噬菌体裂解细菌过程中冷光实时监测活菌方法的建立

Development of a luminescence real-time method for monitoring live bacteria during phage lysis

产毒素的霍乱弧菌Vibrio cholerae可导致严重腹泻,已引起7次全球大流行。对于烈性噬菌体清除霍乱弧菌的效果评价上,一般使用传统的活细胞培养计数及噬菌斑进行观察分析,但操作费时耗力,尤其不能实时获得菌株被裂解及残存细胞的数量变化。进一步探索简便、能够实时监测噬菌体裂解霍乱弧菌的方法是非常必要的。利用荧光报告质粒的策略、将可在霍乱弧菌中高表达生物冷光的质粒转化至O1血清群霍乱弧菌耐药菌株中,通过测定比较生物冷光以及活菌计数,实时分析了噬菌体对液体培养状态下霍乱弧菌的裂解效果,结果显示:冷光值作为监测指标与传统的活细胞计数方法有很高的相关性,通过测定霍乱弧菌耐药株的冷光值监测霍乱弧菌活细胞的数量,可实时分析噬菌体裂解霍乱弧菌过程中细菌残存数量。这种分析方法与菌落计数和噬斑形成观察相比,能够重复对同一样本进行无干扰的连续多时间点检测,没有经过再培养或噬斑形成的时间迟滞,有利于进行噬菌体与宿主菌相互作用的实时监测分析。

The toxin-producing bacterium Vibrio cholerae can cause severe diarrhea and has caused seven global pandemics. Traditional viable cell counts and phage plaques are commonly used to evaluate the efficacy of virulent phage clearance of V. cholerae, but these operations are time-consuming and labor-intensive, and difficult to provide real-time changes. It is desirable to develop a simple and real-time method to monitor V. cholerae during phage lysis. In this study, a luminescence-generating plasmid pBBR-pmdh-luxCDABE was transformed into three O1 serogroup drug-resistant strains of V. cholerae. The results showed that the luminescence value as a monitoring index correlates well with the traditional viable cell count method. Monitoring the number of live cells of V. cholerae by measuring the luminescence allowed real-time analysis of the number of bacteria remaining during phage lysis. This method enables repeated, interference-free, continuous multiple-time-point detection of the same sample without the time delay of re-culture or plaque formation, facilitating real-time monitoring and analysis of the interaction between the phage and the host bacteria.