丁酸钠对嗜水气单胞菌生长及毒力的影响

doi:10.16431/j.cnki.1671-7236.2023.07.038

摘要/Abstract

摘要： 【目的】探究丁酸钠对嗜水气单胞菌生长和毒力的影响，评估丁酸钠对嗜水气单胞菌的抑菌活性，并探索其作用机制。【方法】通过微量肉汤稀释法测定丁酸钠对嗜水气单胞菌的最小抑菌浓度（MIC），采用泳动和集群运动试验检测丁酸钠对嗜水气单胞菌的运动状态的影响，使用酶活性和溶血性试验检测丁酸钠对嗜水气单胞菌的脂肪酶、蛋白酶和溶血性的影响，生物膜试验检测丁酸钠对嗜水气单胞菌生物膜的抑制作用，实时荧光定量PCR技术检测丁酸钠对嗜水气单胞菌毒力和群体感应系统的相关基因表达的影响。【结果】丁酸钠对3株嗜水气单胞菌的MIC均>512 mg/L；丁酸钠浓度≤512 mg/L时，不影响3株嗜水气单胞菌的生长。当丁酸钠浓度≥64 mg/L时，除对GZ-SS2019的集群运动能力无显著影响（P>0.05）外，均能显著抑制3株嗜水气单胞菌的运动能力（P<0.05），此浓度下还显著抑制3株嗜水气单胞菌生物膜的形成（P<0.05）。在丁酸钠浓度为128 mg/L时，对3株嗜水气单胞菌脂肪酶活性无显著影响（P>0.05），却显著抑制3株嗜水气单胞菌蛋白酶活性（P<0.05）；而丁酸钠浓度≥32 mg/L时，除对GZDX-01的抑制效果不显著（P>0.05）外，能显著降低其余2株嗜水气单胞菌溶血率（P<0.05）。实时荧光定量PCR结果显示，丁酸钠浓度≥128 mg/L时能显著降低群体感应系统luxS和ahyR基因表达量（P<0.05）；溶血相关基因aerA表达量显著下降（P<0.05），此外ompW、ompA、fur基因表达量也显著下降（P<0.05）。【结论】丁酸钠浓度≤512 mg/L时不影响嗜水气单胞菌的生长，但能有效干扰嗜水气单胞菌的运动状态、酶活性、溶血性及生物膜的形成，以及毒力基因和群体感应系统相关基因的表达，其中丁酸钠浓度≥128 mg/L时效果较好。

关键词: 丁酸钠; 嗜水气单胞菌; 生物膜; 溶血性; 蛋白酶活性

Abstract: 【Objective】 The experiment was aimed to investigate the effects of sodium butyrate on the growth and virulence of Aeromonas hydrophila, to assess the antibacterial activity of sodium butyrate against Aeromonas hydrophila, and to explore its mechanism of action.【Method】 The minimum inhibitory concentration (MIC) of sodium butyrate on Aeromonas hydrophila was determined by micro-broth dilution method, the effect of sodium butyrate on the motility of Aeromonas hydrophila was examined by swimming and cluster motility assay, the effect of sodium butyrate on lipase, protease and hemolysis of Aeromonas hydrophila was examined by using enzyme activity and hemolysis assay, the effect of sodium butyrate on the biofilm of Aeromonas hydrophila was examined by biofilm assay, and the fluorescence quantitative PCR was used to detect the effect of sodium butyrate on the virulence of Aeromonas hydrophila and the expression of genes related to the population sensing system.【Result】 The MIC of sodium butyrate was >512 mg/L for all three strains of Aeromonas hydrophila, and the growth of the three strains of Aeromonas hydrophila was not affected when the concentration of sodium butyrate was ≤ 512 mg/L.When the concentration of sodium butyrate was ≥ 64 mg/L, the growth of the three strains of Aeromonas hydrophila was not affected.When the concentration of sodium butyrate was ≥ 64 mg/L, it significantly inhibited the motility of the three Aeromonas hydrophila strains (P<0.05), except for the cluster motility of GZ-SS2019 (P>0.05), which also significantly inhibited the biofilm formation of the three Aeromonas hydrophila strains at this concentration (P<0.05).At the concentration of 128 mg/L, sodium butyrate had less effect on the lipase activity of the three Aeromonas hydrophila strains (P>0.05), but significantly inhibited the protease activity of the three Aeromonas hydrophila strains (P<0.05). When the concentration of sodium butyrate was ≥ 32 mg/L, it significantly reduced the hemolysis rate of the remaining two Aeromonas hydrophila strains(P<0.05), except for the inhibition of GZDX-01, which was not significant (P>0.05).The fluorescence quantitative PCR results showed that when the concentration of sodium butyrate ≥ 128 mg/L, it significantly reduced the expression of luxS and ahyR genes in the population induction system (P<0.05), the expression of aerA, ompW, ompA and fur genes were also significantly reduced (P<0.05).【Conclusion】 It was shown that sodium butyrate with the concentration was ≤ 512 mg/L did not affect the growth of Aeromonas hydrophila, but could effectively interfere with the motility status, protease activity, hemolysis, and biofilm formation and expression of virulence genes and population sensing system of Aeromonas hydrophila, and when the concentration was ≥ 128 mg/L, it was more effective.

Key words: sodium butyrate; Aeromonas hydrophila; biofilm; hemolysis; protease activity

中图分类号:

S852.61

曾成容, 刘馨, 毕文文, 梅世慧, 何广霞, 张峻杰, 文明, 周碧君, 陈江凤, 姜海波. 丁酸钠对嗜水气单胞菌生长及毒力的影响[J]. 中国畜牧兽医, 2023, 50(7): 2987-2997.

ZENG Chengrong, LIU Xin, BI Wenwen, MEI Shihui, HE Guangxia, ZHANG Junjie, WEN Ming, ZHOU Bijun, CHEN Jiangfeng, JIANG Haibo. Effect of Sodium Butyrate on the Growth and Virulence of Aeromonas hydrophila[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2987-2997.

参考文献

[1] JIN L, CHEN Y, YANG W, et al.Complete genome sequence of fish-pathogenic Aeromonas hydrophila HX-3 and a comparative analysis:Insights into virulence factors and quorum sensing[J].Scientific Reports, 2020, 10(1):15479.
[2] 赵海晴, 李耘, 梁严内, 等.联合用药对嗜水气单胞菌耐药性影响研究进展[J].生物技术通报, 2022, 38(6):53-65. ZHAO H Q, LI Y, LIANG Y N, et al.Advances in research on the effects of joint medication on the drug resistance of Aeromonas hydrophila[J].Biotechnology Bulletin, 2022, 38(6):53-65.(in Chinese)
[3] ROGES E M, GONÇALVES V D, CARDOSO M D, et al.Virulence-associated genes and antimicrobial resistance of Aeromonas hydrophila isolates from animal, food, and human sources in brazil[J].BioMed Research International, 2020, 2020:1052607.
[4] DE LAZARI M G T, PEREIRA L X, ORELLANO L A A, et al.Sodium butyrate downregulates implant-induced inflammation in mice[J].Inflammation, 2020, 43(4):1259-1268.
[5] 王海瑞, 胡俊茹, 赵红霞, 等.饲料中添加丁酸钠对黄颡鱼幼鱼非特异性免疫、抗氧化指标及肠道黏膜形态的影响[J].动物营养学报, 2021, 33(9):5379-5390. WANG H R, HU J R, ZHAO H X, et al.Effects of dietary sodium butyrate on non-specific immune, antioxidant indices and intestinal mucosal morphology of juvenile y ellow catfish (Pelteobagrus fulvidraco)[J].Chinese Journal of Animal Nutrition, 2021, 33(9):5379-5390.(in Chinese)
[6] YANG C, OUYANG L, WANG W, et al.Sodium butyrate-modified sulfonated polyetheretherketone modulates macrophage behavior and shows enhanced antibacterial and osteogenic functions during implant-associated infections[J].Journal of Materials Chemistry. B, 2019, 7(36):5541-5553.
[7] 秦婷, 习丙文, 谢骏, 等.QseB在嗜水气单胞菌响应去甲肾上腺素中的作用[J].水产学报, 2022, 46(2):270-279. QIN T, XI B W, XIE J, et al.QseB regulates in vitro and in vivo virulence of Aeromonas hydrophila in response to norepinephrine[J].Journal of Fisheries of China, 2022, 46(2):270-279.(in Chinese).
[8] GAO J W, XI B J, CHEN K, et al.The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila[J].Microbiologyopen, 2019, 8(4):e00664.
[9] ZHOU D, PAN Q, XIN F Z, et al.Sodium butyrate attenuates high-fat diet-induced steatohepatitis in mice by improving gut microbiota and gastrointestinal barrier[J].World Journal of Gastroenterology, 2017, 23(1):60-75.
[10] 李祥, 何金环, 潘春梅, 等.丁酸钠对肉鸡肠道形态与消化吸收功能影响的研究进展[J].中国畜牧兽医, 2021, 48(5):1603-1612. LI X, HE J H, PAN C M, et al.Research progress on effect of sodium butyrate on intestinal morphology, digestion and absorption function of broilers[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(5):1603-1612.(in Chinese)
[11] 李祥, 何金环, 潘春梅, 等.丁酸钠对鱼类肠道消化吸收的影响研究进展[J].中国畜牧兽医, 2022, 49(1):131-139. LI X, HE J H, PAN C M, et al.Research progress on effects of sodium butyrate on intestinal digestion and absorption of fish[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(1):131-139.(in Chinese)
[12] ZHOU Z, YANG H, LI H, et al.Sodium butyrate ameliorates Corynebacterium pseudotuberculosis infection in RAW264.7 macrophages and C57BL/6 mice[J].Microbial Pathogenesis, 2019, 131:144-149.
[13] 韩乾杰, 张玲玲, 陈敏, 等.植物精油与丁酸钠的体外协同抑菌效果研究[J].动物营养学报, 2017, 29(2):712-718. HAN Q J, ZHANG L L, CHEN M, et al.Synergistic bacteriostatic effect of plant essential oils and sodium butyrate in vitro[J].Chinese Journal of Animal Nutrition, 2017, 29(2):712-718.(in Chinese)
[14] 朱文秀, 柳红莉, 高静竹, 等.丁酸钠对副溶血性弧菌毒性的影响及机制研究[J].食品安全质量检测学报, 2021, 12(17):6887-6893. ZHU W X, LIU H L, GAO J Z, et al.Effect and mechanisms of sodium butyrate on the virulence of Vibrio parahaemolyticus[J].Journal of Food Safety & Quality, 2021, 12(17):6887-6893.(in Chinese)
[15] ZHAO X L, WU G, CHEN H, et al.Analysis of virulence and immunogenic factors in Aeromonas hydrophila:Towards the development of live vaccines[J].Journal of Fish Diseases, 2020, 43(7):747-755.
[16] DONG J, LIU Y, XU N, et al.Morin protects channel catfish from Aeromonas hydrophila infection by blocking aerolysin activity[J].Frontiers in Microbiology, 2018, 9:2828.
[17] SRINIVASAN R, DEVI K R, SANTHAKUMARI S, et al.Anti-quorum sensing and protective efficacies of naringin against Aeromonas hydrophila infection in Danio rerio[J].Frontiers in Microbiology, 2020, 11:600622.
[18] GUO T, LIU P, WANG Z, et al.Luteolin binds streptolysin o toxin and inhibits its hemolytic effects and cytotoxicity[J].Frontiers in Pharmacology, 2022, 13:942180.
[19] 许腾腾, 赵慧娟, 刘尊英.连翘提取物对嗜水气单胞菌群体感应系统的影响[J].微生物学通报, 2022, 49(1):256-269. XU T T, ZHAO H J, LIU Z Y.Effect of Forsythia suspensa extract on quorum sensing system of Aeromonas hydrophila[J].Microbiology China, 2022, 49(1):256-269.(in Chinese)
[20] TANHAY MANGOUDEHI H, ZAMANI H, SHAHANGIAN S S, et al.Effect of curcumin on the expression of ahyI/R quorum sensing genes and some associated phenotypes in pathogenic Aeromonas hydrophila fish isolates[J].World Journal of Microbiology & Biotechnology, 2020, 36(5):70.
[21] JAHID I K, LEE N Y, KIM A, et al.Influence of glucose concentrations on biofilm formation, motility, exoprotease production, and quorum sensing in Aeromonas hydrophila[J].Journal of Food Protection, 2013, 76(2):239-247.
[22] NAKAGAWA S, HILLEBRAND G G, NUNEZ G.Rosmarinus officinalis L.(rosemary) extracts containing carnosic acid and carnosol are potent quorum sensing inhibitors of Staphylococcus aureus virulence[J]. Antibiotics (Basel, Switzerland), 2020, 9(4):149.