摘要

直接排放抗生素生产废水污染的环境是抗生素抗性基因（ARGs）的重要储库，可能转移到人类病原体。然而，我们对这些污染环境中ARG的身份和多样性的了解仍然有限。我们应用功能性宏基因组学研究了两种克罗地亚抗生素生产废水和废水排放点上游和下游收集的沉积物的抗生素组合。筛选了由阿奇霉素生产位点构建的宏基因组文库对大环内酯抗生素的抗性，而筛选来自产生兽用抗生素的位点的文库以筛选对磺酰胺，四环素，甲氧苄啶和β-内酰胺的抗性。 8个文库的功能分析共鉴定出82个独特的，通常临床相关的ARG，这些ARG经常在簇中发现并且侧面是移动遗传元件。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类抗性基因包括一个与来自Clostridium的23S rRNA甲基转移酶和另一个来自上游未污染沉积物的23S rRNA甲基转移酶最相似的来自Emergencia的GTPase HflX。在来自暴露于较低水平兽用抗生素沉积物的文库中，我们发现了8种潜在的新型ARGs，包括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。总之，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新型和多样化的ARGs，为监测药物流出物排放场等环境热点的ARGs扩散提供了定性依据。

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