Abstract
The biocide triclosan is in many consumer products and is a frequent contaminant of wastewater (WW) such that there is concern that triclosan promotes resistance to important antibiotics. This study identified functional mechanisms of triclosan resistance (TCSR) in WW metagenomes, and assessed the frequency of TCSR in WW-derived and clinical isolates of Escherichia coli and Enterococcus spp. Metagenomic DNA extracted from WW was used to profile the microbiome and construct large-insert cosmid libraries, which were screened for TCSR. Resistant cosmids were sequenced and the TCSR determinant identified by transposon mutagenesis. Wastewater Enterococcus spp. (N = 94) and E. coli (N = 99) and clinical Enterococcus spp. (N = 146) and vancomycin-resistant E. faecium (VRE; N = 149) were collected and tested for resistance to triclosan and a comprehensive drug panel. Functional metagenomic screening revealed diverse FabV homologs as major WW TCSR determinants. Resistant clones harboured sequences likely originating from Aeromonas spp., a common WW microbe. The triclosan MIC90s for E. coli, E. faecalis, and E. faecium isolates were 0.125, 32, and 32 mg/L, respectively. For E. coli, there was no correlation between the triclosan MIC and any drug tested. Negative correlations were detected between the triclosan MIC and levofloxacin resistance for E. faecalis, and between triclosan and vancomycin, teicoplanin, and ampicillin resistance for E. faecium. Thus, FabV homologs were the major contributor to the WW triclosan resistome and high-level TCSR was not observed in WW or clinical isolates. Elevated triclosan MICs were not positively correlated with antimicrobial resistance to any drug tested.
Highlights
Triclosan is a synthetic chlorinated bisphenol antimicrobial drug, commonly referred to as a biocide, and is effective against a wide variety of microorganisms, including bacteria, fungi, and apicomplexan parasites [1, 2]
The two genomic control libraries constructed with DNA from E. coli and E. faecalis were comprised of 22.5K and 20K clones respectively, and with genome sizes of ~5.2 and ~3.0 Mb, it was estimated that 596 and 343 clones provided 99.9% genomic coverage [34]
A total of 48 triclosan resistance (TCSR) colonies were subcultured for isolation and extraction of cosmid DNA, which was digested with EcoRI to profile and eliminate identical clones
Summary
Triclosan is a synthetic chlorinated bisphenol antimicrobial drug, commonly referred to as a biocide, and is effective against a wide variety of microorganisms, including bacteria, fungi, and apicomplexan parasites [1, 2]. The widespread inclusion of triclosan in consumer products is controversial [2], as it is structurally similar to T3 and T4 thyroid hormones [6, 7] and has the potential to promote antimicrobial resistance (AMR) [5, 8]. There are four well-known ENR isozymes: FabI, FabL, FabV, and FabK, with FabI, FabL, and FabV belonging to the short-chain dehydrogenase/reductase superfamily [10]. Most bacteria encode fabI, such that triclosan resistance (TCSR) is conferred by mutations or overexpression of FabI, or via efflux pumps [10]. The other isozymes exhibit varied sensitivity to triclosan, with FabV conferring almost complete insensitivity. Screening of metagenomic libraries has identified novel TCSR genes such as candidate FabG-like and YX7K-type ENRs, AcrAB efflux pump homologs, and other hypothetical and unknown determinants [11]
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