Isolation and Identification of Escherichia coli in Diarrheic Piglets (Sus scrofa domesticus) and the Effects of Huang-lian-jie-du Decoction on Its Pathogenicity
ZHANG Feng-Yu, CAI Yan-Zi, SHI Yan, JI Peng, HUA Yong-Li, YUAN Zi-Wen*, WEI Yan-Ming*
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Eschericha coli is the main pathogen causing diarrhea in piglets (Sus scrofa domesticus), Huanglian-jie-du decoction (HLJDD) can be used to prevent and control E. coli diarrhea in livestock and poultry. However, whether it can act on the pathogenic bacteria and reduce their pathogenicity has rarely been reported.This study aimed to investigate the effect of HLJDD on the pathogenicity of E. coli in diarrheic piglets. A total of 32 anal swab samples were collected from piglets with diarrhea, and bacterial isolation, culture, biochemical and molecular biological identification, and virulence gene detection were performed. Potential pathogenic strains were selected for pathogenicity analysis in mice (Mus musculus), drug sensitivity tests, and antibiotic resistance gene detection. Based on this, HLJDD was used for in vitro intervention, with its MIC determined using a two-fold dilution method, and qPCR was employed to assess the effects of HLJDD on the virulence and antibiotic resistance genes of pathogenic strains. Finally, the impact of HLJDD intervention on mouse survival rates was explored through pathogenicity tests. The result showed that a total of 21 bacterial strains were isolated from the collected samples, and biochemical identification and 16S rRNA gene testing showed that all isolates were E. coli. Virulence gene detection indicated that only 3 isolates carried the high pathogenicity island (HPI) iron-repressible protein 2 (irp2). One strain carrying the irp2 gene was randomly selected for pathogenicity testing in mice, revealing that this strain was pathogenic with an LD50 of 4.4×106 CFU. Further testing showed that this pathogenic strain was resistant to 9 antibiotics, including penicillin, tetracycline, and erythromycin, and carried β-lactam and tetracycline resistance genes temoneira beta-lactamase (TEM) and tetracycline resistance protein A (tetA). The intervention results of HLJDD showed that its MIC against pathogenic bacteria was 250 mg/mL. Further qPCR results showed that HLJDD intervention could significantly inhibit the expression of irp2, TEM, and tetA of the pathogenic bacteria, and reduce the mortality rate of the pathogenic bacteria in mice, prolonging their survival time. This study provides a reference for the prevention and treatment of diarrhea in piglets and lays the foundation for the development of new antimicrobial drugs.
张丰裕, 蔡燕子, 史艳, 纪鹏, 华永丽, 袁子文, 魏彦明. 仔猪腹泻大肠杆菌的分离鉴定及黄连解毒汤对其致病性的影响[J]. 农业生物技术学报, 2025, 33(8): 1800-1811.
ZHANG Feng-Yu, CAI Yan-Zi, SHI Yan, JI Peng, HUA Yong-Li, YUAN Zi-Wen, WEI Yan-Ming. Isolation and Identification of Escherichia coli in Diarrheic Piglets (Sus scrofa domesticus) and the Effects of Huang-lian-jie-du Decoction on Its Pathogenicity. 农业生物技术学报, 2025, 33(8): 1800-1811.
[1] 成大荣, 孙怀昌, 徐建生, 等. 2006. 断奶仔猪源大肠杆菌LEE及HPI毒力岛的检测[J]. 微生物学报, 46(3): 368-372. (Cheng D R, Sun H C, Xu J S, et al.2006. Prevalence of LEE and HPI pathogenicity islands of Escherichia coli isolates from weaned piglets in China[J]. Acta Microbiologica Sinica, 46(3): 368-372.) [2] 董颖, 刘保光, 许二平. 2021. 黄连解毒汤抗炎作用与临床应用研究进展[J]. 中国实验方剂学杂志, 27(12): 245-250. (Dong Y, Liu B G, Xu E P.2021. Anti-inflammatory effect of Huanglian jiedutang and clinical application: A review[J]. Chinese Journal of Experimental Traditional Medical Formulae, 27(12): 245-250) [3] 高玲. 2023. 猪场的仔猪腹泻防治技术[J]. 中国动物保健, 25(05): 19-20. (Gao L.2023. Piglet diarrhea control technology in pig farms[J]. China Animal Health, 25(05): 19-20.) [4] 何金娇, 毛雪飞, 孙国鹏, 等. 2022. 猪源大肠杆菌的分离鉴定及耐药性分析[J]. 家畜生态学报, 43(09): 58-62. (He J J, Mao X F, Sun G P, et al.2022. Isolation, identification and drug resistance analysis of E.coli from pigs[J]. Journal of Domestic Animal Ecology, 43(09): 58-62.) [5] 黄美州, 刘永明, 王慧, 等. 2015. 甘肃地区新生仔猪腹泻细菌性病原的分离鉴定及其耐药性分析[J]. 西北农业学报, 24(12): 35-39. (Huang M Z, Liu Y M, Wang H, et al.2015. Isolation, identification and drug resistance analysis of bacterial etiology in new neonatal piglets diarrhea in Gansu area[J]. Acta Agriculturae Borealioccidentalis Sinica, 24(12): 35-39.) [6] 刘志伟, 王小清, 张清瑞, 等. 2021. 规模化猪场大肠杆菌病防控策略探析[J]. 猪业科学, 38(03): 38-40. (Liu Z W, Wang X Q, Zhang Q R, et al.2021. Exploration of prevention and control strategies of Escherichia coli disease in large-scale pig farms[J]. Swine Science, 38(03): 38-40.) [7] 罗昌俊. 2022. 猪大肠杆菌病的药物治疗及其抗菌素耐药性探讨[J]. 四川畜牧兽医, 49(09): 36-37. (Luo C J.2022. Drug treatment of Escherichia coli disease in swine and its antimicrobial resistance discussed[J]. Sichuan Animal & Veterinary Sciences, 49(09): 36-37.) [8] 吕颜枝, 李华坤, 康永刚, 等. 2021. 白头翁汤和黄连解毒汤对鸡大肠杆菌病的治疗作用[J]. 中兽医医药杂志, 40(01): 65-67. (Lv Y Z, Li H K, Kang Y G, et al.2021. Effect of Baitouweng decoction and Huanglian Jiedu decoction on chicken colibacillosis[J]. Journal of Traditional Chinese Veterinary Medicine, 40(01): 65-67.) [9] 门倩云, 张勇, 李宗帅, 等. 2020. 奶牛乳房炎大肠杆菌的分离鉴定及其主要毒力基因原核表达载体的构建[J]. 甘肃农业大学学报, 55(03): 1-8. (Men Q Y, Zhang Y, Li Z S, et al.2020. Isolation and identification of Escherichia coli from dairy cows with mastitis and construction of prokaryotic expression vector of its main virulence genes[J]. Journal of Gansu Agricultural University, 55(03): 1-8.) [10] 全国兽用抗菌药使用减量化行动方案(2021-2025年)[J]. 四川畜牧兽医, 48(11): 57-59. (Ministry of agriculture and rural affairs of the People's Republic of China. National vet erinary antimicrobial use reduction action plan(2021-2025)[J].Sichuan Animal &Veterinary Sciences, 48(11): 57-59. [11] 宋庆华. 2022. 烟台地区猪源大肠杆菌分离鉴定及耐药性分析[J]. 特种经济动植物, 25(09): 1-3. (Song Q H.2022. Isolation and identification of swine-derived Escherichia coli in Yantai and analysis of drug resistance[J]. Special Economic Animals and Plants, 25(09): 1-3.) [12] 佟盼盼, 黄顺敏, 王芋丹, 等. 2023. 新疆地区腹泻仔猪源大肠杆菌的分群、血清型鉴定及耐药性分析[J]. 畜牧兽医学报, 54(01): 414-420. (Tong P P, Huang S M, Wang Y D, et al.2023. Phylogenetic clustering, serotype and drug resistance analysis of Escherichia coli from diarrhea with piglets in Xinjiang[J]. Acta Veterinaria et Zootechnica Sinica, 54(01): 414-420.) [13] 王维, 李丹, 刘洋, 等. 2016. 黄连解毒汤的体外抑菌试验及拆方研究[J]. 动物医学进展, 37(11): 65-69. (Wang W, Li D, Liu Y, et al.2016. In vitro antibacterial experiment of Huanglian Jiedu decoction and decomposed recipes experiment[J]. Progress in Veterinary Medicine, 37(11): 65-69.) [14] 王艳萍, 郭时金, 徐倩倩, 等. 2018. 禽致病性大肠杆菌HPI毒力岛irp2蛋白表达、纯化及其间接ELISA方法建立[J]. 中国兽医学报, 38(02): 287-292. (Wang Y P, Guo S J, Xu Q Q, et al.2018. Establishment of indirect ELISA method and avian pathogenic E. coli HPI irp2 protein expression and purification[J]. Chinese Journal of Veterinary Science, 38(02): 287-292.) [15] 魏玉芬. 2014. 黄连解毒汤对CLP诱导的脓毒症大鼠的保护作用及其代谢组学研究[D]. 博士学位论文, 第二军医大学, 导师: 张卫东, pp. 52-64. (Wei Y F.2014. Protective effect of Huang-Lian-Jie-Du-Tang against CLP-induced polymicrobial sepsis and its influence on metabolome profile in rats[D]. Thesis for Ph.D., Second Military Medical University, Supervisor: Zhang W D, pp. 52-64.) [16] 吴杨, 闫艳新, 孙虎芝, 等. 2022. 仔猪细菌性腹泻病原菌的分离鉴定与耐药性分析[J]. 中国兽医杂志, 58(11): 95-99. (Wu Y, Yan Y X, Sun H Z, et al.2022. Isolation, identification and drug resistance analysis of pathogenic bacteria in bacterial diarrhea piglets[J]. Chinese Journal of Veterinary Medicine, 58(11): 95-99.) [17] 徐倩倩, 吕素芳, 李峰, 等. 2020. 38味中药对猪大肠杆菌的体外抑菌活性[J]. 中国兽医杂志, 56(05): 66-71. (Xu Q Q, Lu S F, Li F, et al.2020. In vitro bacteriostatic activity of 38 traditional chinese medicines against a swine E.coli[J]. Chinese Journal of Veterinary Medicine, 56(05): 66-71.) [18] 薛盼盼, 薛凤娟. 2020. 猪腹泻的病因分析与防治原则[J]. 畜牧业环境, (08): 67. (Xue P P, Xue F J. 2020. Analysis of the etiology of swine diarrhea and principles of prevention and control[J]. Animal Industry and Environment, (08): 67.) [19] 薛舒文. 2023. 广西某猪场猪大肠杆菌分离鉴定、耐药性分析及联合用药方案筛选[D]. 硕士学位论文, 华中农业大学, 导师: 谭诚, pp. 14-17. (Xue S W.2023. Isolation, identification, drug resistance analysis and selection of drug combination of swine Escherichia coli from a pig farm in Guangxi[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Tan C, pp. 14-17.) [20] 杨丽宏. 2020. 黄连解毒汤中13种活性成分的HPLC检测及其有效部位筛选与物质组方安全性评价[D]. 硕士学位论文, 甘肃农业大学, 导师: 魏彦明, pp. 1-5. (Yang L H.2020. Determination of 13 active compounds in Huang-lian-jie-dudecoction and screening of effective fraction by HPLC andsafety assessment of the active components combination[D]. Thesis for M.S., Gansu Agricultural University, Supervisor: Wei Y M, pp. 1-5.) [21] 元振杰, 王燕, 夏晨阳, 等. 2014. 致犊牛腹泻大肠杆菌对小鼠致病性及LEE、HPI毒力岛的检测[J]. 西南农业学报, 27(05): 2212-2215. (Yuan Z J, Wang Y, Xia C Y, et al.2014. Study on pathogenicity of Escherichia coli strains causing diarrhea calf and detection of LEE and HPI pathogenicity island[J]. Southwest China Journal of Agricultural Sciences, 27(05): 2212-2215.) [22] 袁子文. 2020. 黄连解毒汤治疗小鼠急性溃疡性结肠炎疗效的评价及其作用机制研究[D]. 甘肃农业大学, 博士学位论文, 甘肃农业大学, 导师: 魏彦明, pp. 25-26. (Yuan Z W.2020. Therapeutic effect and mechanism of Huang-lian-jie-du decoction on acute ulcerative colitis in mice[D]. Thesis for Ph.D., Gansu Agricultural University, Supervisor: Wei Y M, pp. 25-26.) [23] 张婷, 宋厚盼, 林也, 等. 2020.黄连解毒汤之“清热解毒”药效与作用机制研究进展[J]. 中华中医药学刊, 38(11): 135-139. (Zhang T, Song H P, Lin Y, et al.2020. Research advances on pharmacological effects and mechanisms underlying heat-clearing and detoxifying effect of Huanglian Jiedu decoction[J]. Chinese Archives of Traditional Chinese Medicine, 38(11): 135-139.) [24] 郑晓风, 张妍, 刘英玉, 等. 2020. 新疆部分地区牛羊源产志贺毒素大肠埃希菌菌株检测与分析[J]. 畜牧兽医学报, 51(10): 2518-2527. (Zheng X F, Zhang Y, Liu Y Y, et al.2020. Detection and analysis of shiga toxin-producing Escherichia coli isolates from cattle and sheep sources in some regions of Xinjiang, China[J]. Acta Veterinaria et Zootechnica Sinica, 51(10): 2518-2527.) [25] Cebula T A, Payne W L, Feng P.1995. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplexPCR[J]. Clinical Microbiology, 33(1): 248-250. [26] Koczura R, Krysiak N, Taraszewska A, et al.2015. Coliform bacteria isolated from recreational lakes carry class 1 and class 2 integrons and virulence-associated genes[J]. Journal of Applied Microbiology 119(2): 594-603. [27] Ojeniyi B, Ahrens P, Meyling A.2010. Detection of fimbrial and toxin genes in Escherichia coli andtheir prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerasechain reaction and phenotypic assays[J]. Zentralblatt Für Veterinärmedizin. Reihe B. Journal of Veterinary Medicine, 41(1-10): 49-59. [28] Perry R D, Pendrak M L, Schuetze P.1990. Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis[J]. Journal of Bacteriology, 172(10): 5929-5937. [29] Vidal R, Vidal M, Lagos R, et al.2004. Multiplex PCR for diagnosis of enteric infections associatedwith diarrheagenic Escherichia coli[J]. Journal of Clinical Microbiology,42(4): 1787-1789.
