All materials and reagents were supplied by Sigma-Aldrich Inc., (St. Louis MO), unless otherwise specified; all microorganisms were supplied by the American Type Culture Collection (Manassas VA).
Lactobacillus crispatus ATCC 33820 was grown in ATCC medium 1490 (Modified chopped meat medium), L. jensenii ATCC 25258 and L. gasseri ATCC were grown in ATCC medium 416 (Lactobacilli MRS broth), L. iners ATCC 55195 was grown in ATCC medium 1685 (NYC III medium). Each species was grown anaerobically without agitation at 37°C for 24 hours before use in an experiment.
Bacteria associated with bacterial vaginosis
Gardnerella vaginalis ATCC 14018 was grown in ATCC medium 1685 (NYC III medium). Prevotella bivia ATCC 29303, Prevotella corporis ATCC 33547, Anaerococcus prevotii ATCC 14952, Fusobacterium nucleatum ATCC 25586 and Porphyromonas levii ATCC 29147 were all grown in ATCC medium 1490 (Modified chopped meat medium); Bacteroides ureolyticus ATCC 33387 was grown in ATCC medium 1490 with formate and fumarate. Peptostreptococcus anaerobius ATCC 27337, Anaerococcus tetradius ATCC 35098, Atopobium vaginae ATCC BAA-55, Megasphaera elsdenii ATCC 25940, and Propionibacterium acnes ATCC 6919 were all grown in ATCC medium 1053 (Reinforced Clostridial medium) supplemented with 5% defibrinated rabbit blood (Colorado Serum Company, Denver CO). Ureaplasma urealyticum ATCC 27618 was grown in ATCC medium 1331 (Urea broth); Mobiluncus curtisii ATCC 35241 and Mobiluncus mulieris ATCC 35239 were grown in BBL™ Schaedler medium (Becton, Dickinson and Company, Sparks MD). Mycoplasma hominis ATCC 23114 was grown in ATCC medium 243 (Mycoplasma medium). Micromonas micros ATCC 33270 was grown in ATCC medium 1102 (Chopped meat medium) supplemented with 0.1% each of cellobiose, maltose, starch, and Tween 80. Each species was grown anaerobically in a 50 mL volume of its recommended growth medium without agitation at 37°C for 24 or 48 hours before use in an experiment, yielding bacterial concentrations between approximately 106 and 109 colony-forming units (cfu) per mL (48 hour incubations were used for bacteria that failed to produce consistently = 106 cfu/mL after 24 hour incubations). The relatively high concentrations of bacteria used were chosen both to increase the dynamic range of the experiments (i.e., large numbers of bacteria permit a more meaningful quantification of observed inactivation), and to reflect the high density of bacteria seen in vivo .
Experimental media for each organism were prepared by adding H2O2, lactic acid, or acetic acid to the appropriate growth medium for that organism; they were not added to control media. For experiments using H2O2, both experimental and control media contained 50 mU/mL human myeloperoxidase (MPO). All growth medium formulations contained at least ten-times more than the 1 mM concentration of chloride ions required for full activity of a myeloperoxidase-halide-H2O2 microbicidal system . Aliquots of each experimental and control medium were titrated with sodium hydroxide or hydrochloric acid as necessary to obtain a pH of either 4.5 or 7.0 (with allowance made for the change in pH that would occur when an aliquot of bacterial culture was added, as described below).
Bacterial cultures were gently agitated immediately before use. A 100 μL aliquot of culture was added to 9.9 mL of each control or experimental medium; media and bacteria were then incubated anaerobically at 37°C. Two replicate samples were removed from control and experimental conditions after ten minutes, thirty minutes, one hour, and two hours exposure. Each sample was then serially diluted with the appropriate growth medium containing 200 mM HEPES (pH 6.8-7.2 depending on growth medium) and track-plated  onto the appropriate growth medium containing 1.5% (w/v) ultrapure agar (USB Corporation, Cleveland OH). The pH of each experimental or control medium was re-measured after the experiment to confirm it had remained within 0.1 pH units of the starting pH. Agar plates were incubated anaerobically at 37°C for 24 or 48 hours, until colonies could be easily distinguished and counted. Colonies on some plates were recounted after a further 48 hours incubation to allow for extended lag-phases in treated cells; however, no further changes in colony-counts were observed. Each experiment was independently repeated at least four times.
Bacterially-depleted vaginal fluid
The study was carried out at the Johns Hopkins University Homewood campus. Each participant gave written informed consent under a protocol approved by the Homewood Institutional Review Board on the Use of Human Subjects at Johns Hopkins University. Participants were required to be between 18 and 45 years old, in good general health, at least three days past the most recent menstruation or unprotected penile-vaginal intercourse, at least three weeks past the most recent use of vaginal or systemic antimicrobials, and free from vaginal symptoms (discharge, odor, itching, or pain). Results from twenty-two samples donated by eight participants are reported here; the group comprised roughly equal numbers of non-Hispanic whites, blacks, and Asians, aged between 21 and 44 years old (mean age 27 ± 4 years).
Collection of vaginal fluid samples
For these experiments, undiluted non-menstrual VF was collected at the laboratory using the non-absorbent disposable Instead® Softcup™ menstrual device (Evofem Inc., San Diego CA) . The Softcup was vaginally inserted, removed, and placed in a conical centrifuge tube. The collected VF was removed from the Softcup by centrifugation for one minute at 500 g; the Softcup was then discarded.
A sterile cotton swab was dipped into the collected fluid, rolled out onto a glass microscope slide, and air-dried for later Gram-staining and Nugent-scoring. A total of eight participants donated VF; all samples had Nugent score ≤ 3 and no evidence of leukorrhea (the mean PMNL/hpf of the samples was 2.3). As reported earlier, VF samples as obtained contain ~ 1% lactic acid and ~ 20 μM H2O2 [9, 29].
To avoid conflating endogenous vaginal bacteria with the cultured bacteria used in these experiments, bacterially-depleted VF was prepared: each collected sample was diluted with a half-volume of sterile saline (0.9% [w/v] sodium chloride), mixed thoroughly, centrifuged at 1000 g for three minutes, and the supernatant was drawn off for immediate use in an experiment. Pilot experiments showed that this centrifugation reduced bacterial concentrations in the diluted VF by a factor of approximately 106, from a pre-centrifugation mean of 5.6 × 107 cfu/mL to a post centrifugation mean of 4.0 × 101 cfu/mL (data not shown). Rather than pooling VF samples for use in experiments, individual samples from at least four different participants were used in conjunction with each treatment (H2O2 or lactic acid) to assess the reproducibility of results across different VF samples.
The effect of VF on the microbicidal activities of H2O2 and lactic acid against seven prevalent species of BV-associated bacteria (G. vaginalis, A. vaginae, P. bivia, P. anaerobius, M. curtisii, M. mulieris, and M. hominis) and four species of vaginal lactobacilli was measured. Each organism was exposed to growth medium containing an inactivating concentration of H2O2 (3.4% w/v [1 M] with 50 mU/mL MPO at pH 7) or lactic acid (1% w/v [111 mM] at pH 4.5), with or without the addition of bacterially depleted VF to a final VF concentration of 1% or 10% (v/v). In all cases, the bacterially depleted VF was added to the experimental media and mixed for five seconds before the addition of bacteria. The pH of the experimental media was also checked before the addition of bacteria, and if necessary readjusted to 4.5 or 7.0. Samples were removed from control and experimental conditions after ten minutes, thirty minutes, one hour, and two hours, serially diluted, plated and enumerated as described above.
Results are reported as means of at least six independently repeated experiments (two replicates performed within each experiment). The difference between three or more means was tested using an ANOVA one-way analysis of variance; difference between two means was tested using a two-tailed Student's t test (comparisons are paired unless otherwise indicated in the results); p values ≤ 0.05 were considered to be statistically significant. Statistical analysis was performed using PHStat2 version 3.0 (Microsoft Excel add-on). Due to the large amount of data presented in the graphs, standard deviations have been omitted for visual clarity; however, there were no significant differences among data from different replicates or repeats of individual experiments (differences were less than a log
unit in all cases).