随着微生物学的发展，细菌的可培养性和活性的概念逐渐分离，紫外线消毒能够通过造成DNA损伤而有效地降低细菌的可培养性，但传统上认为其对细菌的活性直接影响有限。为了更准确地评价紫外线消毒技术，本研究分别以大肠杆菌和金黄色葡萄球菌作为革兰氏阳性菌和阴性菌的代表菌种，基于可培养性和活性研究紫外线对细菌的影响，建立了同时保证活性实验和紫外线辐照实验质量的实验技术体系，设计测量活性指标变化的方法以期能够准确体现紫外线消毒效果，选择出ATP作为用来评价紫外线消毒对细菌活性影响的指标，并基于这种选择构建了紫外线对细菌活性的影响模型，更全面地揭示了紫外线消毒机理。研究结果表明：紫外线能在40 mJ/cm2内就有效地降低细菌的可培养性，但是细菌能够利用暗修复和光修复的机制使可培养性得到一定程度的恢复。利用荧光定量PCR技术能够测量紫外线对DNA造成的损伤时，需要选择较长的基因扩增片段才能进行有效检测；细菌的暗修复对DNA损伤修复效率较低，而光修复则能够有效降低DNA损伤率。紫外线能够对RNA造成非特异性的损伤；通过测量细菌SOS响应系统和程序性细胞死亡系统对应基因的mRNA含量，能够检测细菌的修复系统和衰亡系统的启动程度；细菌在受到紫外线照射后，会更大程度地启动SOS响应系统，但是程序性细胞死亡系统的启动程度与未受到照射的细菌差异不明显。ATP适合作为评价紫外线消毒效果的细菌活性指标。经过紫外线照射的细菌，其ATP衰减速度会高于未受到紫外线照射的细菌，这种加速衰减符合e-△kT形式的指数下降，且衰减速率系数与紫外线剂量呈正比例关系。中压紫外线相比低压紫外线能够更有效地加速细菌活性的衰减，且加速效应在剂量达到40 mJ/cm2后开始饱和。利用ATP在实际工程中评价紫外线消毒效果仍然具有一定局限性。本研究基于活性对紫外线消毒机理进行了全新揭示：细菌受到UV损伤后，除了根据传统的消毒机理会由于DNA受损而失去可培养性，并自然地进行衰亡以外，还会启动SOS响应系统来应对损伤，导致细菌活性加快衰减而引起相关活性指标的降低。此外如果细菌除DNA受损之外，其蛋白质还受到损伤，那么其修复损伤的能力会被进一步破坏。
With the development of microbiology, the concepts of bacterial culturability and viability are gradually uncoupled. Ultraviolet disinfection can effectively reduce bacterial culturability by causing DNA damage, but it is generally considered that its direct impact on bacterial viability is limited. In order to evaluate the ultraviolet disinfection technology more comprehensively, this study used Escherichia coli and Staphylococcus aureus as representative strains of Gram-positive bacteria and Gram-negative bacteria respectively. Based on culturability and viability, the influence of ultraviolet radiation on bacteria was studied. An experimental technology system was established to ensure the quality of both the viability experiment and the ultraviolet radiation experiment, and a method to measure the change of the viability index was designed. In order to accurately reflect the effect of ultraviolet disinfection, ATP was selected as an index to evaluate the effect of ultraviolet disinfection on bacterial viability. Based on this choice, a model of the effect of ultraviolet radiation on bacterial viability was constructed, and the mechanism of ultraviolet disinfection was revealed more comprehensively.The results show that ultraviolet radiation can effectively reduce the culturability of bacteria within 40 mJ/cm2, but bacteria can regain culturability to a certain extent by using the mechanisms of dark and photo repair.Quantitative polymerase chain reaction (qPCR) can detect DNA damage caused by ultraviolet radiation, but long amplified fragments are needed to detect DNA damage effectively; dark repair of bacteria is relatively inefficient for DNA damage repair, while photo repair can reduce DNA damage effectively.Ultraviolet radiation can cause non-specific damage to RNA; by measuring the RNA content of genes corresponding to bacterial SOS response system and programmed cell death system, the initiation of bacterial repair system and decay system can be detected; bacteria will activate SOS response system to a greater extent after exposure to ultraviolet radiation, but the difference of initiation of programmed cell death system between bacteria exposed to UV and those not was not observed.ATP is suitable for evaluating the bacterial viability of ultraviolet disinfection. The ATP degradation rate of the bacteria exposed by ultraviolet is higher than that of the bacteria not exposed. This accelerated degradation conforms to the exponential decrease in the form of e-kT, and the degradation rate coefficient is proportional to the ultraviolet dose. Compared with low-pressure ultraviolet, medium-voltage ultraviolet radiation can accelerate the decline of bacterial viability more efficient, and the acceleration effect starts to saturate after the dose reaches 40 mJ/cm2.In this study, a new mechanism of ultraviolet disinfection based on viability was revealed: after the bacteria was exposed to UV, in addition to the traditional disinfection mechanism where they will lose culturability and decay at a natural rate due to the DNA damage, they will also initiate the SOS response system, leading to accelerated declince of bacterial viability and relevant indicator. And if their protein is damaged besides DNA damage, the ability of bacteria to repair damage for survival will be further reduced.
