石榴皮水提物对猪源多重耐药大肠杆菌的抑菌作用研究

doi:10.16431/j.cnki.1671-7236.2023.01.033

摘要/Abstract

摘要： 【目的】筛选腹泻仔猪中携毒力基因且多重耐药的大肠杆菌菌株，评估中药水提物对该菌株的抑菌活性，并探索中药抑菌机制。【方法】通过PCR方法与Kirby-Bauer （K-B）药敏纸片法评估临床大肠杆菌菌株的肠毒素基因携带情况和耐药性；通过微量肉汤稀释法评估中药水提物对多重耐药大肠杆菌菌株的抑菌活性，确定最小抑菌浓度（minimal inhibitory concentration，MIC）和最小杀菌浓度（minimal bactericidal concentration，MBC）；通过电导率仪以及相关试剂盒检测评估石榴皮水提物对多重耐药菌株作用不同时间后菌液电导率、碱性磷酸酶（alkaline phosphatase，AKP）含量及菌体内ATP含量的变化；通过SDS-PAGE和蛋白含量检测评估菌体内可溶性蛋白的含量变化；通过扫描电镜观察大肠杆菌形态变化。【结果】PCR检测14株临床大肠杆菌中肠毒素基因携带率达到78.57%，抗生素药敏试验结果显示，所有菌株至少对2种抗生素耐药，均属于多重耐药菌株。中药药敏试验结果显示，黄连、黄芩、石榴皮水提物对多重耐药菌株抑菌活性较好，其中石榴皮水提物的MIC和MBC分别为31.25和62.5 mg/mL。石榴皮水提物在1/2MIC和MIC浓度下能显著抑制质控菌株和多重耐药菌株的生长繁殖。在石榴皮水提物作用大肠杆菌3 h内，菌液电导率相比于对照组极显著上升（P<0.01）。AKP与ATP检测结果显示，与对照组相比，石榴皮水提物在作用大肠杆菌6 h内，菌液中AKP含量极显著增加（P<0.01），菌体内ATP含量极显著降低（P<0.01）。SDS-PAGE和蛋白含量检测结果显示，随着石榴皮水提物刺激时间增加，菌体内可溶性蛋白含量减少。扫描电镜结果显示，石榴皮刺激后菌体形态发生碎裂和溶解。【结论】本研究结果表明，石榴皮水提物能显著抑制多重耐药且携带肠毒素基因的大肠杆菌生长，为临床治疗大肠杆菌性仔猪腹泻提供理论参考和药物开发依据。

关键词: 耐药性; 大肠杆菌; 石榴皮水提物; 抑菌机制

Abstract: 【Objective】 The aim of this study was to obtain Escherichia coli (E.coli)strains carrying virulence genes and multi-drug resistance from diarrheic piglets, and evaluate the antibacterial activity of aqueous extract of Chinese herbal medicine against multi-drug resistant E.coli, and explore the antimicrobial mechanism.【Method】 The enterotoxin genes and drug resistance of clinical E.coli isolates were measured with PCR and Kirby-Bauer (K-B) disk diffusion method, respectively.Antibacterial activity of Chinese herbal medicine against multi-drug resistant E.coli was evaluated through broth microdilution method, and then minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of aqueous extract of Chinese herbal medicine were obtained.After aqueous extract of Granati pericarpium exposure for different time, the conductivity meter and related detection kits were used to evaluated the changes of conductivity and alkaline phosphatase (AKP) content in bacterium solution, and intrabacterial ATP content.Intrabacterial soluble proteins changes were examined by SDS-PAGE and protein content detection, and then morphological changes of E.coli were visualized using scanning electron microscopy.【Result】 The prevalence of enterotoxin genes reaches 78.57% in 14 isolated clinical E.coli strains in PCR detection.Antibiotic-susceptibility testing results showed that all strains were resistant to at least two antibiotics, and all belonged to multi-drug resistant strains.The results of Chinese herbal medicine susceptibility testing showed the better antibacterial activity of aqueous extracts of Coptidis rhizoma, Scutellariae radix and Granati pericarpium against multi-drug resistant strains, among which the MIC and MBC values of Granati pericarpium were 31.25 and 62.5 mg/mL, respectively.Aqueous extract of Granati pericarpium could inhibit growth and reproduction of multi-drug resistant and quality control strains at concentrations of 1/2MIC or MIC.Within 3 h post-stimulation of aqueous extract of Granati pericarpium, conductivity of bacterial solution was extremely significantly increased compared with control group (P<0.01).During 6 h treatment of aqueous extract of Granati pericarpium, the results of AKP and ATP detections showed that AKP content in bacterial solution in treated group was extremely significant higher than control group (P<0.01), and intrabacterial ATP content was extremely significantly lower than control group (P<0.01).The results of SDS-PAGE and protein content detection showed that intrabacterial soluble proteins were decreased with the increase of action time.The results of scanning electron microscopy showed fragmentation and lysis in aqueous extract of Granati pericarpium-treated E.coli.【Conclusion】 The present study demonstrated that aqueous extract of Granati pericarpium could significantly inhibit growth of multi-drug resistant E.coli with enterotoxin genes, which provided theoretical reference and drug development basis for clinical treatment of piglet diarrhea infected by multi-drug resistant E.coli.

Key words: drug resistance; Escherichia coli; aqueous extract of Granati pericarpium; antimicrobial mechanism

中图分类号:

S853.74

刘琳, 吴保庆, 郭晓银, 于文会, 田浪. 石榴皮水提物对猪源多重耐药大肠杆菌的抑菌作用研究[J]. 中国畜牧兽医, 2023, 50(1): 328-340.

LIU Lin, WU Baoqing, GUO Xiaoyin, YU Wenhui, TIAN Lang. Study on Bacteriostatic Action of Aqueous Extract of Granati pericarpium Against Multi-drug Resistant Escherichia coli of Swine[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 328-340.

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