T-2毒素压力下大肠杆菌多重耐药和泛耐药模型的建立及其诱导耐药机制的研究

Both antibiotic resistance of bacteria and the ubiquitous presence of mycotoxin contamination in animal husbandry become issues of global concern, posing great threat to healthy breeding of livestock and food safety. The fundamental solution of the difficult issue of antibiotic resistance should be emphasis on seeking the external factors inducing antibiotic resistance in food animal production. In our previous studies, the antibiotic-sensitive Escherichia coli (E. coli) following the incubation with low concentration of T-2 toxin for 6 days, showed resistance to antibiotics including colistin, tigecycline and carbapenems and exhibited up to 512-fold increases in the MIC (minimal inhibitory concentration) of tested antibiotics, indicating T-2 toxin could induce PDR (pan-drug resistance) in E. coli. Currently, data regarding the relationship between mycotoxin and antibiotic resistance are not available, so mycotoxin as a novel factor inducing antibiotic resistance deserves our attention and exploration. In this project, we are going to establish the model of MDR (multi-drug resistance) and PDR in E. coli and study the resistance mechanism under T-2 toxin pressure. To establish the model, method to induce resistance in E. coli by T-2 toxin in vitro will be determined, and then resistance profile among the antibiotic-resistant E. coli following T-2 toxin treatment will be analyzed. To study the resistance mechanism under T-2 toxin pressure, the function and regulation of the critical resistance determinants which involved in MDR ad PDR of E. coli will be illuminated. The findings of the project would provide brand new insights into prevention and control of antibiotic resistance and benefit the healthy development of animal husbandry as the new fundamental theory.

畜牧生产中细菌的耐药性和霉菌毒素的广泛污染，时刻威胁着畜禽健康养殖和食品安全，均成为全球关注的问题。要从根本上解决细菌的耐药性难题，应重点寻找生产中潜在的可诱导细菌耐药的外因。我们前期研究表明，在低浓度的T-2毒素下，大肠杆菌敏感株在培养的第6天以后，对黏菌素、替加环素、碳青霉烯类等抗生素表现耐药，最小抑菌浓度上升高达512倍，提示T-2毒素可诱导大肠杆菌泛耐药的形成。国内外关于霉菌毒素与细菌耐药性的相关性研究尚属空白，霉菌毒素诱导细菌的耐药亟待研究。本项目以霉菌毒素中的T-2毒素模拟外界胁迫压力，旨在确定毒素处理下大肠杆菌体外诱导的方式，建立稳定的耐药模型，分析毒素压力下菌株的耐药特征，找到毒素诱导菌株多重耐药性和泛耐药性的关键耐药因子，阐明该因子在诱导耐药过程中的具体功能，最终厘清T-2毒素诱导大肠杆菌耐药的机制及其特异性，为细菌耐药性防控提供新视野，为畜牧业健康发展提供新的理论依据。

近年来T-2毒素污染谷物状况严峻，针对该现象本研究将大肠杆菌用不同浓度T-2毒素连续诱导处理，通过药敏实验确定耐药表型出现的诱导次数，并研究其耐药机制。药敏试验结果显示，标准菌株ATCC 25922经10-5 ng/mL T-2毒素诱导后第7次出现泛耐药的表型，工程菌株C600经10 ng/mL T-2毒素诱导后第6次出现泛耐药的表型。对耐药菌株进行生物被膜观察，发现相比于敏感菌株，耐药株生物被膜增强,，且通过转录组学发现色氨酸降解酶显著下调，进一步实验验证发现色氨酸降解酶下调后可导致生物被膜形成增强。由此表明生物被膜形成是导致T-2毒素诱导下细菌出现泛耐药表型的主要原因之一。