Background: Lactobacillus Crispatus Is a Part of the Normal Vaginal Microflora of Humans

Background: Lactobacillus crispatus is a part of the normal vaginal microflora of humans.

Goal: The goal of this study was to assess whether a capsule containing an H2O2-producing strain of L crispatus (CTV-05) would alter the vaginal microflora and/or epithelial tissues when applied intravaginally in the pig-tailed macaque model.

Study Design: Ten sexually mature female Macaca nemestrina were assessed at baseline for quantitative vaginal microbiology and vaginal pH and with colposcopy. One capsule containing 108 colony forming units of desiccated L crispatus CTV-05 was inserted into the vaginal fornix of each animal. Vaginal assessments were repeated on days 1 and 2 after capsule insertion. The L crispatus CTV-05 strain was identified with use of a DNA fingerprinting method.

Results: Before product use, four of 10 animals had detectable levels of H2O2-producing lactobacilli. L crispatus CTV-05 was detected in 1 of 10 animals on day 1 and in 3 of 10 animals on day 2 following insertion of the capsule. There were no tissue changes observed by colposcopy. Vaginal pH decreased in two animals colonized by CTV-05, from 7.0 at baseline to 4.5 ± 0.5 on days 1 and 2 after product use.

Conclusions: A single intravaginal application of capsules containing 108L crispatus CTV-05 resulted in vaginal colonization in three of 10 animals 2 days after use. The absence of colposcopic changes in the vagina/cervical tissues indicates that L crispatus capsules are well tolerated.

LACTOBACILLUS SPECIES ARE THE predominant aerobes of the vaginal microflora in women of child-bearing age. While all lactobacilli produce lactic acid, some lactobacilli also produce hydrogen peroxide (H2O2), a known antibacterial compound. 1 Because of this activity, H2O2-producing lactobacilli are believed to act as endogenous microbicides in the vagina. Women having H2O2-producing lactobacilli are less likely to have bacterial vaginosis 1,2 and more likely to be persistently colonized by Lactobacillus species over several months. 3 H2O2-producing lactobacilli may protect against genital infections, including Neisseria gonorrhoeae and HIV infection. 4,5 Of the many species of Lactobacillus described, L crispatus and L jensenii are the most prevalent species among women having Lactobacillus-predominant vaginal microflora, and 94% to 95% of strains of these two species produce H2O2. 2,3,6

Recently, a capsule containing L crispatus has been developed for use in humans to promote and/or increase vaginal colonization by H2O2-producing lactobacilli. Use of this probiotic as a new intravaginal agent is now being evaluated. In a pilot study involving nine women aged 18 to 40 years, vaginal colonization by L crispatus CTV-05 was evaluated at 1 to 3 days and 6 to 8 days following insertion of one capsule intravaginally twice daily for three days. 7L crispatus CTV-05 was detected in five of nine women at the first follow-up and six of nine women at the second follow-up.

The pig-tailed macaque (Macaca nemestrina) was chosen as the model for testing the use of the L crispatus capsule as a topical microbicide, because this macaque has been extensively utilized as a model for human vaginal physiology and sexually transmitted infections (STIs). 8-11 While new vaginal products are usually evaluated for toxicity in the rabbit vaginal irritation model, this model has limited relevance for testing of Lactobacillus species. Rabbit vaginal flora, pH, and anatomy are markedly different from those of humans. 12,13 The vaginal ecosystem of the pig-tailed macaque has been shown to be quite similar to that of humans. 8 The objective of the study was to determine if intravaginal application of capsules containing 108L crispatus significantly altered the epithelial tissues when applied as a topical microbicide. Here we report that the L crispatus CTV-05 strain can colonize the vagina of the pig-tailed macaque, as detected 48 hours after application.

Methods and Materials

Experimental Animals

Ten breeding-age female Macaca nemestrina were obtained from colony animals at the Washington National Primate Research Center for assignment to this study. Prior approval for use of monkeys in this protocol was obtained from the Institutional Animal Care and Use Committee at the University of Washington. Animals were housed and cared for under conditions that meet NIH standards as stated in the Guide for the Care and Use of Laboratory Animals.

Experimental Design

The effects of a single L crispatus gelatin capsule, containing 108 colony forming units of desiccated L crispatus in a maltodextrin matrix, were assessed on vaginal microflora and epithelial tissues in the pig-tailed macaque model. The capsules were manufactured by Gynelogix, Inc. (Louisville, CO) and stored at 4 °C until their use. The animals were sedated with ketamine hydrochloride and atropine. A colposcopic examination was performed and swab specimens were collected for quantitative microbiology and pH assessment before insertion of the capsule in the vaginal fornix. At 4 hours post-insertion, colposcopy was performed. At 24 and 48 hours post-insertion, follow-up colposcopy and swab collection for microbiology and pH assessments were performed.

Colposcopy

Colposcopy was performed under general sedation to assess and document inflammatory reactions of the vagina and cervix before and after challenge with the test product. Vaginal wall and ectocervix were evaluated by visual inspection through a colposcope. Examinations were conducted at baseline and at 4, 24, and 48 hours after L crispatus capsule application. All examinations were conducted by the same observer, and the vaginal epithelium and cervical mucosa were evaluated as described in the Manual for the Standardization of Colposcopy for the Evaluation of Vaginally Administered Products, published by the World Health Organization in 1995.

Vaginal pH

Vaginal pH was measured by swabbing the vaginal wall, proximal to the fornix, with a polyester-tipped swab and transferring the content to 0.5 graduated pH indicator strips (Baker-pHIX, a division of Mallinckrodt Baker, Inc., Phillipsburg, NJ). Readings were recorded while the indicator strips were still moist, and color formation was stabilized.

Microbiology

Vaginal cultures for microbiologic assessment were obtained with polyester-tipped swabs and immediately placed into Port-a-Cul anaerobic transport tubes (Becton-Dickinson, Cockeysville, MD). These were packaged for express-mail transport to the infectious disease laboratory at Magee-Womens Research Institute (Pittsburgh, PA) within 24 hours of collection. This transport mechanism has been shown to preserve the viability of aerobic and anaerobic bacteria in vaginal specimens. 14

Lactobacilli were detected by culture on human blood bilayer Tween agar, Brucella agar supplemented with 5% sheep blood (PML Microbiologicals, Tualitan, OR), and Rogosa agar prepared on-site. Catalase-negative large gram-positive rods were presumptively identified as lactobacilli. After testing for H2O2 production on tetramethylbenzidine agar, 15 all isolates were stocked at -70°C in litmus milk for future DNA fingerprinting studies.

The stocked Lactobacillus isolates were fingerprinted with repetitive element sequence-based polymerase chain reaction (rep PCR) DNA fingerprinting as previously described. 7 The fingerprint pattern for the DNA of the L crispatus CTV-05 strain was used to identify Lactobacillus isolates recovered from the vaginal cultures of the monkeys having identity with the CTV-05, based on having five identical bands in the 1500 to 2400 base-pair range.

Results

Colposcopic Observations

All ten animals were evaluated by colposcopy before exposure to the capsule containing L crispatus. At day 0, before insertion of the capsule, no significant findings were observed. At 4 hours post-insertion, no tissue changes were observed. The capsule, however, had not completely dissolved at this time. At 24 and 48 hours after insertion, there was no evidence of inflammation or disruption of the cervical or vaginal epithelium. The capsule was completely dissolved at 24 hours.

Vaginal Microflora

L crispatus CTV 05 was detected in only 1 of 10 animals 24 hours after vaginal insertion and in 3 of 10 animals 48 hours after insertion (Table 1).

Table 1
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Before product use, 4 of 10 animals had detectable levels of H2O2-producing lactobacilli in vaginal specimens. On days 1 and 2 after product use, 7 animals were culture-positive for these organisms. Colonization by L crispatus CTV-05 occurred in two animals not colonized by H2O2-producing lactobacilli at baseline and in one animal that was already colonized by H2O2-producing lactobacilli at baseline. The geometric mean titer of detected H2O2-producing lactobacilli increased 10-fold by the final time point (day 2) in this study. The other constituents of vaginal flora assessed in this study remained largely unchanged, with the exception of H2O2-producing viridans streptococci, which increased in prevalence from five animals (before product use) to eight animals (day 2 after capsule insertion), although the mean titer did not significantly change (Table 1).

Vaginal pH

Vaginal pH decreased in two of three CTV-05-colonized animals, dropping from precapsule application measurement of 7 to 4.5 on days 1 and 2 after capsule insertion (Figure 1) in one animal and from 5.0 before capsule use to 4.5 on day 2, when vaginal colonization by CTV-05 was detected. This suggested that the L crispatus CTV-05 was metabolically active and producing lactic acid.

Fig. 1
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Discussion

Different products proposed to be used as vaginal microbicides have been under development for nearly a decade. 16 To date, the global need for microbicides continues to escalate, as does the HIV epidemic. 17 A topical microbicide could act as a chemical or biologic barrier to HIV infection.

The development of the L crispatus capsule as a natural, probiotic microbicide offers several advantages for vaginal health. It is likely that a product for use in establishing L crispatus as part of the vaginal microflora would be used for two to three days each month at established intervals or following antibiotic therapy, and not during sexual intercourse. Therefore, the probiotic microbicide approach is unique in that it is focused on promoting innate defenses of the vaginal ecosystem, 18 and its use would not be linked with sexual activity. Women may also choose other chemically based microbicides for use during intercourse, since they have long sought strategies for prevention of STIs both during and after intercourse. 19

In this study, we have shown that the application of a single L crispatus capsule resulted in colonization of the vagina in three of eight nonmenstruating pig-tailed macaques 48 hours after insertion of the capsule. In the pilot study of the L crispatus CTV-05 capsules, five of nine women were colonized 1 to 3 days after using the capsules twice daily for 3 days. The decreased frequency of colonization in the current study may be due to the fact that monkeys were given a single capsule, whereas women in the pilot study received six capsules intravaginally. L crispatus CTV-05 binds to red blood cells (Vallor and Hillier, unpublished observation). Therefore, it was not surprising that the two animals that were menstruating during the experimental time points were not colonized by L crispatus CTV-05.

Vaginal microbiology was not adversely affected by product use. In fact, the only discernable change in microorganism population was the increase in lactobacilli, paralleled by an increase in H2O2-producing viridans streptococci. This overall increase in H2O2-producing bacteria in the vaginal milieu is thought to be advantageous for maintaining healthy flora and protecting against genital infections. The maintenance of acidic vaginal pH and absence of colposcopic changes to cervicovaginal tissues further indicate that use of this L crispatus capsule as a topical microbicide is well tolerated after a single application in the pig-tailed macaque.