Rôle des éléments intégratifs conjugatifs de la famille d’ICE SXT/R391 dans la dissémination des gènes d’antibiorésistance en milieu aquatique

Antibiotic resistance is a major global health issue. In 2016, a report on antibiotic resistance estimated that at least 700,000 deaths per year worldwide, were linked to antibiotic resistance. Projections indicate that by 2050, the number of deaths could reach 10 million per year in the absence of new measures to control the emergence and the spread of antibiotic resistances (O’Neill, 2016). While antibiotic therapy is an important part of the antibiotic resistance selection problem, it is now believed that the spread of resistant bacteria, or their resistance genes in or between human, animal, and environmental spheres, might be the key.There are several families of mobile genetic elements at the origin of the dissemination of antibiotic resistance genes between bacteria, but relatively few studies on their relative abundances, their respective host range, or their dissemination in the environment. In this study, we determined the bacterial host range of the integrative conjugative elements (or ICEs) from the SXT/R391 family by qPCR from environmental strain collections, then from two aquatic environments using an epicPCR approach. The latter allows linking a fragment of the 16S rRNA gene to a fragment of interest, here a specific fragment of the traB gene from SXT/R391 elements. We then identified new environmental hosts, so far never described in the literature (ie Tolumonas auensis,), as a possible vector of SXT/R391 ICEs and their genes. Therefore, our data demonstrated that, contrary to what we find in the literature, the dissemination of SXT/R391 ICEs is not only limited to Vibrio species like Vibrio cholerae.We also identified molecules activating the expression of the ICE SXTMO10 transfer genes in E. coli MG1656, evaluated the real effect in vitro of some of those molecules (like ciprofloxacin) on the transfer frequency of the element, and monitored its dissemination in environmental conditions. Experiments conducted in other hosts likely to have an ICE SXT/R391 in the environment, demonstrated different regulatory patterns ranging from the loss of inducibility to the lack of transfer. Those preliminary results indicate that, in the context of an environmental dissemination, the behavior of SXT/R391 ICEs depends on their host.L’antibiorésistance est un problème de santé publique majeur. En 2016, un rapport sur la résistance aux antibiotiques a estimé que près de 700 000 morts par an étaient liées à la résistance aux antibiotiques dans le monde. Des projections estiment qu’en 2050, ce chiffre pourrait atteindre 10 millions de morts annuelles en absence de nouvelles mesures pour limiter l’émergence et la propagation de résistances (O’Neill, 2016). Si la sélection de l’antibiorésistance par l’antibiothérapie est une composante importante du problème, on estime aujourd’hui que la dissémination de bactéries résistantes ou de leurs gènes de résistance, dans ou entre les sphères humaines animales et environnementales, en est probablement la clé.Il existe plusieurs familles d’éléments génétiques mobiles à l’origine de la dissémination de gènes de résistance aux antibiotiques entre bactéries et finalement, assez peu d’études qui traitent de leurs abondances et spectres d’hôtes respectifs ou de leur dissémination dans l’environnement. Dans ce travail, nous avons notamment déterminé le spectre d’hôtes des éléments intégratifs et conjugatifs (ou ICE) de type SXT/R391, dans des collections d’isolats environnementaux par qPCR, puis dans deux environnements aquatiques grâce à une approche par epicPCR. L’epicPCR est méthode de biologie moléculaire permet de lier un fragment du gène codant pour l’ARNr 16S à un fragment d’intérêt, ici un fragment spécifique au gène traB des éléments SXT/R391. Ainsi, nous avons identifié une batterie d’hôtes environnementaux, encore jamais décrits à ce jour (eg Tolumonas auensis,), comme possible courroie de transmission environnementale des ICE SXT/R391 et de leurs gènes cargo. Ces données tendent à démontrer que, contrairement à ce qui prévaut dans la littérature, la conséquence de la dissémination des ICE SXT/R391 ne se limitent pas à une diffusion de l’antibiorésistance au sein des Vibrio, notamment Vibrio cholerae.Nous avons également identifié des molécules induisant l’expression de gènes de transfert de l’ICE SXTMO10 chez E. coli MG1656, évalué l’effet de plusieurs de ces molécules, dont la ciprofloxacine, sur la fréquence de transfert de l’élément in vitro, et suivi sa dissémination dans des conditions environnementales. Des expériences menées dans d’autres hôtes susceptibles d’héberger un ICE SXT/R391 dans l’environnement montrent des schémas de régulation différents allant de la perte217d’inductibilité à l’absence de transfert. Bien que relativement préliminaires, ces résultats indiquent que, dans le contexte d’une dissémination environnementale, le comportement des ICE SXT/R391 est relativement versatile en fonction de l’hôte qui les héberge

Role of the integrative conjugative elements of the SXT/R391 family in the dissemination of antibiotic resistance genes in aquatic environments

L’antibiorésistance est un problème de santé publique majeur. En 2016, un rapport sur la résistance aux antibiotiques a estimé que près de 700 000 morts par an étaient liées à la résistance aux antibiotiques dans le monde. Des projections estiment qu’en 2050, ce chiffre pourrait atteindre 10 millions de morts annuelles en absence de nouvelles mesures pour limiter l’émergence et la propagation de résistances (O’Neill, 2016). Si la sélection de l’antibiorésistance par l’antibiothérapie est une composante importante du problème, on estime aujourd’hui que la dissémination de bactéries résistantes ou de leurs gènes de résistance, dans ou entre les sphères humaines animales et environnementales, en est probablement la clé.Il existe plusieurs familles d’éléments génétiques mobiles à l’origine de la dissémination de gènes de résistance aux antibiotiques entre bactéries et finalement, assez peu d’études qui traitent de leurs abondances et spectres d’hôtes respectifs ou de leur dissémination dans l’environnement. Dans ce travail, nous avons notamment déterminé le spectre d’hôtes des éléments intégratifs et conjugatifs (ou ICE) de type SXT/R391, dans des collections d’isolats environnementaux par qPCR, puis dans deux environnements aquatiques grâce à une approche par epicPCR. L’epicPCR est méthode de biologie moléculaire permet de lier un fragment du gène codant pour l’ARNr 16S à un fragment d’intérêt, ici un fragment spécifique au gène traB des éléments SXT/R391. Ainsi, nous avons identifié une batterie d’hôtes environnementaux, encore jamais décrits à ce jour (eg Tolumonas auensis,), comme possible courroie de transmission environnementale des ICE SXT/R391 et de leurs gènes cargo. Ces données tendent à démontrer que, contrairement à ce qui prévaut dans la littérature, la conséquence de la dissémination des ICE SXT/R391 ne se limitent pas à une diffusion de l’antibiorésistance au sein des Vibrio, notamment Vibrio cholerae.Nous avons également identifié des molécules induisant l’expression de gènes de transfert de l’ICE SXTMO10 chez E. coli MG1656, évalué l’effet de plusieurs de ces molécules, dont la ciprofloxacine, sur la fréquence de transfert de l’élément in vitro, et suivi sa dissémination dans des conditions environnementales. Des expériences menées dans d’autres hôtes susceptibles d’héberger un ICE SXT/R391 dans l’environnement montrent des schémas de régulation différents allant de la perte217d’inductibilité à l’absence de transfert. Bien que relativement préliminaires, ces résultats indiquent que, dans le contexte d’une dissémination environnementale, le comportement des ICE SXT/R391 est relativement versatile en fonction de l’hôte qui les héberge.Antibiotic resistance is a major global health issue. In 2016, a report on antibiotic resistance estimated that at least 700,000 deaths per year worldwide, were linked to antibiotic resistance. Projections indicate that by 2050, the number of deaths could reach 10 million per year in the absence of new measures to control the emergence and the spread of antibiotic resistances (O’Neill, 2016). While antibiotic therapy is an important part of the antibiotic resistance selection problem, it is now believed that the spread of resistant bacteria, or their resistance genes in or between human, animal, and environmental spheres, might be the key.There are several families of mobile genetic elements at the origin of the dissemination of antibiotic resistance genes between bacteria, but relatively few studies on their relative abundances, their respective host range, or their dissemination in the environment. In this study, we determined the bacterial host range of the integrative conjugative elements (or ICEs) from the SXT/R391 family by qPCR from environmental strain collections, then from two aquatic environments using an epicPCR approach. The latter allows linking a fragment of the 16S rRNA gene to a fragment of interest, here a specific fragment of the traB gene from SXT/R391 elements. We then identified new environmental hosts, so far never described in the literature (ie Tolumonas auensis,), as a possible vector of SXT/R391 ICEs and their genes. Therefore, our data demonstrated that, contrary to what we find in the literature, the dissemination of SXT/R391 ICEs is not only limited to Vibrio species like Vibrio cholerae.We also identified molecules activating the expression of the ICE SXTMO10 transfer genes in E. coli MG1656, evaluated the real effect in vitro of some of those molecules (like ciprofloxacin) on the transfer frequency of the element, and monitored its dissemination in environmental conditions. Experiments conducted in other hosts likely to have an ICE SXT/R391 in the environment, demonstrated different regulatory patterns ranging from the loss of inducibility to the lack of transfer. Those preliminary results indicate that, in the context of an environmental dissemination, the behavior of SXT/R391 ICEs depends on their host

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

International audienceAbstract 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

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies