山东地区肉鸡源产气荚膜梭菌的调查及植物精油对其抑制效果的研究

doi:10.16431/j.cnki.1671-7236.2025.12.046

摘要/Abstract

摘要： 【目的】调查山东地区产气荚膜梭菌(Clostridium perfringens)的流行、耐药、致病性等情况，并探究牛至油、肉桂醛、桉叶油、月桂酸甘油酯、大蒜油5种植物精油对该菌的抑制作用。【方法】从山东地区不同商品肉鸡规模化养殖场采集100份肉鸡肠道样品进行细菌分离，利用生化试验、16S rRNA测序和PCR扩增毒素基因对分离菌株进行鉴定，并对鉴定为产气荚膜梭菌的菌株进行药物敏感性检测和致病试验；利用微量肉汤稀释法检测5种植物精油对产气荚膜梭菌的最小抑菌浓度(MIC)，选用抑菌效果较好的植物精油，利用棋盘法进行两两复配，检测复配后植物精油对产气荚膜梭菌的分级抑菌浓度(FIC)指数。【结果】分离菌在TSC平板上呈现中间黑色、周围有白色浑浊圈的圆形菌落，革兰染色镜检呈革兰阳性，100份样品中共分离获得32株分离菌。生化鉴定结果显示，分离菌对葡糖糖、乳糖、麦芽糖等呈阳性，对甘露醇、吲哚呈阴性。16S rRNA基因PCR扩增、测序比对结果显示，分离菌株与产气荚膜梭菌相似性＞99%。毒素基因多重PCR鉴定结果显示，仅扩增出202 bp的目的条带，鉴定分离菌株均为A型产气荚膜梭菌，分离率为32%。药敏试验结果显示，分离菌株对红霉素、诺氟沙星、环丙沙星耐药率较高。致病性试验显示，5株不同地区来源的分离菌均能引起肉鸡严重坏死性肠炎。体外抑菌结果显示，大蒜油、肉桂醛、月桂酸甘油酯对产气荚膜梭菌的MIC分别为250、125和125 μg/mL，复配组合大蒜油＋肉桂醛、大蒜油＋月桂酸甘油酯、肉桂醛＋月桂酸甘油酯的FIC分别为1、2和2.5。【结论】山东地区肉鸡源产气荚膜梭菌分离率较高且A型菌株占绝对优势，分离菌株耐药性和致病性较强。大蒜油、肉桂醛、月桂酸甘油酯对产气荚膜梭菌的抑菌效果较好，且大蒜油与肉桂醛联合表现相加作用。

关键词: 产气荚膜梭菌; 耐药性; 致病性; 植物精油; 抑菌效果

Abstract: 【Objective】 This experiment was conducted to investigate the prevalence,drug resistance and pathogenicity of Clostridium perfringens in Shandong region,and explore the inhibitory effects of five plant essential oils (oregano oil,cinnamaldehyde,eucalyptus oil,glyceryl laurate,and garlic oil) against this bacterium. 【Method】 One hundred intestinal samples of broilers were collected from different large-scale commercial broiler farms in Shandong region for bacterial isolation.The isolats were identified by biochemical tests,16S rRNA sequencing and PCR amplification of toxin genes.The drug sensitivity detection and pathogenicity tests were conducted on the isolates identified as Clostridium perfringens.The minimum inhibitory concentrations (MIC) of five essential oils against Clostridium perfringens were determined by broth microdilution method.Essential oils with superior antibacterial activity were selected and combined in pairwise formulations using the checkerboard assay,after which the fractional inhibitory concentration (FIC) index of the combined essential oils against Clostridium perfringens was evaluated. 【Result】 The isolated bacteria presented as circular colonies with black center and white turbidity ring around the TSC plate.Gram-staining microscopic examination showed Gram-positive results.A total of 32 isolated bacteria were isolated from 100 samples.Biochemical identification results showed that the isolates were positive for glucose,lactose,maltose,etc.,but negative for mannitol and indole.The results of 16S rRNA gene PCR amplification and sequencing comparison showed that the similarity of the isolates with Clostridium perfringens was greater than 99%.The results of multiplex PCR identification of the toxin genes showed that only a 202 bp target band was amplified.All the isolated strains identified were Clostridium perfringens type A,with an isolation rate of 32%.Antimicrobial susceptibility testing demonstrated high resistance rates to erythromycin,norfloxacin,and ciprofloxacin.The pathogenicity test showed all five isolates from different regions could cause severe necrotizing enteritis in broilers.In vitro antibacterial assays showed that the MIC of garlic oil,cinnamaldehyde and glyceryl laurate against Clostridium perfringens were 250,125 and 125 μg/mL,respectively.The FIC of the compound combinations of garlic oil＋cinnamaldehyde,garlic oil＋glyceryl laurate,and cinnamaldehyde＋glyceryl laurate were 1,2 and 2.5, respectively. 【Conclusion】 The isolation rate of Clostridium perfringens from broilers in Shandong region was relatively high,and type A strains had an absolute advantage.The isolates had strong drug resistance and pathogenicity. Garlic oil,cinnamaldehyde,and glycerol monolaurate demonstrated superior antibacterial effect against Clostridium perfringens.In addition,the combination of garlic oil and cinnamaldehyde showed an additive effect.

Key words: Clostridium perfringens; drug resistance; pathogenicity; plant essential oils; antibacterial effect

中图分类号:

S831.7

孔垂民, 李健, 黄金, 张燊, 王金君. 山东地区肉鸡源产气荚膜梭菌的调查及植物精油对其抑制效果的研究[J]. 中国畜牧兽医, 2025, 52(12): 6042-6051.

KONG Chuimin, LI Jian, HUANG Jin, ZHANG Shen, WANG Jinjun. Investigation and Inhibitory Effects of Plant Essential Oils on Clostridium perfringens in Broilers from Shandong Province[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 6042-6051.

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