C. pseudodiphtheriticum mediates bactericidal activity against S. aureus. (a) C. pseudodiphtheriticum was spotted on agar plates that had been seeded with various S. aureus strains (2014.N, A970377, A950085, COL, LAC, Mu50, MW2, N315, and NRS384) or S. epidermidis 1457 (S. epi). Plates were incubated at 28°C, and images were taken every 24 h (24- and 120-hour images are shown). The agr allele type of each strain is given in parentheses. Bar, 10 mm. (b) The ZOC was defined as the distance between the edge of the C. pseudodiphtheriticum spot and the visible edge of the S. aureus ring of clearance. The ZOC was measured using ImageJ software (NCBI). Each symbol represents the arithmetic mean from 3 independent biological replicates measured temporally; error bars represent ±1 standard deviation. A two-way ANOVA with Dunnett’s correction for multiple comparisons was performed on the ZOC lengths at the 120-hour time point. The symbol **** on the graph indicates a statistically significant difference (P < 0.0001) in ZOC lengths for 2014.N versus all other strains and for Mu50 versus all other sensitive strains. (c) Punches of agar from the ZOC directly adjacent to the C. pseudodiphtheriticum or outside the ZOC were plated and grown for 48 h to recover surviving S. aureus CFU. White arrows indicate rare surviving colonies.