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.

HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance

“…This duplication represents an example of how bacteria can employ common stress response factors as antibiotic resistance genes. In addition, our work is now strengthened by two recently published studies where functional metagenomic databases constructed from antibiotic-rich environments pointed to hflX from Simkania negevensis and Emergencia timonensis as a putative resistance gene (23, 33). Based on our phylogenetic analysis, hflX from Eubacterium spp., which is closely related to E. timonensis , belongs to the hflXr family.…”

“…However, a distinction should be made with hflXr , given that the tmRNA is a general stress factor, while hflXr duplication, specialization, and transcription regulation favor the idea that this gene is dedicated to response to antibiotics. In addition, functional metagenomic proves that hflX from other species that belong to the same subfamily as hflXr confer resistance to macrolides when transferred to other bacteria, which, to our knowledge, is not the case for tmRNA (23, 33). However, it has been shown that the sigma factor sigR induced by antibiotics in Streptomyces coelicolor regulates the transcription of various ribosome-associated genes, and notably tmRNA and HflX (36).…”

Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance

“…identify hflX homologs as putative antibiotic resistance genes belong to both the HflX-r (Emergencia spp) and HflX (Simkania spp.) clades (40,41). A possible explanation is that while all HflX proteins retain their ability to split 70S ribosomes into their subunits, their duplication allows each paralog to recognize ribosomes that are stalled under different physiological conditions.…”

Whole Genome Sequencing of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated From a Wastewater Treatment Plant in China

“…In recent years, the issue of pharmaceuticals in the environment has been gaining more attention worldwide and there has been growing interest in the WWTPs treating effluents from pharmaceutical industries with antimicrobial agents production. For example, several studies revealed the environmental impacts and the resistome of antimicrobial agents contaminated effluents from pharmaceutical industries in India and Croatia (Fick et al, 2009; Rutgersson et al, 2014; Bielen et al, 2017; Razavi et al, 2017; González-Plaza et al, 2018). A recent study in Nigeria has applied a culture-depend method to isolate ARB in wastewater from pharmaceutical facilities (Obayiuwana et al, 2018).…”