芳樟醇诱导肠炎沙门氏菌进入活的不可培养状态的研究

Research on Induction of Salmonella enteritidis into Viable But Nonculturable State by Linalool

芳樟醇可有效降低食品在生产过程中受致病菌污染而引起食源性疾病的概率。鲜有关注由芳樟醇诱导活的不可培养状态(VBNC)细菌的抗性以及其复苏研究。采用二倍稀释法测定芳樟醇对肠炎沙门氏菌的最小抑菌浓度(MIC)为0.4%(V/V),通过PMAxx-qPCR方法对比可培养菌体细胞与VBNC状态的肠炎沙门氏菌对热、柠檬酸、乙酸、乳酸、头孢曲松钠、左氧氟沙星以及过氧化氢的耐受能力差异。采用丙酮酸钠、过氧化氢酶及对数期沙门氏菌无菌上清对VBNC细菌进行复苏。结果表明,芳樟醇诱导的VBNC状态的肠炎沙门氏菌对热、酸、抗生素的耐受能力均弱于可培养状态细胞,而对过氧化氢的耐受能力显著高于可培养沙门氏菌。经过72 h处理,只有过氧化氢酶成功复苏约(5.51±0.08)lg(CFU/mL)的肠炎沙门氏菌。通过测定可培养、VBNC以及复苏菌株内活性氧差异,表明活性氧的积累在菌体VBNC状态形成中的重要作用。本研究为监控天然抑菌物质使用过程中VBNC细菌的形成及其可能产生的食品安全问题提供了研究思路和科学依据。

Linalool can effectively reduce the probability of foodborne diseases caused by pathogen contamination.However,few studies have focused on the resistance and resuscitation of viable but nonculturable(VBNC)state induced by the typical essential oil,linalool.The minimum inhibitory concentration(MIC)of linalool against Salmonella enteritidis was determined by twofold dilution test(0.4%)and differences of resistances between culturable and VBNC S.enteritidis against heat,citric acid,acetic acid and lactic acid,ceftriaxone sodium,levofloxacin and hydrogen peroxide was compared by PMAxx-qPCR method.VBNC bacteria was resuscitated for 72 h incubation of sodium pyruvate,catalase and logarithmic Salmonella cell-free supernatant dilution.The results showed that resistances of VBNC S.enteritidis to heat,acid and antibiotics were weaker than those of the culturable,but resistance to hydrogen peroxide was significantly higher than that of the culturable.After 72 h treatment,only catalase successfully revived VBNC S.enteritidis with concentration of(5.51±0.08)lg(CFU/mL).Reactive oxygen species(ROS)levels among culturable,VBNC and resuscitated S.enteritidis were measured,from which the role of ROS accumulation of the formation of VBNC state was demonstrated.This study may provide ideas and scientific basis for the effective supervision of VBNC state under the application of natural antibacterial substances.