Fig 9. CNS sp restore resistance to ocular P. aerugiunosa 6294–induced keratitis.

A. P. aeruginosa 6294 CFU/cornea (left) and pathology scores (right) 24 h following infection. Groups of GF (n = 8), SPF-SW (n = 10), HMB (n = 10), and MMB (n = 10) mice were infected with 10⁷ CFU per eye. Data were pooled from two performed experiments. The bacterial burden data and pathology scores were analyzed with one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparisons test. The individual circles represent individual animals. Data suggest that reconstituting GF mice with either human-derived or mouse-derived gut commensals restores resistance to infection. B. Monocolonizing GF mice with CNS sp. by topical application of CNS sp. suspension onto the eye restores resistance to infection. Quantification of the levels of CNS sp recovered from the conjunctival swabs one week and 12 days post-reconstitution. Quantification of the CNS sp. in the fecal pellets, demonstrating stable monoassociation. C. Neutrophils derived from monocolonized GF mice show comparable bactericidal activity against P. aeruginosa PAO1 as SPF-derived PMNs. p-values by one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparisons test. The individual sample values are plotted as circles. D. Conjunctival CNS sp. presence promotes IL-1ß transcription. Conjunctival tissues from GF (n = 5) and monocolonized GF mice with CNS sp. (n = 3) were collected, total RNA purified, and the levels of IL-1ß transcripts quantified with RT-PCR. The levels of transcripts were compared using one-way ANOVA followed by Turkey’s multiple comparisons test. Significant p<0.05 values are indicated by asterisks. The tissues harvested from the monocolonized mice showed significantly increased presence of IL-1ß transcripts 12 days post-colonization. E. P. aeruginosa 6294 CFU/cornea (left) and pathology scores (right) 24 h following infection in monocolonized GF with CNS sp. (n = 6), SPF SW (n = 5), or GF (n = 10) mice. Monocolonizing of GF mice with CNS sp. restores the resistance to infection. The values for the individual animals are represented as circles. Significant differences are denoted with asterisks. p-values by one-way ANOVA followed (p = 0.0007) by ordinary one-way ANOVA multiple comparisons test. Pathology scores were compared using one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparison test.