氧化压力下沙门氏菌可培养性检测及其活的非可培养状态形成情况

Detection of culturable Salmonella and its formation of a viable but non-culturable state under oxidative stresses

摘要:

【背景】氧化压力会导致细菌进入活的非可培养(viable but non-culturable，VBNC)状态，菌落形成能力可能受到亚致死损伤的影响。目前对于VBNC态细菌的定量检测是基于活菌数与可培养数的差值，因此可培养数的检测对于VBNC态定量研究很关键，培养基组成不合适可能会造成漏检。【目的】分析培养基组成对氧化压力下亚致死损伤细菌检测的重要影响；探究常见食源性致病菌肠炎沙门氏菌在氧化压力下形成VBNC态的情况。【方法】分别采用Luria-Bertani (LB)、beef peptone yeast (BPY)和Salmonella shigella (SS)培养基检测并比较肠炎沙门氏菌的可培养数；采用RT-qPCR、荧光染色-激光共聚焦显微镜观测氧化压力下肠炎沙门氏菌形成VBNC态的情况。【结果】非选择性培养基LB和BPY能检出亚致死细菌，SS培养基中牛胆盐导致可培养数减少；肠炎沙门氏菌经53℃过氧化氢处理1.5 h后进入VBNC态的比例显著高于53℃过氧化氢+亚铁离子和过氧化氢+柠檬酸处理(P<0.05)。【结论】在对VBNC态的检测中应选择合适的固体培养基检测可培养菌；氧化压力下沙门氏菌进入VBNC态，这对以过氧化氢为消毒剂的处理提出了挑战，应尽量避免该特殊亚致死状态的形成。

Abstract:

[Background] Bacteria would enter a viable but non-culturable (VBNC) state under oxidative stress, and their colony forming ability may be affected by sublethal injury that imposed on bacterial cells. Currently, the quantitative detection of VBNC bacteria is based on the difference between the viable and culturable counts. Therefore, the accurate detection of culturable counts is critical to quantitating the number of VBNC cells. In addition, a proper growth medium might avoid the missing detection of viable pathogenic bacteria. [Objective] To analyze the effect of medium composition on the detection of bacteria exposed to sublethal injury caused by oxidative stresses, and to explore the formation of VBNC state of Salmonella enterica serovar Enteritidis under oxidative stresses. [Methods] The culturable cells in Luria-Bertani (LB), beef peptone yeast (BPY), and Salmonella Shigella (SS) medium were counted and compared. The formation of VBNC S. enterica serovar Enteritidis was quantified respectively with RT-qPCR and fluorescent staining under confocal laser microscope. [Results] The sublethal injured cells under oxidative stress could be detected with non-selective LB and BPY medium, and the counts of culturable cells reduced in SS medium because of bile salt. The formation rate of VBNC S. enterica serovar Enteritidis exposed to H₂O₂at 53 °C for 1.5 h was significantly higher than that in the other two cases (P<0.05). [Conclusion] For the detection of VBNC state, a suitable medium should be selected to detect culturable cells. Oxidative stress would induce the VBNC state of food-borne pathogens, which deserves special attention as H₂O₂ is widely used as a disinfectant in food processing and medical treatment.