唾液乳杆菌无细胞上清液对金黄色葡萄球菌溶血活性的抑制作用研究

doi:10.16431/j.cnki.1671-7236.2025.04.040

摘要/Abstract

摘要： 【目的】探究唾液乳杆菌无细胞上清液(CFCS)对金黄色葡萄球菌溶血活性的影响，并比较不同培养条件对唾液乳杆菌CFCS抑菌效果的影响。【方法】通过肉汤稀释法测定唾液乳杆菌CFCS对金黄色葡萄球菌的最小抑菌浓度(MIC)与亚抑菌浓度(SIC)；利用溶血活性半定量法检测唾液乳杆菌CFCS对金黄色葡萄球菌溶血活性的影响，利用实时荧光定量PCR检测金黄色葡萄球菌α-溶血素(hla)和AraC/XylS家族转录调控因子rbf基因相对表达量；通过小鼠致病性试验，检测唾液乳杆菌CFCS对小鼠死亡率、脏器载菌量和组织病理变化的影响；使用牛津杯双层琼脂扩散法分别测定不同培养基和培养条件对唾液乳杆菌CFCS抑菌活性的影响。【结果】唾液乳杆菌CFCS对金黄色葡萄球菌的MIC为31.25 mg/mL，SIC为3.906 mg/mL。经MIC唾液乳杆菌CFCS处理后，与阳性对照组相比，金黄色葡萄球菌的溶血活性极显著下降(P＜0.01)，hla基因相对表达量显著降低(P＜0.05)，rbf基因相对表达量极显著升高(P＜0.01)。小鼠致病性试验结果显示，经MIC唾液乳杆菌CFCS处理后，金黄色葡萄球菌感染小鼠肝脏、肾脏和脾脏载菌量极显著降低(P＜0.01)，肝脏、肾脏和脾脏脏器组织病变明显减轻。MRS培养基中发酵的唾液乳杆菌CFCS对金黄色葡萄球菌的抑菌效果最好，抑菌圈直径为17.64 mm，TPY培养基次之(12.83 mm)。以MRS培养基为基础，在初始葡萄糖含量为4%、初始氮源含量为1.1%、初始pH 4.0、发酵时间48 h时，唾液乳杆菌CFCS对金黄色葡萄球菌的抑菌效果最好。【结论】唾液乳杆菌CFCS可抑制金黄色葡萄球菌的溶血活性，降低其致病性。培养条件的改变可对唾液乳杆菌CFCS抑菌活性产生显著影响。

关键词: 唾液乳杆菌; 无细胞上清液(CFCS); 金黄色葡萄球菌; 溶血活性; 致病性

Abstract: 【Objective】 The objective of this experiment was to investigate the effect of cell-free culture supernatant (CFCS) of Lactobacillus salivarius (L.salivarius) on the hemolytic activity of Staphylococcus aureus (S.aureus)and to compare the effect of different culture conditions on the bacteriostatic effect of CFCS of L.salivarius.【Method】 The minimum inhibitory concentration (MIC) and subinhibitory concentration (SIC) of CFCS of L.salivarius on S.aureus were determined by broth dilution method.The influence of CFCS of L.salivarius on the hemolytic activity of S.aureus was detected by semi-quantitative method.The relative expression levels of α hemolysin (hla) and AraC/XylS family transcriptional regulators rbf gene in S.aureus were determined by Real-time quantitative PCR.The effects of CFCS of L.salivarius on mortality,organ bacteria load and histopathological changes of mice were detected by pathogenicity test.The effects of different medium and culture conditions on the antibacterial activity of CFCS of L.salivarius were determined by Oxford Cup double-layer agar diffusion method,respectively.【Result】 The MIC and SIC of CFCS of L.salivarius against S.aureus were 31.25 and 3.906 mg/mL,respectively.After treatment with CFCS of L.salivarius with MIC,compared with positive control group,the hemolytic activity of S.aureus was extremely significantly decreased (P＜0.01),the relative expression level of hla gene was significantly decreased (P ＜ 0.05),and the relative expression level of rbf gene was extremely significantly increased (P＜0.01).The results of pathogenicity test in mice showed that after treatment with MIC of CFCS of L.salivarius,the bacteria load in liver,kidney and spleen of mice infected with S.aureus was extremely significantly decreased (P＜0.01),and the lesions of liver,kidney and spleen tissues were significantly reduced.The fermentation of CFCS of L.salivarius in MRS medium had the best inhibitory effect on S.aureus,and the inhibitory zone diameter was 17.64 mm,followed by TPY medium (12.83 mm).Based on MRS medium,when the initial glucose content was 4%,the initial nitrogen source content was 1.1%,the initial pH was 4.0 and the fermentation time was 48 h,CFCS of L.salivarius had the best bacteriostatic effect on S.aureus.【Conclusion】 CFCS of L.salivarius could inhibit the hemolytic activity of S.aureus and reduce its pathogenicity.The change of culture conditions could significantly affect the antibacterial activity of CFCS of L.salivarius.

Key words: Lactobacillus salivarious; cell-free culture supernatant (CFCS); Staphylococcus aureus; hemolytic activity; pathogenicity

中图分类号:

S831.5

邢国锋, 党乙凡, 赵耀, 苏帆帆, 吴自豪, 李涛. 唾液乳杆菌无细胞上清液对金黄色葡萄球菌溶血活性的抑制作用研究[J]. 中国畜牧兽医, 2025, 52(4): 1884-1894.

XING Guofeng, DANG Yifan, ZHAO Yao, SU Fanfan, WU Zihao, LI Tao. Inhibition of CFCS of Lactobacillus salivarius on Hemolytic Activity of Staphylococcus aureus[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1884-1894.

参考文献

[1] SERGELIDIS D,ANGELIDIS A S.Methicillin-resistant Staphylococcus aureus:A controversial food-borne pathogen[J].Letters in Applied Microbiology,2017,64(6):409-418.
[2] ZHOU W X,JIN Y,LIU X,et al.Comparison of genetic features and evolution of global and Chinese strains of community-associated methicillin-resistant Staphylococcus aureus ST22[J].Microbiology Spectrum,2022,10(1):e0203721.
[3] 李佳婷,柳雅馨,宋琪,等.金黄色葡萄球菌毒力因子研究进展[J].东北农业科学,2020,45(3):124-128.LI J T,LIU Y X,SONG Q,et al.Research progress of virulence factors in Staphylococcus aureus[J].Journal of Northeast Agricultural Sciences,2020,45(3):124-128.(in Chinese)
[4] 周陆红.产α-溶血素的食物中毒金黄色葡萄球菌特性研究[D].杨凌:西北农林科技大学,2019.ZHOU L H.The characterization of alpha-hemolysin producing Staphylococcus aureus in food poisoning[D].Yangling:Northwest A&F University,2019.(in Chinese)
[5] FANG B,LIU B,SUN B.Transcriptional regulation of virulence factors Hla and phenol-soluble modulins α by AraC-type regulator Rbf in Staphylococcus aureus[J].International Journal of Medical Microbiology,2020,310(5):151436.
[6] DELEO F R,KENNEDY A D,CHEN L,et al.Molecular differentiation of historic phage-type 80/81 and contemporary epidemic Staphylococcus aureus[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(44):18091-18096.
[7] WARDENBURG J B,PATEL R J,SCHNEEWIND O.Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia[J].Infection and immunity,2007,75(2):1040-1044.
[8] 张莎.后抗生素时代饲料行业发展现状及未来展望[J].中国饲料,2022,33(6):117-120.ZHANG S.Current situation and future prospect of feed industry in the post-antibiotic era[J].China Feed,2022,33(6):117-120.(in Chinese)
[9] 欧阳喜光,邓强,仝钰洁,等.我国金黄色葡萄球菌耐药性现状、产生机制及防治措施初探[J].中国奶牛,2021,39(11):27-31.OUYANG X G,DENG Q,TONG Y J,et al.A preliminary study on the current situation,mechanism and control measures of Staphylococcus aureus drug resistance in China[J].China Dairy Cattle,2021,39(11):27-31.(in Chinese)
[10] 尹树磊,杜香,张小希,等.乳酸菌抗氧化机制研究进展及在食品领域的应用[J].食品科学,2024,45(13):345-355.YIN S L,DU X,ZHANG X X,et al.Research progress on antioxidant mechanism of lactic acid bacteria and its application in food[J].Food Science,2024,45(13):345-355.(in Chinese)
[11] MACIEJ K,HANIA S.Lactobacillus reuteri DSM 17938 in the prevention of antibiotic-associated diarrhoea in children:Protocol of a randomised controlled trial[J].BMJ Open,2017,7(1):e013928.
[12] 崔磊,郭伟国.乳酸菌产生的抑菌物质及其作用机制[J].食品安全质量检测学报,2018,9(11):2578-2584.CUI L,GUO W G.Antibacterial substances produced by lactic acid bacteria and their mechanism[J].Journal of Food Safety&Quality,2018,9(11):2578-2584.(in Chinese)
[13] ESPECHE C M,PELLEGRINO M,FROLA I,et al.Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis[J].Anaerobe,2012,18(1):103-109.
[14] 杜琨.乳酸菌产抑菌物质抑制金黄色葡萄球菌作用机理研究[J].中国乳品工业,2022,50(12):17-22.DU K.Study on the inhibitory mechanism of antibacterial substances produced by lactic acid bacterial against Staphylococcus aureus[J].China Dairy Industry,2022,50(12):17-22.(in Chinese)
[15] 李平,李涛,齐萌,等.1株唾液乳杆菌的抑菌物质及益生作用[J].中国畜牧兽医,2023,50(9):3771-3780.LI P,LI T,QI M,et al.Bacteriostatic substances and probiotics of a strain of Lactobacillus salivarius[J].China Animal Husbandry&Veterinary Medicine,2023,50(9):3771-3780.(in Chinese)
[16] 吴自豪,吕俊凡,廖光华,等.南疆巴州地区奶牛隐性乳腺炎性金黄色葡萄球菌的MLST分型及其药物敏感性分析[J].塔里木大学学报,2020,32(2):10-16.WU Z H,LYU J F,LIAO G H,et al.Antimicrobial resistance features and MLST typing of Staphylococcus aureus isolates from subclinical bovine mastitis in Bayingol Mongolian Autonomous Prefecture of Southern Xinjiang,China[J].Journal of Tarim University,2020,32(2):10-16.(in Chinese)
[17] 阮紫涵,黄安雄,王秀娟,等.CLSI、EUCAST和中国耐药判定标准概述[J].生物技术通报,2022,38(9):47-58.RUAN Z H,HUANG A X,WANG X J,et al.Overview of CLSI,EUCAST,and susceptibility breakpoints in China[J].Biotechnology Bulletin,2022,38(9):47-58.(in Chinese)
[18] 吴自豪,蔡依龙,陀海欣,等.1株马乳源PVL+ST22型金黄色葡萄球菌致病性分析[J].畜牧兽医学报,2024,55(2):718-726.WU Z H,CAI Y L,TUO H X,et al.Pathogenicity analysis of a PVL+ST22Staphylococcus aureus isolated from equine raw milk[J].Acta Veterinaria et Zootechnica Sinica,2024,55(2):718-726.(in Chinese)
[19] 宁柯铭,周汝顺,黎满香.湖南部分地区乳源金黄色葡萄球菌耐药性及溶血性分析[J].动物医学进展,2023,44(5):57-64.NING K M,ZHOU R S,LI M X.Analyses of antimicrobial resistance and hemolysis of Staphylococcus aureus from raw milk in Hunan province[J].Progress in Veterinary Medicine,2023,44(5):57-64.(in Chinese)
[20] 郭凯旋,王湘如,赵月,等.我国动物源细菌耐药性监测及预警的研究进展[J].中国兽药杂志,2021,55(4):79-85.GUO K X,WANG X R,ZHAO Y,et al.Progress in the research of drug resistance monitoring and early warning system of animal-derived bacteria in China[J].Chinese Journal of Veterinary Drug,2021,55(4):79-85.(in Chinese)
[21] 孙庆申,王钰涵,韩德权,等.酸菜中具有抑菌活性乳酸菌的分离鉴定及其产细菌素特性[J].食品科学技术学报,2022,40(4):64-73.SUN Q S,WANG Y H,HAN D Q,et al.Isolation and identification of lactic acid bacteria with antibacterial activity in pickles and their bacteriocin production characteristics[J].Journal of Food Science and Technology,2022,40(4):64-73.(in Chinese)
[22] ANNA T,INGA B,ARMEN T.The effects of different carbon sources on the antifungal activity by lactic acid bacteria[J].Current Microbiology,2017,74(2):168-174.
[23] 姜铁民,隋欣,王建,等.直投式发酵剂制备中乳酸菌增殖条件的探讨[J].食品与发酵工业,2005,8:112-114.JIANG T M,SUI X,WANG J,et al.Discuss on the propagation of lactic acid bacteria for preparation of direct vat inoculation[J]. Food and Fermentation Industries,2005,8:112-114.(in Chinese)
[24] 于翠芳,王鹏,朱英莲.乳酸菌发酵液对金黄色葡萄球菌的抑菌效果[J].生物加工过程,2019,17(6):662-666.YU C F,WANG P,ZHU Y L.Bacteriostatic effect of lactobacillus fermented broth on Staphylococcus aureus[J].Chinese Journal of Bioprocess Engineering,2019,17(6):662-666.(in Chinese)
[25] HUGAS M,GARRIGA M,PASCUAL M,et al.Enhancement of sakacin K activity against Listeria monocytogenes in fermented sausages with pepper or manganese as ingredients[J].Food Microbiology,2002,19(5):519-528.
[26] 杜琨.响应面法优化乳酸乳球菌Z103菌株产抑菌物质的发酵条件[J].江苏农业科学,2021,10:155-161.DU K.Optimization of fermentation conditions of bacteriostatic substance production in Lactococcus lactis Z103 by response surface methodology[J].Jiangsu Agricultural Sciences,2021,10:155-161.(in Chinese)