农产品越来越被认为是人类病原体和可转移抗生素抗性基因的储库。本研究旨在探索表征与产品相关的细菌可转移抗性组的方法。混合沙拉,芝麻菜,通过培养和基于DNA的方法分析了从德国超市购买的香菜。在非选择性富集步骤之前和之后,分离四环素(TET)抗性大肠杆菌,并通过外源质粒分离捕获赋予TET抗性的质粒。抗TET大肠杆菌分离株,移情者,和总群落DNA(TC-DNA)从叶片分离或富集后的微生物部分进行了分析的抗性基因的存在,1类积分子,和各种质粒通过实时PCR和PCR-Southern印迹杂交。开发了用于IncI和IncF质粒的实时PCR引物。从芝麻菜和香菜中分离出的抗TET大肠杆菌携带IncF,IncI1,IncN,IncHI1,IncU,和IncX1质粒。来自香菜的三个分离株对IncN质粒和混合沙拉和香菜的blaCTX-M-1呈阳性,IncF,外源捕获IncI1和IncP-1β质粒。重要的是,而直接检测TC-DNA中的IncI和IncF质粒失败,这些质粒在从富集培养物中提取的DNA中变得可检测到。这证实了不依赖于培养的基于DNA的方法并不总是足够灵敏以检测稀有微生物组中的可转移抗性组。总之,这项研究表明,在与生食相关的细菌中检测到令人印象深刻的可自我传播的多重抗性质粒多样性,外源捕获大肠杆菌表明它们也可以转移到肠道细菌中。重要农产品是最受欢迎的食品之一。不幸的是,绿叶蔬菜可以是可转移的抗生素抗性基因的储库。我们发现IncF和IncI质粒是来自农产品的大肠杆菌分离物中最普遍的质粒类型。这项研究强调了与生产相关的罕见微生物组作为抗生素抗性基因来源的重要性,这些基因可能会逃避与培养无关的检测。但可能会转移到人类病原体或共生体。
Produce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistant Escherichia coli were isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistant E. coli isolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for
IncI and IncF plasmids. TET-resistant E. coli isolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids and blaCTX-M-1 From mixed salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured exogenously. Importantly, whereas direct detection of
IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing into E. coli suggests that they could transfer to gut bacteria as well.IMPORTANCE Produce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and
IncI plasmids were the most prevalent plasmid types in E. coli isolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.