摘要

       直接排放抗生素生产废水所污染的环境是抗生素抗性基因 (ARG) 的重要储存库，这些基因可能会转移给人类病原体。然而，我们对这种污染环境中 ARGs 的特性和多样性的了解仍然有限。我们应用功能宏基因组学来探索在废水排放点上游和排放点收集的两种克罗地亚抗生素生产废水和沉积物的抵抗力。对从阿奇霉素生产地点构建的宏基因组文库进行了大环内酯类抗生素耐药性筛选，而对来自兽用抗生素生产地点的文库进行了对磺胺类、四环素、甲氧苄啶和β-内酰胺类抗生素的筛选。对 8 个文库的功能分析总共确定了 82 个独特的、通常与临床相关的 ARG，这些 ARG 经常在集群中发现，并且两侧是可移动的遗传元件。从暴露于高水平大环内酯的基质中鉴定的大多数大环内酯抗性基因与编码核糖体保护蛋白、大环内酯磷酸转移酶和转运蛋白的已知基因相似。潜在的新型大环内酯抗性基因包括一个与来自梭菌的 23S rRNA 甲基转移酶最相似的基因，另一个来自上游未受污染的沉积物，与来自 Emergencia 的 GTPase HflX 相似。在源自暴露于较低水平兽用抗生素的沉积物的文库中，我们发现了 8 种潜在的新型 ARG，包括来自 A、B 和 D 类的二氢叶酸还原酶和 β-内酰胺酶。此外，我们在上游沉积物中检测到 7 种潜在的新型 ARG，包括胸苷酸合酶、二氢叶酸还原酶和 D 类β-内酰胺酶。综上所述，除了发现已知的基因类型外，我们还报告了在抗生素污染的工业废水和沉积物中发现了新颖多样的 ARG，为监测 ARG 从环境热点（如制药废水排放点）的扩散提供了定性基础。

       Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.

https://www.frontiersin.org/articles/10.3389/fmicb.2017.02675/full