Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high producing costs. The rise of antibiotic selective pressure together with on-going limitation on their use demands news strategies to tackle this pathology. The pertinence of using bacteriophages to tackle this problematic is being explored, and in this work, the efficacy of vB_EcoM_FJ1 (FJ1) in reducing the load of the ETEC EC43-Ph (O9:H9, with STa, F5 and F41) was assessed. Envisaging the oral application to piglets, FJ1 was previously encapsulated on CaCO3/alginate. Encapsulated FJ1 better tolerate the adverse conditions of the simulated gastric fluid (pH 3), compared to the free phage (93.3% vs 29.7%, p-value<0.001) and was about 94% released in simulated intestinal fluid (pH 6.5) after 1 h. Furthermore, a single-dose of encapsulated FJ1 administered to the IPEC-1 swine cell cultures infected by EC43 provided maximum bacterial reductions of about 99.9% of the ETEC load after 6 h. Bacteriophage-insensitive mutants (BIMs) emerged from the treatment, but with fitness costs comparatively to the originating strain. This was supported by the higher ability of the pig complement system to decrease BIMs’ viability, by their lower level of intestinal cells colonization of the piglet intestinal cells, and by their increased survival rates and health index in infected Galleria mellonella larvae. Overall, FJ1 enabled to establish a proof of concept of the efficiency of phages to fight against ETEC in intestinal cultured cells.