Polymyxin E, also known as colistin, was used initially in humans for treatment of infections caused by Gram negative bacteria. Because of its nephrotoxicity, colistin was withdrawn from therapeutic use in humans. Nevertheless, with increasing microbial resistance to current antibiotics and the lack of new drug candidates in the pipeline, colistin has now been reintroduced into human therapy as a drug of last resort to treat multi-drug resistant Gram negative bacteria. Importantly, colistin is also used in pig farming and in overall veterinary medicine to control Escherichia coli post-weaning diarrhea, which could lead to major economic losses. Colistin is clearly of major importance for human and animal welfare and its utilization requires a better management in order to avoid selection of resistant strains. The main purpose of the Sincolistin project is to reduce drastically the amount of colistin used in pig farming through the development of novel, sustainable and innovative antibiotic products based on increasing the potency of colistin by addition of bacteriocins. Indeed, recent data from the coordinator's group have shown that colistin and bacteriocins, such as nisin and pediocin PA-1, can act synergistically against E. coli and other Gram negative bacteria. Taking advantage of this finding, formulations based on the use of colistin and bacteriocins will be developed and incorporated into chitosan nanoparticles (50-100 nm) and microspheres (5-20 µm), which will survive the harsh gastrointestinal environment and then be delivered on the appropriate infection site. Bacteriocins are safe and natural antimicrobials. They are heat stable and insensitive to pH variations, though liable to hydrolysis by proteases. Bacteriocins foreseen to be used in the Sincolistin project are pentocin LB3F2, pentocin LB2F2, bavaricin LB1F2, bavaricin LB14F1 and bavaricin LB15F1, recently isolated from lactic acid bacteria and characterized for their E. coli inhibitory activity. These bacteriocins will be assessed against a set of fully characterized colistin-susceptible and colistin-resistant E. coli isolates of swine origin obtained from the RESAPATH network, which is headed by ANSES. The bacteriocin with the higher anti-E. coli activity, designated bacteriocin X, will be characterized for its: a) Mode of action against E. coli, in order to determine the mechanism that provokes cell death. b) Ability to generate resistant mutants (frequencies of mutation vs. colistin) c) Mechanism of synergy with colistin d) Cytoxicity to porcine and human cells Bacteriocin X will be produced at larger scale after optimizing the growth conditions and determining the best parameters of production. The release and bio-accessibility of the different formulations (nanoparticles and microspheres) developed within the framework of this project will be studied in the TIM in vitro model of upper gastrointestinal tract of pig. Further, the impact of these formulations on the pig gut microbiota and the survival of colistin-susceptible and colistin-resistant E. coli isolates from swine origin will be determined in the ARCOL in vitro model of pig colon thereby establishing whether the formulations destabilize the microbiota to a lesser extent than colistin alone. Finally, the best formulation will be tested in vivo under controlled conditions in pigs, inoculated with colistin-resistant E. coli, to validate the concept and strategies developed within the framework of the Sincolistin project.

Intestinal microbiota is now recognized as playing a major role in human health by conferring a protection against pathogens and contributing to human nutrition. A disequilibrium of this ecosystem (dysbiosis) can lead to the development of diseases (inflammatory bowel diseases, obesity or colorectal cancer). Throughout life, the total consumption of food by an individual will reach 60 tons on average, corresponding to a large ingestion of bacteria (bacteria used in food industry or pathogens). These bacteria brought by food will be able to interact with symbiotic and commensal bacteria and in particular exchange genes (horizontal gene transfer) with them. This acquisition of genes can confer novel properties to bacteria (antibiotic resistance, colonization factors, novel catabolic properties, synthesis of bacteriocin, stress responses…). This can threat the equilibrium of digestive microbiota or lead to the emergence of new pathogens. Conjugation is the main mechanism of gene acquisition and can lead not only to gene transfers between strains belonging to the same species but also between very distantly related bacteria. It is encoded by several classes of mobile genetic elements, in particular chromosomal elements called Integrative and Conjugative Elements or ICEs, a widespread but poorly known class of elements. This project aims at evaluating gene transfers occurring through ICE between bacteria brought by dietary intake and commensal bacteria of the digestive tract. Work will mainly focus on gene transfers within Streptococcus, an interesting bacterial genus since it includes various species that live or transit in the human gastrointestinal tract (commensal, pathogenic or food bacteria). The objectives of the project are to characterize the factors and the mechanisms of induction of transfer of ICEs and to give a first insight on the incidence of gene transfers mediated by these ICEs in the human digestive ecosystem. Approaches will include in vitro analyses, biorelevant in vitro studies (in artificial digester and in artificial colon inoculated with human microbiota) and in vivo experiments (axenic mice inoculated with strains of interest). This project will help to propose strategies to fight against bacterial gene transfers mediated by conjugation.