Extended Data Fig. 4: RI-vaccination with the toxin and NTA formula eliminates C. difficile from tissue.

a–d, Enumeration of total C. difficile R20291 bacteria (vegetative cells and spores) (a,c) and spores (b,d) from macerated colons and caeca of vaccinated mice fifteen days post-R20291 infection. Limit of detection (dotted line) is 500 CFU/g faeces. e, PCR of C. difficile 16S gene from bulk DNA extracted from macerated colons and caeca from a–e, with each lane representing one mouse. Desired amplicon is 175 bp (expected size indicated by red arrow; solid line indicates bands are present whereas dotted shows lack of bands). Distinct bands can be noted for certain mice; smeared bands are background DNA from extracted tissues. f, Normalized reporter values from C. difficile 16S qPCR of bulk DNA extracted from macerated colons and caeca from a–d. qPCR was performed in triplicate per sample. Data reported as the mean of all samples’ experimental averages, normalized from baseline. Standard threshold (dotted line) is 30 cycles. g, Cycle threshold from C. difficile 16S qPCR of bulk DNA extracted from macerated colons and caeca from a–d. qPCR was performed in triplicate per sample. Mean +/− SEM. Standard threshold (dotted line) is 30 cycles. h, Total colonization burden (vegetative and spore counts combined; closed circle) versus spore counts (open circle) in faeces of naive, unvaccinated mice infected with WT C. difficile R20291. Limit of detection (dotted line) is 500 CFU/g faeces. 4a-g: n = 2-10 per group, dependent on survival at 15 days post-infection; 4h: n = 3 per group. CFU, colonization forming units; RI, rectal instillation; IP, intraperitoneal injection; L, ladder; bp, base-pairs; ∆Rn, normalized reporter signal above baseline; gDNA, C. difficile R20291 genomic DNA. For source gel data, see Supplementary Information.

Source data