F. OECHSLIN¹, P. PICCARDI¹, M. GIDDEY¹, S. MANCINI¹, J. GABARD², P. MOREILLON¹, J.M. ENTENZA¹, G. RESCH¹, Y-A. QUE<sup>3

1</sup>Dept. of Fundamental Microbiology, University of Lausanne, Switzerland, ²Pherecydes Pharma, Romainville, France, ³Dept. of Intensive Care Medicine, Bern University Hospital, Switzerland

The continuing development of antibiotic resistance stresses the need for alternative treatment. Infective endocarditis due to P. aeruginosa is an archetype of highly lethal valve infection in human. Therefore we used in vitro and in vivo models of P. aeruginosa experimental endocarditis (fibrin clots and rats with catheter-induced aortic vegetations, respectively) to study the efficacy of an anti-pseudomonas phage cocktail and ciprofloxacin administered alone or in combination.
In fibrin clots, phage therapy decreased bacterial density by 6 log10 CFU/g in 6h. Bacterial regrowth due to phage resistance was observed after 24h, but was prevented by addition of ciprofloxacin (2x MIC). In rats, phage therapy alone decreased vegetation bacterial density by 2.5 log10 CFU/g after 6h (P<0.001), compared to 2.2 log10 CFU/g with ciprofloxacin (P<0.05). Moreover, combining phages with ciprofloxacin appeared highly synergistic with a >6 log10 CFU/g decrease in 6h, and successful treatment in 64% (7/11) of the animals.
Phage-induced killing correlated with in situ phage multiplication - as also confirmed by histology and transmission electron microscopy examination of the vegetations - and cytokines production compared to antibiotherapy alone. Importantly, no phage-resistant mutants were detected in vivo, which was most likely due to altered fitness. Indeed, two phage-resistant clones isolated in vitro were 50-70% less infective in rats with experimental endocarditis (P<0.01 versus parent strain). This infectivity decrease was either due to a 362 kb deletion encompassing the galU gene, resulting in impaired LPS synthesis, or a 15 bp deletion in the pilT gene resulting in impaired motility.
In conclusion, phage therapy significantly reduced P. aeruginosa experimental endocarditis and was highly synergistic with ciprofloxacin. Phage-resistant mutants selected in vitro resulted in impaired infectivity, due to reduced in vivo fitness. Phage therapy alone or combined with antibiotics represents a promising alternative in the treatment of P. aeruginosa infections and merits further consideration.