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'Get in Early'; Biofilm and Wax Moth (Galleria mellonella) Models Reveal New Insights into the Therapeutic Potential of Clostridium difficile Bacteriophages.

Clostridium difficile infection (CDI) is a global health threat associated with high rates of morbidity and mortality. Conventional antibiotic CDI therapy can result in treatment failure and recurrent infection. C. difficile produces biofilms which contribute to its virulence and impair antimicrobial activity. Some bacteriophages (phages) can penetrate biofilms and thus could be developed to either replace or supplement antibiotics. Here, we determined the impact of a previously optimized 4-phage cocktail on C. difficile ribotype 014/020 biofilms, and additionally as adjunct to vancomycin treatment in Galleria mellonella larva CDI model. The phages were applied before or after biofilm establishment in vitro, and the impact was analyzed according to turbidity, viability counts and topography as observed using scanning electron and confocal microscopy. The infectivity profiles and efficacies of orally administered phages and/or vancomycin were ascertained by monitoring colonization levels and larval survival rates. Phages prevented biofilm formation, and penetrated established biofilms. A single phage application reduced colonization causing extended longevity in the remedial treatment and prevented disease in the prophylaxis group. Multiple phage doses significantly improved the larval remedial regimen, and this treatment is comparable to vancomycin and the combined treatments. Taken together, our data suggest that the phages significantly reduce C. difficile biofilms, and prevent colonization in the G. mellonella model when used alone or in combination with vancomycin. The phages appear to be highly promising therapeutics in the targeted eradication of CDI and the use of these models has revealed that prophylactic use could be a propitious therapeutic option.

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