Modified microdilution assays.

To determine whether Cz in vaginal secretions had a direct effect on gonococcal viability, modified microdilution assays were performed using N. gonorrhoeae strain WHO Z. Pooled vaginal fluid from control participants (CBP03-05) was added to select wells of a 384-well plate. Cz (100 μM) was added to the first well after which Cz-containing vaginal fluid was serially diluted 10-fold (as above), yielding a dose range of 10 nM to 100 μM (2.4 ng/mL to 23 μg/mL), with the last well corresponding to a no Cz control. Gonococci were added to each well at a culture density of 10⁷ bacterial per milliliter. Microtiter plates were incubated (3% O₂, 37°C) for 1 h or 24 h. At each time point, serial dilutions of the vaginal fluid-gonococcal suspension were plated. Viable gonococci were enumerated by counting CFU after a 48-h incubation (37°C, 5% CO₂). The percentage of viable N. gonorrhoeae was determined as a function of the inoculum. Additional assay controls included 0.1% DMSO (vehicle), 0.5 μg/mL (902 nM) ceftriaxone, 100 μg/mL (209 μM) gentamicin, and an untreated-uninfected, vaginal fluid sterility control. Assays were performed on three separate occasions. Statistical significance of data obtained was determined using a paired Student's t test (GraphPad).