High concentrations of antibiotics which are above the minimum inhibitory concentrations could cause death of many susceptible environmental microorganisms and enrich those genetically adapted, while sub-inhibitory concentrations exert a selective pressure, which act as a moving force in horizontal dissemination of ARGs ( Baker-Austin et al., 2006 Tacão et al., 2014 Di Cesare et al., 2016 Navon-Venezia et al., 2017). Understanding these reservoirs and behaviors of ARGs is crucial to control the emergence of resistant pathogens at a global scale.ĭirect discharge of pharmaceutical effluents in receiving water bodies has been recognized as an important source of pollution, as they may contain high concentrations of antibiotics, ARB, heavy metals, and other hazardous materials ( Babić et al., 2007 Larsson et al., 2007 Li et al., 2009 Sim et al., 2011 Larsson, 2014 Bielen et al., 2017). The selection pressure imposed by antibiotics and other selective agents has promoted the propagation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) (collectively known as the resistome) in the environment, creating vast reservoirs of ARGs with potential to be transferred to pathogens ( Bengtsson-Palme et al., 2014 Cabello et al., 2016 Tao et al., 2016 Peng et al., 2017 Su et al., 2017). However, there is a growing awareness that anthropogenic inputs of antibiotics into the environment through effluents, use of manure and biosolids in agriculture, and aquaculture contribute to this problem. This phenomenon is strongly associated with hospitals and other clinical environment ( Brown et al., 2006 Rodriguez-Mozaz et al., 2015), because the extensive use of antibiotics in clinical settings is the driving force for increasing antibiotic resistance. 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.Īntibiotic resistance is one of the most serious global public health threats of the twenty-first century ( Carlet et al., 2012 ECDC, 2016 O'neill, 2016). ![]() In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. 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. 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. 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. 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. 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. 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. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. 4Independent Researcher, Lausanne, SwitzerlandĮnvironments 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.3Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia.2Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia. ![]() ![]()
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