Determination of the MIC and minimum bactericidal concentration (MBC).

Results for NMII MIC were taken from Clay et al. (12) and had been determined as the lowest concentration of an antimicrobial agent that prevented visible growth (measured via optical density [OD] at 590 nm) in a broth dilution susceptibility test (in accordance with the Clinical and Laboratory Standard Institute guidelines). Briefly, a range of increasingly concentrated antibiotic broths were made, inoculated with approximately 5 × 10⁶/ml (final concentration) of culture, and then incubated for 6 days (in 5% CO₂ and 2.5% O₂) when the OD was measured. The MIC for NMI was determined both by measuring the OD and then also plating out each broth to obtain a viable bacterial count to make it comparable to the THP-1 intracellular assay. The MBC was determined by plating out at the neat broth onto ACCM-2 plates, and no visible bacterial growth appeared following incubation after 10 days. Significant inhibition of bacterial growth compared with the bacterial count after 7 days in the presence of no antibiotics (0 μg/ml) was assessed using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons. All tests were done in triplicate.