Identification of antibiotics triggering the dissemination of antibiotic resistance genes by SXT/R391 elements using a dedicated high-throughput whole-cell biosensor assay.

BACKGROUND: Mobile genetic elements (MGEs) are widely involved in the dissemination of antibiotic resistance genes and some of them, such as the integrative and conjugative element SXT, are even induced by specific antibiotics at sub-lethal concentrations. OBJECTIVES: This work explores collateral effects of a broad range of antibiotics on the mobility of the SXTMO10 element using a specifically designed high-throughput screening test. METHODS: Twenty-five promoters involved in the mobility of SXT and six artificial constitutive promoters were transcriptionally fused to luxCDABE bioluminescent genes and introduced into Escherichia coli strains with or without SXT to build whole-cell biosensors for a large-scale screening involving 48 antibiotics. A bioluminescent assay implementing a classical agar diffusion approach was coupled to an automated data processing pipeline developed to extract and analyse luminescence data from over 2000 antibiotic/biosensor combination profiles. RESULTS: In addition to quinolones previously reported as inducing the expression of SXT mobility genes, we found that specific antibiotics belonging to other classes, such as imipenem and azithromycin, also behave as inducers. The use of a control set of constitutive biosensors also revealed an unexpected intricate relationship between cell respiration and light production that allowed the identification of antibiotics interfering with the respiration process. CONCLUSIONS: The effect of antibiotics goes beyond the interaction with their primary cell targets and may lead to adverse effects such as triggering the dissemination of resistance by MGEs, sometimes in unpredictable ways. Identifying such MGE-triggering antibiotics is of prime importance for better controlling collateral effects during therapy.

EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach.

EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

Abundance and environmental host range of the SXT/R391 ICEs in aquatic environmental communities.

Mobile genetic elements (MGEs) such as plasmids or integrative conjugative elements (ICEs) are widely involved in the horizontal transfer of antibiotic resistant genes (ARGs), but their environmental host-range and reservoirs remain poorly known, as mainly assessed through the analysis of culturable and clinical bacterial isolates. In this study, we used a gradual approach for determining the environmental abundance and host-range of ICEs belonging to the SXT/R391 family, otherwise well known to bring ARGs in Vibrio spp. epidemic clones and other pathogens. First, by screening a set of aquatic bacteria libraries covering 1794 strains, we found that almost 1% of the isolates hosted an SXT/R391 element, all belonging to a narrow group of non-O1/non-O139 Vibrio cholerae. However, when SXT/R391 ICEs were then quantified in various aquatic communities, they appeared to be ubiquitous and relatively abundant, from 10-6 to 10-3 ICE copies per 16 S rDNA. Finally, the molecular exploration of the SXT/R391 host-range in two river ecosystems impacted by anthropogenic activities, using the single-cell genomic approach epicPCR, revealed several new SXT/R391 hosts mostly in the Proteobacteria phylum. Some, such as the pathogen Arcobacter cryaerophilus (Campylobacteraceae), have only been encountered in discharged treated wastewaters and downstream river waters, thus revealing a likely anthropogenic origin. Others, such as the non-pathogenic bacterium Neptunomonas acidivorans (Oceanospirillaceae), were solely identified in rivers waters upstream and downstream the treated wastewaters discharge points and may intrinsically belong to the SXT/R391 environmental reservoir. This work points out that not only the ICEs of the SXT/R391 family are more abundant in the environment than anticipated, but also that a variety of unsuspected hosts may well represent a missing link in the environmental dissemination of MGEs from and to bacteria of anthropogenic origin.