Kanamycin

Spn 23F strain, P2499, was used in experimental work unless otherwise indicated. P2499 is a streptomycin-resistant derivative of P1121, an ST33 strain obtained from a study of experimental human Spn colonization, and previously tested to colonize mice efficiently^{14 (link),24 (link),43 (link)–45 (link)}. A mutant lacking the fratricidal genes (ΔcibABΔcbpD, P2576; kanamycin-resistant) was also used in this work^{24 (link)}. A streptomycin-resistant derivative of TIGR4, P2406, was used for competition experiments^{14 (link)}. All bacterial strains used in this experimental work are listed in Table S1. Colonies were grown from frozen stocks by streaking on TSA-II agar plates supplemented with 5% sheep blood (BD BBL, NJ, USA). Unless otherwise stated, starter cultures were prepared by inoculating streaked colonies in tryptic soy (TS) broth statically at 37°C until they reached an optical density at 620 nm (OD₆₂₀) of 1.0. The cells were then pelleted, washed and resuspended in sterile phosphate-buffered saline (PBS) for mouse inoculations. Bacterial numbers were enumerated by plating serial dilutions on TSA plates supplemented with 100 μl of catalase (38,000 U/ml; Worthington Biochemical Corporation, NJ) and the desired antibiotic (250 μg/ml kanamycin, 200 μg/ml streptomycin, or 200 μg/ml spectinomycin) and incubated overnight at 37°C with 5% CO₂.