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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The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

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Sheath blight and brown spot disease of rice caused by Rhizoctonia solani and Bipolaris oryzae causes significant yield loss in rice production worldwide. The present study was undertaken to examine the effect of two essential oils (EOs) from Lippia geminata and Cymbopogon jwarancusa on in vitro growth and sporulation of these two pathogens. The fungal radial growth was inhibited at very low concentration (25 ppm) of the EOs. Similarly, fungal spore production was also inhibited up to C80% at 500 ppm of EOs. However, fungal sporulation was completely retarded at 1,000 ppm of L. geminata EO. Very low oil concentrations (10 ppm) accelerated the radial growth (0-5 mm) and spore germination (3.5-8.5%) of the pathogens. At higher oil concentrations, the mycelial growth and spore productions were completely inhibited. The IC 50 value of the EO of C. jwarancusa was 365.45 and 336.74 ppm and for L. geminata, it was 420.16 and 481.47 ppm against B. oryzae and R. solani, respectively. GC-MS analysis of the oils showed 54.36% piperitone and 30.86% a-phellandrene as major compounds in C. jwarancusa whereas 25.9% geranial and 14.6% neral in L. geminata oil. Essential oils from Lippia geminata and Cymbopogon jwarancusa appear to be good candidates for the in vitro control of these two rice pathogens and can be successfully utilized in management strategies of pathogens in appropriate formulation.