Fig. 2: r.i. of the toxin + NTA formula clears colonization while preventing weight loss, epithelial injury and mortality.

a, Experimental schematic. Mice were infected for 10 or 15 days, with no differences observed between cohorts. The diagram was created using BioRender. b,c, The survival of infected with WT C. difficile R20291 after r.i. (b) and i.p. (c) vaccination. The bracket refers to the lines of both the toxin + dmLT and toxin + NTA + dmLT cohorts. d,e, Weight loss at 2 d.p.i. (d) and 3 d.p.i. (e) relative to day 0. Data are mean ± s.e.m. f–q, Enumeration of total C. difficile R20291 bacteria (vegetative cells and spores (f–h and l–n)) and spores (i–k and o–q) in faeces after vaccination with dmLT only (f,i,l,o), toxins + dmLT (g,j,m,p) and toxins + NTAs + dmLT (h,k,n,q) through the r.i. (f–k) and i.p. (l–q) routes. P values were calculated relative to the control at the same timepoint. Individual lines correspond to individual mice. The limit of detection is shown by the dotted line: 500 CFU per g faeces. Statistical significance was calculated using log-rank Mantel–Cox tests (b and c) and one-way ANOVA with Tukey’s correction (d–g and f–q (each day analysed individually)). For b–q, n = 20 per group; decreases in n are due to animal mortality during infection. Individual datapoints are represented and were pooled from two independent experiments.

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