The emergence and the dissemination of antibiotic resistance genes among bacteria are recognized to follow antimicrobial consumption. Demonstrating gene transfer in environmental matrices is technically difficult, therefore limiting their investigation. Here we propose to push the limits of our knowledge regarding gene transfer and bacterial adaptation to antibiotics, using an approach based on the regulation of genes involved in the transfer of mobile genetic elements.
The aim of the ReguloMobile project is to understand the dynamics of mobile genetic elements responsible for antibiotic resistance gene transfer in the environment. The emphasis is given to a couple of parameters repeatedly pointed out in the dissemination of antibiotic resistance: the antibiotics at sub-inhibitory concentrations and the biofilm life style. In the current period where much emphasis is given to retrospective evidences to study and understand the dissemination antibiotic resistance genes in the environment, we clearly wish to explore a gene regulation-based approach to understand how environmental parameters directly modulate the mobility functions of mobile genetic elements. <br /> The objectives of the ReguloMobile project are multiple: (1) Elaborating a set of biosensors dedicated to the identification of conditions activating transfer functions in mobile genetic elements; (2) Screening known antibiotics for their implication in stimulating transfer function of mobile elements at low concentrations; (3) Localizing the expression of transfer genes in biofilm structures; (4) Correlating the expression of mobility functions and gene dissemination in environmental matrices using a model mobile element ; (5) Setting up the basis of a new biosensor-based screen tool for the identification of environmental hot spots of gene dissemination.
In the context of the ReguloMobile project, we propose to evaluate different antibiotics for their ability to modulate the activity of promoters from mobile genetic elements fused to luminescence genes. Once modulating antibiotics are identified (and their active concentrations defined), they are further examined in combination with an environmental matrix in order to evaluate their effectiveness in an environmental context.
Microbial biofilms have been repeatedly proposed as possible hot spots able to promote the dissemination of antibiotic resistance genes among microorganisms. The promoter-lux fusions constructed are also being used to identify the promoters of mobile genetic elements that are specifically modulated by the biofilm mode of life. Next, the expression of the modulated promoters is further studied in dedicated biofilm reactors allowing the imaging of the promoter expression in the different parts of the biofilm.
Detailed sequence analyses with dedicated bioinformatics tools are used to provide basic information regarding the boundaries of the promoters and to identify predictive regulatory boxes/loops able to explain how antibiotics could promote the mobility of mobile genetic elements at the molecular level.
Sequences from mobile genetic elements of Gram positive and Gram negative bacteria were analyzed allowing the identification of 76 promoters possibly involved in the expression of mobility functions. Of these, about 70 were successfully cloned upstream of lux genes into vectors allowing the expression of a bioluminescence according to the level of activity of the promoters involved. These constructs were introduced into a panel of suitable bacterial hosts leading to the construction of 133 biosensors so far.
These biosensors were exposed to different concentrations of a panel of 48 antibiotics from all known classes these antimicrobials. Promoters activities, evaluated by measuring the bioluminescence emitted by the biosensors, have already allowed establishing two types of profiles: (1) groups of promoters having the same induction profile for one or more antibiotics, and (2) series of antibiotics with the same effects on promoter induction.
Following the first results obtained regarding the promoters responsiveness to antibiotics, the immediate perspectives will consist in analyzing the promoting sequences for identifying common patterns suggesting the binding of related regulators.
The project is now entering a second phase consisting in evaluating the activity of the regulated promoters in the different depth of bacterial biofilms.
The emergence and the dissemination of antibiotic resistances genes among bacteria have been recognized long ago to follow antimicrobial consumption, but only recently the seriousness of the situation has been considered by various authorities. This led to series of recommendations in the public health and the farming domains, for a better control of antibiotic prescriptions, hoping to reduce the adaptation of bacteria to antibiotics. Nevertheless the continuous release of antibiotics and resistant bacteria in the environment is currently raising concerns regarding possible environmental hot spots of resistance genes dissemination.
Demonstrating gene transfer in environmental matrices is technically difficult and always tedious, therefore limiting the range of what can be done when exploring environments for their propensity to support antibiotic resistance dissemination. In this project, we propose to push the limits of our knowledge regarding gene transfer and bacterial adaptation to antibiotics, using an approach based on the regulation of gene involved in the mobility of mobile genetic elements. Three basic questions will be addressed: (1) can we identify environmentally relevant concentrations of antibiotics modulating the transfer/mobility of mobile genetic elements? (2) Are those antibiotics effective in environmentally relevant microbial structures such as biofilms? (3) Are those antibiotics effective in stimulating the transfer of mobile genetic elements in the complexity of environmental conditions?
To answer these questions, we propose to develop a new biosensor-based approach for the rapid identification of conditions modulating the mobility functions of an array of mobile genetic elements. The scientific program proposed is divided in three interconnected tasks. (1) Fifty promoters, governing the expression of mobility functions (transposition, integration, conjugation,...) in various kinds of genetic mobile elements, will be selected for the construction of a biosensors array based on promoter-lux fusions. Detailed sequence analyses with dedicated bioinformatics tools will provide basic information regarding the boundaries of the promoters and predictive regulatory boxes/loops. (2) The biosensors obtained will be exposed to various antibiotics in order to identify which of those are able to modulate the expression of the selected mobility functions and at which concentrations. (3) The modulation of biosensors will also be studied in biofilm, a lifestyle supposed to favor the transfer of numerous mobile elements. Conditions found to modulate the activity of the cognate biosensors will finally be assessed for their influence on the dissemination of our model plasmid pB10 in environmental matrices maintained in microcosms. The dissemination of the model plasmid pB10 will be monitored using molecular-based approach a recently developed by one of the partner. The results obtained should validate or not, the relevance/usefulness of the biosensor approach developed.
Apart from the acquisition of fundamental knowledge on the regulation of gene transfer and its significance in environmentally-relevant conditions, the ReguloMobile project should set the basis of new biosensor-based approach to screen various environments/conditions for their propensity to support gene dissemination, and then therefore for the identification of environmental hot spots of gene dissemination.
Monsieur Merlin Christophe (Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR 7564 CNRS - Université de Lorraine)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMR Inserm 1092 UMR Inserm1092 Anti-Infectieux : Supports moléculaires des résistances et Innovations thérapeutiques
LCPME Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), UMR 7564 CNRS - Université de Lorraine
Help of the ANR 277,908 euros
Beginning and duration of the scientific project: December 2013 - 42 Months