为研究不同金黄色葡萄球菌对低温(4 ℃)的耐受性差异,及其与细胞膜完整性及相关基因表达调节之间的关系,首先通过涂布平板计数法对4株金黄色葡萄球菌食品分离菌株的低温抗性进行比较,发现经过4周的低温胁迫后,与其他菌株相比,BB-1菌株可形成小菌落变种(small colony variants, SCVs),且低温耐受能力最强,存活率达75.50%。而低温耐受能力最弱,且不能形成小菌落变种的BH-25菌株存活率仅为45.68%。随后通过测定胞内容物泄露量、菌悬液电导率和结晶紫染色分析,研究低温对不同金黄色葡萄球菌细胞膜通透性的影响,发现BB-1细胞膜的完整性在低温胁迫下强于BH-25。采用实时荧光定量PCR分析金黄色葡萄球菌胁迫耐受的主要基因(rsbV、rsbW和sigB)和细胞膜脂肪酸合成的主要编码基因(fabD、fabF、fabG、fabH和fabI)表达量的变化,发现BB-1的抗性调控基因和细胞膜脂肪酸调控基因表达水平显著高于BH-25(P<0.05)。可形成SCVs细胞的BB-1菌株在低温下存活能力更强,并且细胞膜更完整,细胞内容物泄露量最少,而且在低温环境下rsbV-rsbW/sigB调节系统基因和调控细胞膜脂肪酸fab家族基因的转录表达水平均显著高于不能形成SCVs细胞的其他菌株(P<0.05)。该研究对深入分析金黄色葡萄球菌的低温抗性机制,以及确立冷链食品安全控制技术具有一定的理论指导意义。
In order to study the heterogeneity of tolerance to low temperature (4 °C) of different Staphylococcus aureus strains, as well as its relationship between the cell membrane integrity and related gene expression. The low temperature tolerances of four isolated S. aureus strains were compared by plating counting prior to viable cell counting. Results showed that after four weeks of low temperature treatment, the BB-1 strain could form small colony variants (SCVs) compared to other strains, which also exhibited the strongest tolerance to low temperature and the survival rate reached 75.50 %. Comparatively, the BH-25 strain with the weakest tolerance was unable to form SCVs and the survival rate was only 45.68 %. Subsequently, the leakage of intracellular contents, the conductivity of the bacterial suspension, and the crystal violet staining analysis were performed to study the effect of low temperature on the cell membrane permeability. The results indicated that the integrity of the BB-1 cell membrane is stronger than BH-25. Thereafter, the quantitative real-time PCR was employed to analyze the regulation patterns of the stress tolerance related genes (rsbV, rsbW and sigB) and the cell membrane fatty acid biosynthesis related genes (fabD, fabF, fabG, fabH and fabI). The result indicated that the transcriptional levels of the tested genes were significantly higher in BB-1 strain than those in BH-25 (P<0.05). The tested four S. aureus strains exhibited significant difference in the tolerance to low temperature, BB-1 that can form SCVs has a stronger survival capability at low temperature, while the cell membrane is more intact to inhibit the leakage of cellular content. Moreover, the expression levels of rsbV-rsbW/sigB system genes and fab series genes that regulate cell membrane fatty acid biosynthesis in SCVs cells were significantly higher than the strain that could not form SCVs. The present study offers a newly basis for clarifying the mechanism of S. aureus low temperature tolerance, as well as for guiding the development of safety control technology for cold-chain food.
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