González-Plaza Juan J, Šimatović Ana, Milaković Milena, Bielen Ana, Wichmann Fabienne, Udiković-Kolić Nikolina
Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia.
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
Front Microbiol. 2018 Jan 17;8:2675. doi: 10.3389/fmicb.2017.02675. eCollection 2017.
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 and another, derived from upstream unpolluted sediment, to a GTPase HflX from . 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.
抗生素生产过程中直接排放废水所污染的环境是抗生素抗性基因（ARGs）的重要储存库，这些抗性基因有可能转移到人类病原体中。然而，我们对这类污染环境中ARGs的种类和多样性的了解仍然有限。我们应用功能宏基因组学来探究克罗地亚两个抗生素生产废水以及废水排放点上游和排放点处采集的沉积物中的抗性组。从阿奇霉素生产场地构建的宏基因组文库筛选对大环内酯类抗生素的抗性，而从生产兽用抗生素场地构建的文库筛选对磺胺类药物、四环素类、甲氧苄啶和β-内酰胺类的抗性。对八个文库的功能分析共鉴定出82个独特的、通常与临床相关的ARGs，这些基因经常成簇出现并侧翼伴有可移动遗传元件。从暴露于高水平大环内酯类物质的样本中鉴定出的大多数大环内酯抗性基因与已知的编码核糖体保护蛋白、大环内酯磷酸转移酶和转运蛋白的基因相似。潜在的新型大环内酯抗性基因包括一个与来自[具体来源]的23S rRNA甲基转移酶最相似的基因，以及另一个来自上游未污染沉积物、与来自[具体来源]的GTPase HflX相似的基因。在来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个潜在的新型ARGs，包括A、B和D类二氢叶酸还原酶和β-内酰胺酶。此外，我们在上游沉积物中检测到7个潜在的新型ARGs，包括胸苷酸合成酶、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型且多样的ARGs，为监测ARGs从环境热点（如制药废水排放点）的扩散提供了定性依据。
