16S rRNA测序分析饮用含氯水对大鼠和小鼠肠道菌群的影响差异

doi:10.12300/j.issn.1674-5817.2025.135

摘要/Abstract

摘要：

目的评估饮用含氯水对大鼠、小鼠肠道菌群的影响，探讨模式动物肠道微生态对含氯饮用水中氯刺激的响应差异。方法 6只8周龄雄性SD大鼠与6只8周龄雄性C57BL/6小鼠，适应性喂养5 d后，将大鼠和小鼠的饮用水由纯水换为含25.4～32.4 nmol/L游离氯的水，持续干预8周，实验期间饲喂相同的饲料。分别于干预前（第0周，作为对照组）和第3周、第8周的最后一天（作为饮用含氯水组）收集粪便样本，通过16S rRNA测序分析菌群组成，并进行α多样性（Chao1和Shannon指数）、β多样性［主成分分析（principal component analysis，PCA）］及线性判别分析效应量（linear discriminant analysis effect size，LEfSe）分析。结果干预3周后，饮用含氯水组大鼠和小鼠肠道菌群的α多样性均显著低于对照组（P<0.01）；干预8周后，大鼠菌群的α多样性与对照组无显著差异，而小鼠的α多样性仍显著低于对照组（P<0.01）。β多样性分析显示，与对照组相比，饮用含氯水大鼠和小鼠组的菌群结构发生显著改变。LEfSe分析表明，与对照组相比，饮用含氯水组（大鼠和小鼠）的拟杆菌属和瘤胃球菌属丰度均显著降低，且对小鼠的菌群扰动更显著［线性判别分析（linear discriminant analysis，LDA）＞4.0］，菌群多样性的降幅更大，差异菌属的数量更多。结论饮用含氯水可改变大鼠、小鼠肠道菌群的多样性及结构，且小鼠对此更为敏感，提示在动物实验饮水管理及模型选择中，应关注氯对肠道微生态的潜在影响。

关键词: 16S rRNA测序, 肠道菌群, 含氯水, 纯化水, SD大鼠, C57BL/6J小鼠

Abstract:

Objective To evaluate the effects of drinking chlorinated water on the intestinal flora of rats and mice and to explore differences in the intestinal microecological responses of model animals to chlorine stimulation in chlorinated drinking water systems. Methods Six 8-week-old male SD rats and six 8-week-old male C57BL/6J mice were acclimated for 5 days. Subsequently, the drinking water for both rats and mice was changed from pure water to water containing free chlorine at a concentration of 25.4–32.4 nmol/L. The intervention lasted for 8 weeks, during which all animals were fed the same diet. Fecal samples were collected before the intervention (week 0, as the control group) and on the last day of week 3 and week 8 (as the chlorinated water group), and microbial composition was analyzed by 16S rRNA sequencing, including α diversity (Chao1 and Shannon indices), β diversity [principal component analysis (PCA)], and linear discriminant analysis effect size (LEfSe) analysis. Results After 3 weeks of intervention, α diversity of the intestinal flora in both rats and mice in the chlorinated water group was significantly lower than that in the control group (P < 0.01). After 8 weeks of intervention, no significant difference in α diversity was observed between rats in the chlorinated water group and those in the control group, whereas α diversity in mice remained significantly lower than that in the control group (P < 0.01). β diversity analysis showed significant alterations in microbial structure in the chlorinated water group. LEfSe analysis indicated that, compared with the control group, the abundances of Bacteroides and Ruminococcus were significantly reduced in the chlorinated water group, and microbial disturbance was more pronounced in mice [linear discriminant analysis (LDA) > 4.0], with a greater decrease in microbial diversity and a larger number of differentially abundant genera. Conclusion Consumption of chlorinated water can alter the diversity and structure of the intestinal flora in both rats and mice, with mice being more sensitive to this exposure. These findings suggest that the potential effects of chlorine on gut microecology should be considered in drinking water management and model selection for animal experiments.

Key words: 16S rRNA sequencing, Intestinal flora, Chlorinated water, Purified water, SD rats, C57BL/6J mice

中图分类号:

Q95-33

艾秀峰,张利棕,方明笋,等. 16S rRNA测序分析饮用含氯水对大鼠和小鼠肠道菌群的影响差异[J]. 实验动物与比较医学, 2026, 46(3): 437-445. DOI: 10.12300/j.issn.1674-5817.2025.135.

AI Xiufeng,ZHANG Lizong,FANG Mingsun,et al. Analysis of Differences in the Intestinal Flora of Rats and Mice after Drinking Chlorinated Water Based on 16S rRNA Sequencing[J]. Laboratory Animal and Comparative Medicine, 2026, 46(3): 437-445. DOI: 10.12300/j.issn.1674-5817.2025.135.

图/表 5

注：A～B，饮用含氯水0周、3周、8周大鼠之间粪便菌群的Chao1指数和Shannon指数；C～D，饮用含氯水0周、3周、8周小鼠之间粪便菌群的Chao1指数和Shannon指数。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^*P<0.05。
">

图1 大鼠和小鼠肠道菌群的α多样性分析
注：A～B，饮用含氯水0周、3周、8周大鼠之间粪便菌群的Chao1指数和Shannon指数；C～D，饮用含氯水0周、3周、8周小鼠之间粪便菌群的Chao1指数和Shannon指数。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^*P<0.05。

Figure 1 Analysis of intestinal flora α diversity in rats and mice
Note： A-B， Chao1 and Shannon indices of fecal microbiota among rats that drank chlorinated water for 0， 3， and 8 weeks； C-D， Chao1 and Shannon indices of fecal microbiota among mice that drank chlorinated water for 0， 3， and 8 weeks. R0L， R3L， and R8L are the rat groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； M0L， M3L， and M8L are the mouse groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； n=6， ^*P<0.05.

注：A～B，饮用含氯水0周、3周、8周大鼠之间的主成分分析（PCA）结果以及基于线性判别分析效应量（LEfSe）的支系图（从门级到种级）；C～D，饮用含氯水0周、3周、8周小鼠之间的PCA结果以及基于LEfSe的支系图（从门级到种级）。p，门水平；c，纲水平；o，目水平；f，科水平；g，属水平；s，种水平；R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6。
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图2 大鼠和小鼠肠道菌群β多样性分析与LDA差异菌群分类树分析
注：A～B，饮用含氯水0周、3周、8周大鼠之间的主成分分析（PCA）结果以及基于线性判别分析效应量（LEfSe）的支系图（从门级到种级）；C～D，饮用含氯水0周、3周、8周小鼠之间的PCA结果以及基于LEfSe的支系图（从门级到种级）。p，门水平；c，纲水平；o，目水平；f，科水平；g，属水平；s，种水平；R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6。

Figure 2 Intestinal flora β diversity and LEfSe differential taxa analysis in rats and mice
Note：A-B， Principal component analysis （PCA） of rats at 0， 3， and 8 weeks of chlorinated water consumption and LEfSe-based cladogram showing taxonomic results from phylum to species level； C-D， PCA of mice at 0， 3， and 8 weeks of chlorinated water consumption and LEfSe-based cladogram showing taxonomic results from phylum to species level. p， phylum； c， class； o， order； f， family； g， genus； s， species； R0L， R3L， and R8L are the rat groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； M0L， M3L， and M8L are the mouse groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； n=6.

注：A～B，饮用含氯水0周、3周、8周后，大鼠粪便微生物在门水平上的群落组成的直方图（A）及其丰度最大菌［变形杆菌门（Proteobacteria）］的相对丰度（B）；C～D，饮用含氯水0周、3周、8周小鼠粪便微生物在门水平上的群落组成的直方图（C）及其丰度最大菌［厚壁菌门（Firmicutes）］的相对丰度（D）。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^*P＜0.05。
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图 3 大鼠与小鼠微生物菌群在门水平上的相对丰度差异
注：A～B，饮用含氯水0周、3周、8周后，大鼠粪便微生物在门水平上的群落组成的直方图（A）及其丰度最大菌［变形杆菌门（Proteobacteria）］的相对丰度（B）；C～D，饮用含氯水0周、3周、8周小鼠粪便微生物在门水平上的群落组成的直方图（C）及其丰度最大菌［厚壁菌门（Firmicutes）］的相对丰度（D）。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^*P＜0.05。

Figure 3 Comparison of the relative abundance of microbial communities at the phylum level between rats and mice
Note：A-B， Histograms of phylum-level community composition of fecal microbiota in rats after 0， 3， and 8 weeks of chlorinated water consumption （A） and the relative abundance of the most abundant phylum （Proteobacteria）（B）； C-D， Histograms of phylum-level community composition of fecal microbiota in mice after 0， 3， and 8 weeks of chlorinated water consumption （C） and the relative abundance of the most abundant phylum （Firmicutes）（D）. R0L， R3L， and R8L represent rat groups drinking chlorinated water for 0， 3， and 8 weeks， respectively； M0L， M3L， and M8L represent mouse groups drinking chlorinated water for 0， 3， and 8 weeks， respectively； n = 6， ^*P< 0.05.

注：A～D，饮用含氯水0周、3周、8周大鼠组粪便微生物中拉奇诺梭菌属、多尔氏菌属、脱硫弧菌属和异杆菌属的相对丰度；E，饮用含氯水0周、3周、8周大鼠组粪便微生物中前30种优势菌属的相对丰度热图。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；n=6，^*P<0.05，^**P<0.01，^***P<0.001。
">

图4 大鼠肠道菌群关键菌属及热图分析
注：A～D，饮用含氯水0周、3周、8周大鼠组粪便微生物中拉奇诺梭菌属、多尔氏菌属、脱硫弧菌属和异杆菌属的相对丰度；E，饮用含氯水0周、3周、8周大鼠组粪便微生物中前30种优势菌属的相对丰度热图。R0L、R3L、R8L分别为饮用含氯水0周、3周和8周的大鼠组；n=6，^*P<0.05，^**P<0.01，^***P<0.001。

Figure 4 Key genus and heatmap analysis of intestinal flora in rats
Note： A-D， Relative abundances of Lachnoclostridium， Dorea， Desulfovibrio， and Allobaculum in the fecal microbiota of rat groups after 0， 3， and 8 weeks of chlorinated water consumption； E， Heatmap showing the relative abundances of the top 30 dominant genera in the fecal microbiota of rat groups after 0， 3， and 8 weeks of chlorinated water consumption. R0L， R3L， and R8L are the rat groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； n = 6， ^*P< 0.05， ^**P< 0.01， ^***P< 0.001.

注：A～D，肠杆状菌属、鼠杆菌属、瘤胃球菌属和拟杆菌属的相对丰度；E，饮用含氯水0周、3周、8周小鼠组粪便微生物中前30种优势物种的相对丰度热图。M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^***P<0.001。
">

图5 小鼠肠道菌群关键菌属及热图分析
注：A～D，肠杆状菌属、鼠杆菌属、瘤胃球菌属和拟杆菌属的相对丰度；E，饮用含氯水0周、3周、8周小鼠组粪便微生物中前30种优势物种的相对丰度热图。M0L、M3L、M8L分别为饮用含氯水0周、3周和8周的小鼠组；n=6，^***P<0.001。

Figure 5 Key genus and heatmap analysis of intestinal flora in mice
Note： A-D， Relative abundances of Enterorhabdus， Muribaculum， Ruminococcus， and Bacteroides； E， Heatmap showing the relative abundances of the top 30 dominant species in the fecal microbiota of mouse groups after 0， 3， and 8 weeks of chlorinated water consumption. M0L， M3L， and M8L are the mouse groups that drank chlorinated water for 0， 3， and 8 weeks， respectively； n = 6， ^***P< 0.001.

参考文献 31

[1]	NGWENYA N, NCUBE E J, PARSONS J. Recent advances in drinking water disinfection: successes and challenges[J]. Rev Environ ContamToxicol, 2013, 222: 111-170. DOI: 10.1007/978-1-4614-4717-7_4 .
[2]	TAYLOR K, POPE R. A new form of automatic watering system for laboratory animals[J]. Lab Anim, 1978, 12(3): 129-131. DOI: 10.1258/002367778780936241 .
[3]	SNELL J A, JANDOVA J, WONDRAK G T. Hypochlorous acid: from innate immune factor and environmental toxicant to chemopreventive agent targeting solar UV-induced skin cancer[J]. Front Oncol, 2022, 12: 887220. DOI: 10.3389/fonc. 2022.887220 .
[4]	SNELL J A, VAISHAMPAYAN P, DICKINSON S E, et al. The drinking water and swimming pool disinfectant trichloroisocyanuric acid causes chlorination stress enhancing solar UV-induced inflammatory gene expression in AP-1 transgenic SKH-1 luciferase reporter mouse skin[J]. Photochem Photobiol, 2023, 99(2): 835-843. DOI: 10.1111/php. 13675 .
[5]	JANDOVA J, SCHIRO G, DUCA F A, et al. Exposure to chlorinated drinking water alters the murine fecal microbiota[J]. Sci Total Environ, 2024, 914: 169933. DOI: 10.1016/j.scitotenv. 2024.169933 .
[6]	ROOKS M G, GARRETT W S. Gut microbiota, metabolites and host immunity[J]. Nat Rev Immunol, 2016, 16(6): 341-352. DOI: 10.1038/nri.2016.42 .
[7]	WATSON K, SHAW G, LEUSCH F D L, et al. Chlorine disinfection by-products in wastewater effluent: Bioassay-based assessment of toxicological impact[J]. Water Res, 2012, 46(18): 6069-6083. DOI: 10.1016/j.watres.2012.08.026 .
[8]	MOUDGAL C J, LIPSCOMB J C, BRUCE R M. Potential health effects of drinking water disinfection by-products using quantitative structure toxicity relationship[J]. Toxicology, 2000, 147(2): 109-131. DOI: 10.1016/s0300-483x(00)00188-8 .
[9]	SHI J Y, ZHANG K, XIAO T S, et al. Exposure to disinfection by-products and risk of cancer: a systematic review and dose-response meta-analysis[J]. Ecotoxicol Environ Saf, 2024, 270: 115925. DOI: 10.1016/j.ecoenv.2023.115925 .
[10]	NADIMPALLI M L, LANZA V F, MONTEALEGRE M C, et al. Drinking water chlorination has minor effects on the intestinal flora and resistomes of Bangladeshi children[J]. Nat Microbiol, 2022, 7(5): 620-629. DOI: 10.1038/s41564-022-01101-3 .
[11]	DIAS M F, REIS M P, ACURCIO L B, et al. Changes in mouse gut bacterial community in response to different types of drinking water[J]. Water Res, 2018, 132: 79-89. DOI: 10.1016/j.watres.2017.12.052 .
[12]	CHIU K, WARNER G, NOWAK R A, et al. The impact of environmental chemicals on the gut microbiome[J]. Toxicol Sci, 2020, 176(2): 253-284. DOI: 10.1093/toxsci/kfaa065 .
[13]	NGUYEN T L A, VIEIRA-SILVA S, LISTON A, et al. How informative is the mouse for human gut microbiota research[J]. Dis Model Mech, 2015, 8(1): 1-16. DOI: 10.1242/dmm.017400 .
[14]	FAN J H, QU Y J, QU L L, et al. Oral exposure to PLA microplastics induces time-dependent nanotoxicity via the gut-liver axis[J]. J Hazard Mater, 2025, 495: 138931. DOI: 10.1016/j.jhazmat.2025.138931 .
[15]	BELKAID Y, HAND T W. Role of the microbiota in immunity and inflammation[J]. Cell, 2014, 157(1): 121-141. DOI: 10.1016/j.cell.2014.03.011 .
[16]	DE FILIPPIS F, VALENTINO V, SEQUINO G, et al. Exposure to environmental pollutants selects for xenobiotic-degrading functions in the human gut microbiome[J]. Nat Commun, 2024, 15(1): 4482. DOI: 10.1038/s41467-024-48739-7 .
[17]	ERICSSON A C, FRANKLIN C L. The gut microbiome of laboratory mice: considerations and best practices for translational research[J]. Mamm Genome, 2021, 32(4): 239-250. DOI: 10.1007/s00335-021-09863-7 .
[18]	NAGPAL R, WANG S H, SOLBERG WOODS L C, et al. Comparative microbiome signatures and short-chain fatty acids in mouse, rat, non-human primate, and human feces[J]. Front Microbiol, 2018, 9: 2897. DOI: 10.3389/fmicb.2018.02897 .
[19]	RAVINDRAN S, HOPKINS B, BOVA S, et al. In vivo metabolism of norbormide in rats and mice[J]. Environ Toxicol Pharmacol, 2009, 28(1): 147-151. DOI: 10.1016/j.etap. 2009.03.013 .
[20]	DE VOS W M, TILG H, VAN HUL M, et al. Gut microbiome and health: mechanistic insights[J]. Gut, 2022, 71(5): 1020-1032. DOI: 10.1136/gutjnl-2021-326789 .
[21]	KRAVITZ A, LAWTON S, BUCKMASTER C A, et al. Influence of water delivery method on the gut microbiome in laboratory mice (Mus musculus)[J]. J Am Assoc Lab Anim Sci, 2025, 64(4): 1-12. DOI: 10.30802/AALAS-JAALAS-24-085 .
[22]	WANG Z X, CHEN G, SUN X T, et al. Multi-omics integration reveals the impact of Mediterranean diet on hepatic metabolism and gut microbiota in mice with metabolic dysfunction-associated steatotic liver disease[J]. Front Nutr, 2025, 12: 1644014. DOI: 10.3389/fnut.2025.1644014 .
[23]	MEDINA-LARQUÉ A S, RODRÍGUEZ-DAZA M C, ROQUIM M, et al. Cranberry polyphenols and agave agavins impact gut immune response and microbiota composition while improving gut barrier function, inflammation, and glucose metabolism in mice fed an obesogenic diet[J]. Front Immunol, 2022, 13: 871080. DOI: 10.3389/fimmu.2022.871080 .
[24]	WEI W, LIU Y L, HOU Y L, et al. Psychological stress-induced microbial metabolite indole-3-acetate disrupts intestinal cell lineage commitment[J]. Cell Metab, 2024, 36(3): 466-483.e7. DOI: 10.1016/j.cmet.2023.12.026 .
[25]	HEMARAJATA P, VERSALOVIC J. Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation[J]. Therap Adv Gastroenterol, 2013, 6(1): 39-51. DOI: 10.1177/1756283X12459294 .
[26]	HAYS K E, PFAFFINGER J M, RYZNAR R. The interplay between gut microbiota, short-chain fatty acids, and implications for host health and disease[J]. Gut Microbes, 2024, 16(1): 2393270. DOI: 10.1080/19490976.2024.2393270 .
[27]	SONG C Y, CHAI Z L, CHEN S, et al. Intestinal mucus components and secretion mechanisms: what we do and do not know[J]. Exp Mol Med, 2023, 55(4): 681-691. DOI: 10.1038/s12276-023-00960-y .
[28]	MUKHOPADHYA I, LOUIS P. Gut microbiota-derived short-chain fatty acids and their role in human health and disease[J]. Nat Rev Microbiol, 2025, 23(10): 635-651. DOI: 10.1038/s41579-025-01183-w .
[29]	SHIN N R, WHON T W, BAE J W. Proteobacteria: microbial signature of dysbiosis in gut microbiota[J]. Trends Biotechnol, 2015, 33(9): 496-503. DOI: 10.1016/j.tibtech.2015. 06.011 .
[30]	ROSSHART S P, HERZ J, VASSALLO B G, et al. Laboratory mice born to wild mice have natural microbiota and model human immune responses[J]. Science, 2019, 365(6452): eaaw4361. DOI: 10.1126/science.aaw4361 .
[31]	王珍, 周亚洲, 王立坤, 等. 持续低氧条件下大鼠肠道微生物变化及其与心肌损伤的关联研究[J]. 微生物学报, 2023, 63(8): 3054-3067. DOI: 10.13343/j.cnki.wsxb.20220862 .
	WANG Z, ZHOU Y Z, WANG L K, et al. Alterations in gut microbiota and their associations with cardiac injury in rats after exposure to continuous normobaric hypoxia[J]. Acta Microbiol Sin, 2023, 63(8): 3054-3067. DOI: 10.13343/j.cnki.wsxb.20220862 .