河北省奶牛源大肠杆菌的分离鉴定、致病性及中草药体外抑菌效果研究

doi:10.16431/j.cnki.1671-7236.2022.09.042

摘要/Abstract

摘要： 【目的】分析河北省奶牛源大肠杆菌的致病性及中草药体外抑菌效果。【方法】于河北省收集30份具有典型临床症状的奶牛粪便样品，对样品进行细菌培养观察、生化鉴定、血清型鉴定、透射电镜观察、PCR扩增及致病性研究。采用传统煎煮法与醇提法提取9种中草药，通过96孔板-二倍稀释平板法测定最小抑菌浓度（minimum inhibitory concentration，MIC）及最低杀菌浓度（minimum bactericidal concentration，MBC），筛选出抑菌效果较强的中草药；将中草药进行两两联合，采用牛津杯法测定抑菌圈直径（inhibition zone diameter，IZD），并通过96孔板-二倍稀释平板法测定MIC和MBC，评价药物的抑菌效果。【结果】分离获得12株奶牛源大肠杆菌，检出率为40%，均具有致病性，其优势血清型为O55，检出率为58.3%。在中草药水提取液中，五倍子、五味子抑菌效果最强，MIC和MBC均为7.80 mg/mL，其次为乌梅、夏枯草、石榴皮，具有较好的抑菌效果，MIC和MBC均为15.63 mg/mL；在中草药醇提取液中，五倍子抑菌效果最强，MIC和MBC均为3.97 mg/mL，其次为五味子抑菌效果较好，MIC和MBC均为7.80 mg/mL。在中草药两两联合中，五味子与乌梅联合用药表现出协同作用，抑菌效果最强，IZD为26.00 mm，MIC和MBC均为3.97 mg/mL，其次为五倍子与乌梅，IZD为23.66 mm，MIC和MBC均为7.80 mg/mL。【结论】本研究成功分离到具有致病性的大肠杆菌，优势血清型为O55型，筛选出抑菌效果较好的单味及复方中草药，为大肠杆菌引起的奶牛腹泻临床应用中药防治提供科学依据。

关键词: 奶牛; 大肠杆菌; 致病性; 中草药; 抑菌作用

Abstract: 【Objective】 This study was aimed to analyze the pathogenicity of Escherichia coli(E.coli) from dairy cows in Hebei province and the antibacterial effect of Chinese herbal medicine in vitro.【Method】 30 feces samples with typical clinical symptoms were collected from dairy cows in Hebei province.The collected samples were cultured and observed, and the biochemical identification, serotype identification, transmission electron microscope observation, PCR amplification and pathogenicity study were carried out.9 kinds of Chinese herbal medicines were extracted by traditional decoction and alcohol extraction.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by 96 well plate double dilution plate method.Chinese herbal medicines with strong antibacterial effect were selected and combined in two, the inhibition zone diameter(IZD) was measured by Oxford cup method, MIC and MBC were measured by 96 well plate double dilution plate method, and the antibacterial effect of the drug was evaluated.【Result】 12 strains of E.coli isolated from dairy cows were pathogenic, with a detection rate of 40%.The dominant serotype was O55, with a detection rate of 58.3%.In the liquid extract of Chinese herbal medicine, Phus chinensis and Schisandra chinensis had the strongest antibacterial effect, MIC and MBC were both 7.80 mg/mL, followed by Pructus mume, Prunella vulgaris and pomegranate peel, with a better antibacterial effect, MIC and MBC were both 15.63 mg/mL.In the alcohol extract of Chinese herbal medicine, Phus chinensis had the strongest antibacterial effect, MIC and MBC were both 3.97 mg/mL, followed by Schisandra chinensis, MIC and MBC were both 7.80 mg/mL.In the combination of two drugs, the combination of Schisandra chinensis and Pructus mume showed synergistic effect, and the antibacterial effect was the strongest, IZD was 26.00 mm, MIC and MBC were both 3.97 mg/mL.Secondly, the antibacterial effect of Phus chinensis and Pructus mume were better, IZD was 23.66 mm, MIC and MBC were both 7.80 mg/mL.【Conclusion】 The pathogenic Escherichia coli was successfully isolated in this study, and the dominant serotype was O55.Single and compound Chinese herbal medicines with good antibacterial effect were screened, which provided a scientific basis for the clinical application of traditional Chinese medicine in the prevention and treatment of cow diarrhea caused by Escherichia coli.

Key words: dairy cows; Escherichia coli; pathogenicity; Chinese herbal medicine; antibacterial effect

中图分类号:

S852.61⁺2

邵明珠, 赵允清, 李松励, 侯绍华, 张艳英. 河北省奶牛源大肠杆菌的分离鉴定、致病性及中草药体外抑菌效果研究[J]. 中国畜牧兽医, 2022, 49(9): 3683-3691.

SHAO Mingzhu, ZHAO Yunqing, LI Songli, HOU Shaohua, ZHANG Yanying. Isolation and Identification of Escherichia coli from Dairy Cows in Hebei Province and Its Pathogenicity and Antibacterial Effect of Chinese Herbal Medicine in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3683-3691.

参考文献

[1] GAO R J, WANG C J, HAN A C, et al.Emodin improves intestinal health and immunity through modulation of gut microbiota in mice infected by pathogenic Escherichia coli O1[J].Animals, 2021, 11(11):3314.
[2] HIRUDKAR J R, PARMAR K M, PRASAD R S, et al.The antidiarrhoeal evaluation of Psidium guajava L.against enteropathogenic Escherichia coli induced infectious diarrhoea[J].Journal of Ethnopharmacology, 2020, 251:112561.
[3] GUERRA S T, ORSI H, JOAQUIM S F, et al.Short communication:Investigation of extra-intestinal pathogenic Escherichia coli virulence genes, bacterial motility, and multidrug resistance pattern of strains isolated from dairy cows with different severity scores of clinical mastitis[J].Journal of Dairy Science, 2020, 103(4):3606-3614.
[4] ALOMARI M, DEC M, NOWACZEK A, et al.Therapeutic and prophylactic effect of the experimental bacteriophage treatment to control diarrhea caused by E.coli in newborn calves[J].ACS Infectious Diseases, 2021, 7:2093-2101.
[5] 高海慧, 王建东, 高小斐, 等.奶牛犊牛腹泻源大肠埃希氏菌耐药情况及LEE相关基因的检测[J].动物医学进展, 2021, 42(6):19-23. GAO H H, WANG J D, GAO X F, et al.Drug resistance and detection of LEE related genes in E.coli from dairy calves with diarrhea[J].Progress in Veterinary Medicine, 2021, 42(6):19-23.(in Chinese)
[6] 岳山.黑龙江部分地区肉牛大肠杆菌耐药基因与毒力基因检测及耐药性分析[D].大庆:黑龙江八一农垦大学, 2017. YUE S.Detection of drug resistance gene and virulence gene and drug resistance analysis of beef cattle Escherichia coli in some areas of Heilongjiang province[D].Daqing:Heilongjiang Bayi Agricultural University, 2017.(in Chinese)
[7] 肖跃强, 莫玲, 崔平, 等.黄河三角洲部分地区肉犊牛致病性大肠杆菌血清型调查及耐药性分析[J].黑龙江畜牧兽医, 2020, 10:93-96. XIAO Y Q, MO L, CUI P, et al.Investigation and analysis of pathogenic Escherichia coli serotypes in Yellow River delta[J].Heilongjiang Animal Science and Veterinary Medicine, 2020, 10:93-96.(in Chinese)
[8] ALIA A, LIAQATA S, TARIQ H.Neonatal calf diarrhea:Apotentreservoir of multi-drug resistant bacteria, environmental contamination and public health hazard in Pakista[J].The Science of the Total Environment, 2021, 799:149450.
[9] ZHU F.A review on the application of herbal medicines in the disease control of aquatic animals[J].Aquaculture, 2020, 526:735422.
[10] 段广靖, 谢锋, 王斌.兽用中药作用机理及应用研究进展[J].特产研究, 2020, 42(6):73-78. DUAN G J, XIE F, WANG B.Research progress on action mechanism and application of veterinary traditional Chinese medicine[J].Specialty Research, 2020, 42(6):73-78.(in Chinese)
[11] ZHANG S, ZHAO Q, XUE W, et al.The Isolation and identification of Candida Glabrata from avian species and a study of the antibacterial activities of Chinese herbal medicine in vitro[J].Poultry Science, 2021, 100(4):101003.
[12] LI E, SUN N, ZHAO J X, SUN Y G, et al. In vitro evaluation of antiviral activity of tea seed saponins against Porcine reproductive and respiratory syndrome virus[J].Antiviral Therapy, 2015, 20(7):743-752.
[13] 王国华.泰安地区部分规模化奶牛场大肠杆菌的流行特点及耐药特点分析[D].泰安:山东农业大学, 2019. WANG G H.Epidemiological characteristics and drug resistance of Escherichia coli in some large-scale dairy farms in Tai'an area[D].Tai'an:Shandong Agricultural University, 2019.(in Chinese)
[14] 方光远, 茅慧华, 蒋加进, 等.不同病例动物源大肠杆菌的分离鉴定及16S rDNA同源性分析[J].中国畜牧兽医, 2015, 42(12):312-323. FANG G Y, MAO H H, JIANG J J, et al.Isolation, identification and 16S rDNA homology analysis of Escherichia coli from animals in different cases[J].China Animal Husbandry & Veterinary Medicine, 2015, 42(12):312-323.(in Chinese)
[15] PENG J J, BALASUBRAMANIAN B, MING Y Y, et al.Identification of antimicrobial resistance genes and drug resistance analysis of Escherichia coli in the animal farm environment[J].Journal of Infection and Public Health, 2021, 14(12):1788-1795.
[16] GIUFRE M, MAZZOLINI E, CERQUETTI M.Extended-spectrum β-lactamase-producing Escherichia coli from extraintestinal infections in humans and from food-producing animals in Italy:A ‘One Health’ study[J].International Journal of Antimicrobial Agents, 2021, 58(5):1788-1795.
[17] FU M.Drug discovery from traditional Chinese herbal medicine using high content imaging technology[J].Journal of Traditional Chinese Medical Sciences, 2021, 8:198-204.
[18] WANG J, HU X F, YANG C L, et al.Growth restriction of Rhizoctonia solani via breakage of intracellular organelles by crude extract of gallnut and clove[J].Molecules, 2021, 26(6):1667.
[19] 李仲兴, 王秀华, 张明明, 等.五倍子乙醇提取物对金葡菌的体外抗菌研究[J].中药新药与临床药理, 2005, 16(2):103-105. LI Z X, WANG X H, ZHANG M M, et al.In-vitro antibacterial activity of Galla chinensis ethanol extract against Staphylococcus aureus[J].Traditional Chinese Drug Research & Clinical Pharmacology, 2005, 16(2):103-105.(in Chinese)
[20] 洪军, 李文玉, 卫夏怡, 等.石榴皮提取物的抑菌活性及其对冷却鸡肉保鲜效果[J].中国调味品, 2020, 45(3):73-82. HONG J, LI W Y, WEI X Y, et al.Antibacterial activity of pomegranate peel extract and their preservation effect on cooled chicken[J].China Condiment, 2020, 45(3):73-82.(in Chinese)
[21] 刘伟, 刘军海.石榴皮中鞣花酸的提取工艺优化研究[J].粮食与油脂, 2022, 35(3):149-154. LIU W, LIU J H.Optimization of extraction process of ellagic acid from pomegranate peel[J].Grain and Oil, 2022, 35(3):149-154.(in Chinese)
[22] PRABUSEENIVASAN S, JAYAKUMAR M, IGNACIMUTHU S.In vitro antibacterial activity of some plant ssential oils[J].BMC Complementary and Alternative Medicine, 2006, 6(1):39.
[23] GUI S M, LI T, WANG Q.Antibacterial efects of Schisandra chinensis extract on Escherichia coli and its applications in cosmetic[J].Current Microbiology, 2020, 77(5):865-874.
[24] 王放银, 段林东, 赵良, 等.夏枯草抗菌成分提取工艺及其抑菌效果研究[J].黑龙江畜牧兽医, 2004, 10:61-62. WANG F Y, DUAN L D, ZHAO L, et al.Study on extraction technology and antibacterial effect of antibacterial components from Prunella vulgari[J].Heilongjiang Animal Science and Veterinary Medicine, 2004, 10:61-62.(in Chinese)