Fig 5. Effect of mucus on colonisation C. perfringens.

(A) Competition assay of C. perfringens wild-type strain CP56 and mutant strains CP56ΔchiA1 or CP56ΔchiB1 in either in vitro or in vivo growth conditions. In vitro: An equal mix of wild-type and mutant strain was grown in either nutrient rich medium or nutrient poor medium supplemented with 5% chicken intestinal mucus. Samples were taken at the exponential growth phase and at saturation after overnight incubation. In vivo: 18-days old broiler chickens were inoculated with an equal mix of wild-type and CP56ΔchiA1 mutant strain. After 24 hours, samples were taken from the intestinal content (jejunum or ileum). The amount of wild-type or mutant strain in the samples was determined using dPCR. The competition index is defined as the ratio of the mutant on wild-type strain, divided by the respective ratio in the inoculum. Lines indicate the means. (B) Mucus binding assay of wild-type CP56 C. perfringens strain in media supplemented with recombinant chitinases, either ChiA or ChiB. Washed C. perfringens overnight culture was added to the wells containing a mucus agar layer, supplemented with either 50 μg of recombinant enzyme (ChiA or ChiB) or an equal volume of PBS as a negative control. Wells were anaerobically incubated for 90 min at 37°C after which the bound bacteria were quantified through titration. The binding ratio is defined as the percentage of bacteria bound to the mucus in supplemented media as compared to non-supplemented conditions. Bars indicate the means with their respective standard deviations.