were subjected to the determination of the leakage of cell contents,the conductivity of bacterial suspension and the analysis of crystal violet staining.The results showed that the integrity of the cell membrane of BB-1 was stronger than the other three strains under low temperature stress.Finally,the expression changes of the main genes(rsb V,rsb W and sig B)and the main genes of cell membrane fatty acid synthesis(fab D,fab F,fab G,fab H and fab I)were analyzed by real-time quantitative PCR,it was found that the expression levels of resistance-regulated genes and cell membrane fatty acid synthesis genes of BB-1 were significantly higher than those of the other three strains(p<0.05).(2)Based on the results of part(1),2 strains of S.aureus(BB-11 and BA-26)with different low temperature survival strategies were selected for subsequent experiments.Using flow cytometry and confocal laser scanning microscope to detect the damage of S.aureus cell membrane macroscopically,it was found that the cell membrane integrity of BB-11 was significantly better than that of BA-26(p<0.05).The changes in the cell structure of S.aureus were visually observed by transmission electron microscope,and it was found that the damage of the BB-11 cell membrane was less,while the BA-26 cell membrane was more shrunken and more numerous.Finally,the microplate spectrophotometer was used to determine the content of trace glutaraldehyde,superoxide dismutase and reduced glutathione of S.aureus in low temperature treatment to evaluate the oxidative stress of S.aureus at low temperature.It was found that the oxidative stress capacity of BB-11 was significantly higher than that of BA-26 under low temperature treatment(p<0.05).(3)Transcriptome sequencing technology(RNA-seq)was used to analyze the differences in transcriptional expression ability of two S.aureus strains with different survival strategies at low temperature,the results showed that a total of 833 genes were significantly differentially expressed in the BB-11 group,of which 424 genes were upregulated and 409 genes were down-regulated;A total of 527 genes were significantly differentially expressed in the BA-26 group,of which 292 genes were up-regulated and235 genes were down-regulated.In the comparison between the BB-11 groups,70 DEGs were annotated in biological processes,337 DEGs were annotated in molecular functions,and 448 DEGs were annotated in cellular components.In the comparison between the BA-26 groups,46 DEGs were annotated in biological processes,219 DEGs were annotated in molecular functions,and 294 DEGs were annotated in cellular components.Subsequently,the RNA-seq data were verified using real-time PCR and found that the q RT-PCR data results were roughly the same as the RNA-seq data results(BB-11 group fitting correlation result: R2=0.79582;BA-26 group fitting correlation result: R2=0.85071).Further analysis of RNA-seq results revealed that the expression of membrane fatty acid biosynthesis(fab G)was significantly up-regulated in BB-11 compared to BA-26 due to membrane damage.Then,it was found that catalase(kat A),reduced glutathione(grx C),and peroxidase(ahp C)were significantly upregulated in BB-11,resulting in increased oxidative stress responsiveness,but BA-26 was associated with gene downregulation.NADH dehydrogenase(nad E)and α-glucosidase(mal A)were up-regulated in cold-tolerant strain BB-11,but down-regulated in coldsensitive strain BA-26,suggesting that energy metabolism may be involved in coldstressed S.aureus.Furthermore,defense mechanisms involving asp23,gre A,and yaf Y play a key role in BB-11’s response to stress. |
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