Strategic alternatives to reduce the use of colistin in pig farming – Sincolistin

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 Because of its nephrotoxicity, colistin was withdrawn from therapeutic use in humans.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.