Differentiation of DNA or membrane damage of the cells in disinfection by flow cytometry
Graphical Abstract
Introduction
The common pathogenic microorganisms in drinking water include bacteria, fungi, alga, viruses, and protists, and these will cause many environmental problems and threaten the health of individuals (Chen et al., 2021, Novak Babič et al., 2017, Sylvestre et al., 2021, Xu et al., 2021). Fungi are ubiquitously found in the water environments, such as groundwater, surface water, tap water, and non-mineral bottled water (Bai et al., 2017, Jiang et al., 2021, Novak Babič et al., 2016, Oliveira et al., 2013). In addition, fungi also enrich certain water-related indoor environments (showering, washing dishes, and laundry) for their extensive consumption in daily hygiene, which affects human health (Babič et al., 2015, Moat et al., 2016, Novak Babič et al., 2016, Zupancic et al., 2016). Therefore, it is necessary to regulate the presence of fungi in drinking water, especially for immunocompromised people.
Methods to control fungi contamination include physical disinfection (e.g. ultraviolet light-emitting diode (UV-LED) and solar disinfection) and chemical disinfection (e.g. chlorine, chloramine, chlorine dioxide, ozone) (Hageskal et al., 2012, Luo et al., 2021, Ma and Bibby, 2017, Pereira et al., 2012, Wen et al., 2017b, Xia et al., 2021). Generally, the inactivation mechanism of the physical disinfection process mainly involves damage to cell DNA, while the inactivation mechanism of chemical disinfection mainly involves the destruction of the cell membrane. As observed in previous studies, nitrogenous organic compounds leaked out and the extracellular ATP concentration increased after fungal spores were treated with chemical disinfectants such as chlorine dioxide, O3, PAA (Liang et al., 2021, Wen et al., 2020, Wen et al., 2017a, Zuo et al., 2022). For the microbial cells treated with UV irradiation, their inactivation was ascribed to the formation of cyclobutane pyrimidine dimers (CPDs) and 6‐4 pyrimidine dimers on the DNA strands of the cells, which would prevent DNA transcription and replication (Braga et al., 2015, Nebot-Sanz et al., 2007).
Disinfection processes inactivate microorganisms by attacking their cellular structure, resulting in damage to the microbial cell membrane, changes in nucleic acids and the physiological state of cells. Flow cytometry (FCM) has been applied as an efficient means to assess fungal viability in water, including membrane integrity, esterase activities, and intercellular ROS levels (Cao et al., 2020, Oliveira et al., 2020, Vanhauteghem et al., 2017, Wan et al., 2022a, Wan et al., 2022b). For example, the effect of metalworking fluids (MWF) on Fusarium solani spores with FCM was assessed, and it was found that the percentage of intact conidia respectively decreased to 76% and 23% after 5% and 10% MWF treatments (Vanhauteghem et al., 2017). Wan et al. reported that the percentage of membrane permeabilized spores of Aspergillus niger (A. niger) was nearly 60% after UV265 irradiation (80% for UV265/Cl2) based on the results of FCM (Wan et al., 2020a, Wan et al., 2020b). The viability of fungal spores treated with chlorine-based disinfectants was evaluated by FCM, and the results demonstrated that the percentages of intact cells, esterase positive cells, and HL-ROS cells for A. niger spores after chlorine treatment were 20%, 60%, 35%, respectively (Cao et al., 2020).
SYBR Green I (SG), a nucleic acid stain, binds with the minor groove in dsDNA where its green fluorescence is enhanced, and SG stains all cells with intact cellular DNA irrespective of cell viability (Berney et al., 2008, Lee et al., 2016). Some studies demonstrated that cells before and after ozone and UV/chlorine treatment were stained with SG, and the decrease in the fluorescence intensity of the cells can reflect the degree of DNA damage (Ding et al., 2019, Lee et al., 2016, Wu et al., 2018, Wu et al., 2021). A decrease in fluorescence intensity of SG-stained cells would thus be indicative of severe nucleic acid (dissociation of dsDNA to a single strand or disappearance of the minor groove structure). However, the above DNA damage detection method is only suitable for UV disinfection and not for chemical disinfection, because the chemical disinfectant will penetrate the cell and continue to damage the biological molecules (DNA, etc.) inside the cell after the reaction is stopped. Considering that SG was possibly consumed by residual disinfectants inside the cell especially for the penetrating disinfectants, resulting in a decrease in fluorescence intensity and causing a decrease in fluorescence intensity, it was unreasonable to quantitatively determine the DNA damage percentage by using a single SG staining method.
Therefore, this study aimed to 1) optimize the SG staining method to quantify the DNA damage degree of spores; 2) establish a new method by combining improved SG staining and PI staining, and use it to distinguish DNA damage and membrane damage during the chemical disinfection; 3) measure the membrane permeability of three genera of fungal spores by the new method and common method (SG/PI double staining), respectively, and compare the two results to assess the accuracy of the new one; 4) apply the new method to analyze inactivation mechanism of chemical disinfection processes.
Section snippets
Preparation of suspensions
In this study, A. niger, Trichoderma harzianum (T. harzianum), and Penicillium polonicum (P. polonicum) selected from groundwater were chosen as the test microorganisms, and incubated with Dichloran Rose Bengal Chloramphenicol (DRBC) (Wen et al., 2019b, Wen et al., 2017b). Escherichia coli (E. coli, ATCC25922) was chosen as a representative of bacteria. The fungal spores and E. coli were prepared in phosphate buffer solution (PBS) at pH 7.2, and the initial concentration was approximately 105-10
Differentiation of DNA damage or membrane damage in fungal spores’ disinfection
In this study, fungal spores were stained with SG after 20 min contact with chloramine, and it was found that the fluorescence intensity of these fungal spores (FL1) decreased by 71.6% compared to those untreated with chloramine (Fig. S1). It can be speculated that the decrease in FL1 intensity was related to DNA damage to cells. As shown in Fig. S2, the reaction between the SG and the disinfectant was studied through the change in fluorescence intensity of fungal spores stained with the
Conclusions
- (1)
This study established a new staining method by SG re-staining and subsequent PI staining. FCM combined with SG-SG-PI staining method provided valuable information on the inactivation mechanism of chemical disinfectants and successfully differentiated DNA damage and membrane damage of fungal spores during the chemical disinfection. The new method could be briefly described as follows: 1) the fungal spores were stained with SG; 2) the stained spores were centrifugated and washed with PBS; 3) the
CRediT authorship contribution statement
Ruihua Cao: Writing – review & editing, Methodology, Investigation, Data curation, Conceptualization. Qiqi Wan: Data Curation, Supervision. Xiangqian Xu: Visualization, Investigation. Shiqi Tian: Supervision, Formal analysis. Gehui Wu: Software, Validation. Jingyi Wang: Resources, Project administration. Tinglin Huang: Supervision, Funding acquisition. Gang Wen: Validation, Supervision, Funding acquisition, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was supported by the Natural Science Foundation of China (Grant No. 51978557, 22076225), The Youth Innovation Team of Shaanxi Universities, Foundation of Guangdong Natural Science (2021A1515010365) and Shaanxi Provincial Key Research and Development Project (2020ZDLSF06-05).
Statement of novelty
Fungi regarded as "hazardous materials" are ubiquitously found in the water environments, such as groundwater, surface water, tap water, and non-mineral bottled water, causing many environmental problems and
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