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Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

ABSTRACT: 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.

SUBMITTER: Gonzalez-Plaza JJ

PROVIDER: S-EPMC5776109 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

González-Plaza Juan J JJ Šimatović Ana A Milaković Milena M Bielen Ana A Wichmann Fabienne F Udiković-Kolić Nikolina N

Frontiers in microbiology 20180117

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 si ...[more]

PMID: 29387045

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Project description:BackgroundAntibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate.ResultsWe evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and β-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1 × e-5 and query coverage of ≥ 60% were employed in the Resfams database, the novel β-lactamases blaHRV-1 and blaHRVM-1 were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 β-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring blaHRV-1 and blaHRVM-1 and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended-spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages.ConclusionOur results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Video abstract.

Project description:The sewer environment is a potential hotspot for the proliferation of antibiotic resistance genes (ARGs) and other hazardous microbial agents. Understanding the potential for ARG proliferation and retardation and/or accumulation in sewer sediments is of interest for protecting the health of sewage workers and the broader community in the event of sewer overflows as well as for interpreting sewage epidemiology data. To better understand this understudied environment for antibiotic resistance, a field survey was conducted to identify the factors that may control ARGs in sewer sediments and sewage. qPCR was performed for select ARGs and amplicon sequencing was performed for paired samples from combined and separate sanitary sewer systems. Metagenomic sequencing was performed on combined sewer sediments. The relative abundances of sul1, tet(O), tet(W), ermF, and vanA were higher in wastewater compared to sewer sediments, while NDM-1 was greater in sewer sediment and ermF was similar between the two matrices. NDM-1 was observed in sewer sediment but rarely above detection in wastewater in this study. This may indicate that larger/more frequent wastewater samples are needed for detection and/or that retardation and/or accumulation in sewage sediment may need to be considered when interpreting wastewater-based epidemiology data for ARGs. Random forest analyses indicated that season and conductivity were important variables and to a lesser extent so were pH, TSS, heavy metals, and sewer type for explaining the variance of the ARGs. These variables explained the 19-61% of the variance of sul1, tet(O), tet(G), and tet(W) quantified in wastewater. These variables performed less well for explaining the variance in sewer sediments (0.2-24%). Sewer sediment and wastewater had distinct microbial community structures and biomarkers for each are described. Metagenomics indicated that a high diversity of ARGs, including several of medical importance, were observed in the combined sewer sediment. This work provides insight into the complex sewer microbiome and the potential hazard posed by different sewer matrices.

Project description:Microbes belonging to Candidate Phyla Radiation (CPR) have joined the tree of life as a new branch, thanks to the intensive application of metagenomics and sequencing technologies. CPR have been eventually identified by 16S rRNA analysis, and they represent more than 26% of microbial diversity. Despite their ultrasmall size, reduced genome, and metabolic pathways which mainly depend on exosymbiotic or exoparasitic relationships with the bacterial host, CPR microbes were found to be abundant in almost all environments. They can be considered survivors in highly competitive circumstances within microbial communities. However, their defense mechanisms and phenotypic characteristic remain poorly explored. Here, we conducted a thorough in silico analysis on 4,062 CPR genomes to search for antibiotic resistance (AR)-like enzymes using BLASTp and functional domain predictions against an exhaustive consensus AR database and conserved domain database (CDD), respectively. Our findings showed that a rich reservoir of divergent AR-like genes (n = 30,545 hits, mean = 7.5 hits/genome [0 to 41]) were distributed across the 13 CPR superphyla. These AR-like genes encode 89 different enzymes that are associated with 14 different chemical classes of antimicrobials. Most hits found (93.6%) were linked to glycopeptide, beta-lactam, macrolide-lincosamide-streptogramin (MLS), tetracycline, and aminoglycoside resistance. Moreover, two AR profiles were discerned for the Microgenomates group and "Candidatus Parcubacteria," which were distinct between them and differed from all other CPR superphyla. CPR cells seem to be active players during microbial competitive interactions; they are well equipped for microbial combat in different habitats, which ensures their natural survival and continued existence. IMPORTANCE To our knowledge, this study is one of the few studies that characterize the defense systems in the CPR group and describes the first repertoire of antibiotic resistance (AR) genes. The use of a BLAST approach with lenient criteria followed by a careful examination of the functional domains has yielded a variety of enzymes that mainly give three different mechanisms of action of resistance. Our genome analysis showed the existence of a rich reservoir of CPR resistome, which is associated with different antibiotic families. Moreover, this analysis revealed the hidden face of the reduced-genome CPR, particularly their weaponry with AR genes. These data suggest that CPR are competitive players in the microbial war, and they can be distinguished by specific AR profiles.

Dataset Information

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

Publications

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

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