探討長期暴露殺真菌劑對食品中常見病原菌仙人掌桿菌之生物膜形成、抗生素抗藥性與致病力之影響及其調控機制

Abstract

食源性病原菌所導致的食源性疾病為重大的食品安全與公共衛生議題，其中 仙人掌桿菌 (Bacillus cereus) 為全球常見的食源性病原菌，容易污染乳製品、肉類、蔬菜、穀物及澱粉類食品；而近年來隨全球人口迅速增長，為穩定糧食供應與維持食品品質，農藥使用日益頻繁，其中殺真菌劑為常用的農藥類別之一，而這些農業化學品大部分進入土壤、水及食品中，增加食源性病原菌與殺真菌劑在環境與食品中共存的機會。研究指出環境壓力如外來化學物質的暴露可促使細菌產生適應性與抗藥性，然而，殺真菌劑與病原菌間之交互作用仍鮮少受到重視。因此，本研究針對市面上常用之8種殺真菌劑 (Chlorothalonil、Propineb、Tebuconazole、Azoxystrobin、Propiconazole、Mancozeb、Carbendazim及 Triadimefon)，探討其對B. cereus生長之影響，以及長期暴露對生物膜形成、抗生素抗藥性之潛在影響，並使用秀麗隱桿線蟲 (Caenorhabditis elegans) 評估其感染致病力之變化。研究結果顯示Chlorothalonil、Propineb及Tebuconazole可顯著抑制病原菌B. cereus之生長，且30天長期重複暴露Chlorothalonil 8 µM、Propineb 175 µM及Tebuconazole 500 µM可提升其對殺真菌劑之適應力，並顯著提升其生物膜形成能力、對Gentamycin與Tetracycline之抗藥性，以及對C. elegans之感染致死率。進一步探討殺真菌劑 (Tebuconazole 500 µM) 所誘導的生物膜形成與抗生素抗藥性改變之相關機制，由qRT-PCR結果顯示長期暴露Tebuconazole會影響生物膜形成相關基因purC、purL、calY、抗生素抗藥性相關之多重藥物外排幫浦蛋白編碼基因smr及mate，與毒力相關基因nheA、nheB、nheC、hblA、hblC、hblD之表達，進而提升B. cereus生物膜形成、抗生素抗藥性與致病力。 總結上述，本研究指出長期暴露殺真菌劑會提升病原菌生物膜形成能力、抗生素抗藥性與感染致病力，並揭示其相關機制，突顯殺真菌劑在使用與管理上控管的重要性，同時，本研究也提供相關學理資訊，可供後續殺真菌劑與病原菌間交互作用相關研究之參考。

Foodborne illnesses caused by foodborne pathogens are a major global concern in food safety and public health. Among them, Bacillus cereus is one of the most common foodborne pathogens worldwide and is prone to contaminating dairy products, meats, vegetables, grains, and starchy foods. In recent years, in response to growing global demands for stable food supply and maintain product quality, the use of pesticides has increased significantly. Fungicides, in particular, are among the most commonly used pesticide categories. As a large proportion of these agrochemicals eventually enter the soil, water, and food, the potential for fungicides and foodborne pathogens to co-exist in environmental and food systems increases. Previous studies have shown that environmental stressors, such as exposure to xenobiotic compounds, can promote bacterial adaptation and antibiotic resistance. However, the interactions between fungicides and bacterial pathogens remain largely underexplored. Therefore, this study investigated the effects of eight commonly used fungicides (Chlorothalonil, Propineb, Tebuconazole, Azoxystrobin, Propiconazole, Mancozeb, Carbendazim, and Triadimefon) on the growth of B. cereus, as well as the potential impacts of long-term exposure on biofilm formation, antibiotic resistance, and pathogenicity using the nematode Caenorhabditis elegans as a model host. Results indicated that Chlorothalonil, Propineb, and Tebuconazole significantly inhibited the growth of B. cereus. Moreover, 30-day repeated long-term exposure to Chlorothalonil 8 µM, Propineb 175 µM, and Tebuconazole 500 µM promoted bacterial adaptation to these fungicides and significantly increased biofilm formation, resistance to Gentamycin and Tetracycline, and infection-induced mortality in C. elegans. To further explore the mechanisms underlying the fungicide Tebuconazole-induced enhancement of biofilm formation and antibiotic resistance, qRT-PCR analysis revealed that long-term exposure to Tebuconazole affected the expression of biofilm-related genes (purC, purl, and calY), the antibiotic resistance-related multidrug efflux pump genes (smr and mate), and virulence-associated genes (nheA, nheB, nheC, hblA, hblC, and hblD), thereby promoting increased biofilm formation, antibiotic resistance, and pathogenicity in B. cereus. In conclusion, this study demonstrates that long-term exposure to fungicides can enhance the biofilm-forming ability, antibiotic resistance, and pathogenicity of foodborne pathogens, and elucidates the underlying mechanisms. These findings highlight the importance of careful use and management of fungicides. Moreover, this study provides fundamental insights that may serve as a reference for future investigations into the fungicide–pathogen interactions.