Mouse Nasopharyngeal Colonization Assay

Mouse colonization was performed as described in reference 25 (link). Briefly, dual or single-strain mixtures of Sp9-BS68 were inoculated into the nasopharynx of unanaesthetized 5- to 7-week-old female BALB/c mice (Taconic)with 1.0 × 10⁶ to 3.0 × 10⁶ CFU/mouse in 10 μl of sterile phosphate-buffered saline (PBS). For dual inoculated mice, the ratio of the kanamycin-resistant strain to the spectinomycin-resistant strain was between 0.25 and 0.6. Mice were euthanized with CO₂ overdose after 72 h, and nasopharyngeal colonization was sampled by nasal wash. See methods in Text S1 for IACUC approval and details on how colonization density and competitive indices were calculated. Differences in colonization densities and competitive indices between groups were evaluated by the Mann-Whitney (2 groups) and Kruskal-Wallis (>2 groups) tests using the wilcox.test() and kruskal.test() functions in R 3.5.1.