The Identification of Vaginal Lactobacillus Species and the Demographic and Microbiologic Characteristics of Women Colonized by These Species (original) (raw)

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Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh

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Pennsylvania

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Human Papillomavirus Research Group, University of Washington

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Seattle

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Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh

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Pennsylvania

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Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh

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Pittsburgh, Pennsylvania

Reprints or correspondence: Dr. Sharon L. Hillier, University of Pittsburgh, Dept. of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Hospital, 300 Halket St., Pittsburgh, PA 15213-3180 (slh6+@pitt.edu).

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03 August 1999

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01 December 1999

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May A. D. Antonio, Stephen E. Hawes, Sharon L. Hillier, The Identification of Vaginal Lactobacillus Species and the Demographic and Microbiologic Characteristics of Women Colonized by These Species, The Journal of Infectious Diseases, Volume 180, Issue 6, December 1999, Pages 1950–1956, https://doi.org/10.1086/315109
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Abstract

Lactobacillus acidophilus has been reported to be the predominant vaginal species. Vaginal lactobacilli isolated from 215 sexually active women were identified using whole-chromosomal DNA probes to 20 American Type Culture Collection Lactobacillus strains. Most women were colonized by L. crispatus (32%), followed by L. jensenii (23%), a previously undescribed species designated L. 1086V (15%), L. gasseri (5%), L. fermentum (0.3%), L. oris (0.3%), L. reuteri (0.3%), L. ruminis (0.3%), and L. vaginalis (0.3%). H2O2 was produced by 95% of L. crispatus and 94% of L. jensenii isolates, compared with only 9% of L. 1086V. Colonization by L. crispatus or L. jensenii was positively associated with being white (P<.001),age ⩾20 years (P = .05), barrier contraceptive usage (P = .008), and lower frequency of bacterial vaginosis (P<.001) and gonorrhea (P = .03). L. crispatus and L. jensenii, not L. acidophilus, are the most common species of vaginal lactobacilli.

Establishing the identity of Lactobacillus species colonizing the vagina of women is of importance, because clinical studies have demonstrated an association between the presence of H2O2-producing strains of Lactobacillus and a decreased prevalence of gonorrhea, bacterial vaginosis (BV) [1], and human immunodeficiency virus (HIV) infection [2–5]. However, in these studies lactobacilli have usually been identified only to the genus level because of the technical difficulties in the species identification of Lactobacillus.

The identity of the predominant Lactobacillus species colonizing the vagina has been uncertain because of the unreliability of classic identification methods, which employ sugar fermentation and other phenotypic assays. From these methods, various lists of vaginal Lactobacillus species have been developed, including any of the following: L. acidophilus, L. fermentum, L. plantarum, L. brevis, L. jensenii, L. casei, L. cellobiosus, L. leichmanii, L. delbrueckii, and L. salivarius [6–8]. Of all these species, L. acidophilus has been the vaginal lactobacillus most widely accepted to be predominant. However, the general use of L. acidophilus to identify human oral, intestinal, and vaginal Lactobacillus has been more a historic than a scientific designation because of the poor reliability of existing tests used to differentiate Lactobacillus species [9]. Even 80 years ago, there was uncertainty whether L. acidophilus characterized a group of related species or a single group of organisms that “undergoes transformation” [10].

Several investigators who were questioning the reliability and reproducibility of classic identification methods for Lactobacillus species sought more dependable identification protocols [11, 12]. On the basis of DNA homology studies, the taxonomy of lactobacilli has been under revision [12, 13]. Formerly, the species group of L. acidophilus comprised 6 DNA homology groups that could not be distinguished biochemically [12]. Two of these homology groups are now L. crispatus and L. gasseri. When DNA homology methods were used to evaluate the lactobacilli from a group of 27 asymptomatic women, Giorgi et al. [14] identified L. gasseri, L. jensenii, and L. crispatus, not L. acidophilus, as the predominant vaginal Lactobacillus species colonizing asymptomatic women.

The present study was undertaken to identify which species of lactobacilli were present in a cross-sectional sample of 302 women, using DNA homology to American Type Culture Collection (ATCC) strains of lactobacilli. H2O2 production and the species specificity of this characteristic were determined. The distribution of Lactobacillus species colonizing women was also assessed demographically and microbiologically.

Methods

In this study, 319 women visiting an adolescent medicine clinic and 2 sexually transmitted disease clinics in Seattle were enrolled. A standardized questionnaire concerning demographic characteristics and contraceptive history was administered. Two vaginal swabs were used to obtain samplings from the lateral vaginal wall. One swab was rolled onto a slide for a vaginal smear, and the other swab for culture was placed into an Amies transport medium (MML Diagnostics, Troutdale, OR). The slide and the transport medium were delivered to the research laboratory within 12 h. Calcium alginate swabs or Dacron swabs were inserted into the cervix to obtain material for gonococcal and chlamydial cultures. Seventeen women were excluded because samples were not obtained for Gram staining or culture, leaving 302 evaluable subjects.

Specimens for Chlamydia trachomatis cultures were transported to the laboratory in 0.2 mL of sucrose-phosphate containing 2% fetal calf serum, 100,000 U of penicillin, 1 mg/mL of gentamicin, 25 mg/mL of vancomycin, and 25 U/mL of nystatin. C. trachomatis was cultured both in vials and in 96-well microtiter plates of cycloheximide-treated McCoy cells. The cell layers were stained with fluorescein-conjugated monoclonal antibodies to a species-specific C. trachomatis antigen and were examined for inclusions at 48–72 h with an epifluorescence microscope [15].

Kellogg's medium and either modified Thayer-Martin medium or enriched chocolate agar were streaked for the isolation of Neisseria gonorrhoeae. Gonococci were identified by sugar utilization, oxidase tests, and examination of Gram-stained preparations [16].

Slides were Gram stained and evaluated by use of the Nugent criteria [17]. A score of 0–10 was assigned in light of the relative proportions of large gram-positive rods (lactobacilli), small gram-negative or gram-variable rods (Bacteroides, Prevotella, or Gardnerella species), and curved gram-variable rods (Mobiluncus species). A score of 0–3 was interpreted as consistent with normal flora, a score of 4–6 was considered consistent with intermediately disturbed flora, and a score of 7–10 was considered consistent with BV [17].

Vaginal swabs were removed from the transport medium and used to inoculate Rogosa agar (Difco, Detroit), Columbia 5% sheep blood agar, prereduced brucella agar with 5% sheep blood, vitamin K, hemin, prereduced laked-blood kanamycin agar, and 2 human blood bilayer Tween (HBT) agar plates (Prepared Media Laboratories, Tualitin, OR). The Columbia agar and 1 HBT plate were incubated at 36°C in 5%–7% CO for a minimum of 48 h. The remaining plates were incubated within an anaerobic glove box at 36°C for a minimum of 5 days. An A7B agar plate and broths for isolation of Ureaplasma urealyticum and Mycoplasma hominis [18], all prepared in-house, were also inoculated and incubated at 36°C in 5%–7% CO2 for a minimum of 72 h. The broths were further subcultured onto A7B agar.

Aerobic bacteria were first identified by use of Gram stain and colony morphologies and catalase, and they were further identified by use of gas chromatography and biochemical tests [19]. Anaerobic gram-negative rods were identified by use of Gram stain and colony morphologies, pigment production on HBT, and their inability to grow aerobically [19]. The mycoplasmas were identified by their characteristic morphology on the A7B agar plate [18].

Lactobacilli were identified to the genus level by Gram stain and colony morphologies, negative catalase test, and production of predominant lactic acid peak as assessed by gas chromatographic analysis [20]. All lactobacilli were tested for the production of H2O2 in a qualitative assay on a tetramethylbenzidine (TMB) agar plate [21]. After 48 h of incubation in an anaerobic glove box at 36°C–37°C, the agar plates were exposed to ambient air. The H2O2 that was formed reacted with the horseradish peroxidase in the agar to oxidize the TMB, causing the colonies of lactobacilli to turn blue. Lactobacillus isolates were stored at −70°C in litmus milk until they were transferred to the Infectious Disease Laboratory at the Magee-Womens Research Institute for DNA studies.

For the DNA studies, each Lactobacillus isolate was grown in PYTSG broth (PY basal medium [20], 1% [wt/vol] dextrose, 1% [wt/vol] soluble starch, and 0.02% [vol/vol] Tween 80) incubated for 24–48 h in 6% CO2 at 37°C. Growth in broth was checked for purity by plating a drop onto a Columbia 5% sheep blood agar plate (Prepared Media Laboratories) and incubating as described above. The following DNA isolation procedure was modified from Luchansky et al. [22]. The cells were washed in TES buffer (50 m_M_ NaCI, 5 m_M_ EDTA, 30 m_M_ Tris, pH 8.0). The pellet was resuspended in lysis buffer (25% ultrapure sucrose, 50 m_M_ Tris, 1 m_M_ EDTA, pH 8.0) containing lysozyme and incubated at 37°C for at least 1 h. About 35 U of RNase A (Sigma, St. Louis) was added to the solution, which was then incubated at 60°C for 30 min. Sixteen units of a nonspecific protease type XIV from Streptomyces griseus (Sigma) was added and was incubated at 37°C for at least 1 h. After the addition of 1 mL of 0.25 M EDTA, pH 8.0, the solution sat in ambient temperature for 5 min. A volume of 0.4 mL of 20% SDS was added and then incubated at 60°C for 1 h. The solution was further incubated at 60°C for 30 min, with the addition of 10 U of proteinase K (Sigma). The lysate was extracted once with an equal volume of buffered phenol and once with an equal volume of chloroform. A double volume of 95% ethanol was added to precipitate the DNA. The DNA pellet was dissolved in a nominal amount of TE (10 m_M_ Tris, 1 m_M_ EDTA, pH 8.0) and stored at 4°C. DNA concentration was determined using absorbance readings at a wavelength of 260 nm. Gel electrophoresis and ethidium bromide staining were done to evidence genomic DNA isolation.

The Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis) was used to make whole-chromosomal probes according to the manufacturer's protocol. The specificity of this method has been demonstrated for identification of Mobiluncus [23] and Campylobacter [24] species. For each whole-chromosomal probe, a 20-µL reaction volume (consisting of 20 µ_M_ each dCTP, dGTP, and dTTP; 2 µL of a reaction mixture containing random hexanucleotide primers; 25 ng denatured target genomic DNA; 2 U of Klenow enzyme; and ∼50 µCi [α32P] dATP [NEN Life Science Products, Boston]) was incubated at 37°C for at least 30 min. The addition of 2 µL of 0.2 M EDTA stopped the reaction. Whole-chromosomal probes were made from 20 ATCC Lactobacillus strains: L. acidophilus 521 and 4356, L. brevis 11577, L. buchneri 11579, L. casei subspecies casei 4646, L. catenaformis 25536, L. confusus 10811, L. crispatus 33197, L. delbrueckii subspecies delbrueckii 9649, L. delbrueckii subspecies bulgaricus 11842, L. fermentum 23271, L. gasseri 4963, L. jensenii 25258, L. minutus 33267, L. oris 49062, L. parabuchneri 49374, L. rhamnosus 21052, L. ruminis 25644, L. salivarius subspecies salivarius 11741, and L. vaginalis 49540. A probe was also made to a previously unidentified species of Lactobacillus derived from a vaginal specimen, designated 1086V The estimated specific activity of each probe was 2 × 109 disintegrations/min/µg.

All DNA from patient isolates and ATCC Lactobacillus strains were slot-blotted onto nylon membranes of a Minifold II slot blotter (Schleicher & Schuell, Keene, OH) according to the manufacturer's instructions. In brief, to a 400-µL total volume of TE (pH 7.0) containing 5 µg of DNA, a 1 : 10 volume of 3 N NaOH was added. The sample was incubated at 60°C for 45 min. After the sample was cooled to ambient temperature, an equal volume of 1 M ammonium acetate was added. The control filters consisted of DNA from the ATCC strains listed above and from ATCC strains L. acidophilus 4357, L. brevis 14869, L. buchneri 4005, L. casei subspecies casei 393, L. delbrueckii subspecies lactis 4797 and 12315, L. fermentum 11739 and 14931, L. gasseri 9857, L. johnsonii 33200, L. minutus 33267, L. paracasei subspecies paracasei 27216, L. plantarum 14917, L. rhamnosus 21052, L. ruminis 25644, and L. salivarius subspecies salicinius 11742. The control filters were included with each whole-chromosomal probe hybridization. About 100 unknown isolates were tested against probes to all of the control strains. The specificity of the probe technique was excellent, with none of the unknown isolates having homology to >1 of the control species.

The wells of the slot blotter are arranged in a 3 × 24 fashion, allowing 24 samples to be vertically arranged with 3 wells per sample. The slot blotter was connected to a vacuum, and during vacuuming, each sample was aliquoted into 3 wells. About 1.5 µg of sample DNA was aliquoted into each slot. After vacuuming was complete, the slot blotter was disassembled. Before removing the membrane from the slot blotter template piece, we marked the width of each well on the backside of the membrane, using a black permanent ink marker. Each 24-sample membrane was given a number and baked for 2 h at 80°C. After being baked, each membrane was cut into 3 strips and then cut again in half, dividing the wells in 2, resulting in 6 strips, each with an identical sequence of samples and filter number.

For each probe, sample and control strips were laid flat, not overlapping one another, and placed in an 8 × 12 inch heat-sealable pouch (Kapak, Minneapolis). Prehybridization solution [25] was added to equilibrate the nylon membranes. The heat-sealed pouch was incubated at 42°C in a rocking incubator for at least 1 h before the addition of whole-chromosomal probes. A corner of the pouch was cut open, and a 20-µL volume of random primed—labeled whole-chromosomal probe was added to the equilibrated membranes.

After the addition of a probe, the membrane was incubated overnight at 42°C. Three 45-min washes in 0.5 × standard saline citrate (0.15 _M_NaCl, 0.015 M sodium citrate) and 0.1% SDS were done at 65°C. If more than the intended species turned positive on the control filter of ATCC Lactobacillus strains, higher-stringency washes were performed. A dark band on the autoradiograph was read as positive if its intensity was similar to or greater than that of the control positive band. Two individuals analyzed the autoradiographs; one was blinded to the identity of all unknown samples and control strains. The “blinded” individual did not work with the processing of DNA samples onto the nylon membranes. The location of a sample, unknown or control, was not revealed to this individual. Agreement between the 2 readers was 100%, suggesting that visual interpretation of a positive hybridization was not subjective. Examples of 2 autoradiographs used in the DNA hybridization to L. crispatus and L. gasseri whole-chromosomal probes are shown in figure 1.

Figure 1

Identical sample portions of slot blot hybridization autoradiographs, using whole-chromosomal probes made from American Type Culture Collection (ATCC) Lactobacillus crispatus strain 33197 (A) and ATCC L. gasseri strain 4963 (B). Same samples were used and arranged in a similar fashion for each autoradiograph shown. Columns a–c include DNA samples of unknown vaginal lactobacilli. Column d consists of DNA samples of ATCC Lactobacillus strains: L. crispatus 33197 (d1), L. vaginalis 49540 (d2), L. plantarum 14917 (d3), L. catenaformis 25536 (d4), L. buchneri 11579 (d5), L. buchneri 4005 (d6), L. gasseri 4963 (d7), and L. gasseri 9857 (d8). Specificity of each whole-chromosomal probe was evidenced when DNA samples of ATCC strains (identical to probe species) were positive. In A, L. crispatus ATCC 33197 whole-chromosomal probe hybridized only to L. crispatus ATCC 33197 sample DNA (d1) on control filter. Clinical vaginal lactobacilli homologous to probe were identified at a1–a4, a6, a7, b4, b5, b7, b8, c6, and c7. In B, L. gasseri ATCC 4963 whole-chromosomal probe hybridized only to DNA of ATCC L. gasseri strains (d7, d8) on control filter. One unknown vaginal Lactobacillus isolate was homologous to probe (c4).

Pearson χ2 tests were utilized to compare discrete variables with respect to the presence of L. crispatus or L. jensenii. P values ⩽.05 were considered statistically significant.

Results

Lactobacilli were recovered from 215 (71%) of the 302 women (table 1). About one-third of the women were colonized by L. crispatus, and one-fourth were colonized by L. jensenii. A Lactobacillus strain we designated L. 1086V colonized 15% of the women. L. gasseri was recovered from only 5% of the women, and only 1 woman each was colonized by L. ruminis, L. reuteri, L. fermentum, L. oris, or L. vaginalis. The following species were not recovered from the vagina of any of the women in this group: L. acidophilus, L. brevis, L. buchneri, L. casei, L. catenaformis, L. confusus, L. delbrueckii, L. parabuchneri, L. rhamnosus, and L. salivarius.

Table 1

Lactobacillus species detected among 302 women with or without bacterial vaginosis (BV), and H2O2 production by the lactobacilli.

At least 2 different Lactobacillus species simultaneously colonized 25 of the women. Both L. crispatus and L. jensenii colonized 11 women, including 1 who was also colonized by L. 1086V. Both L. crispatus and L. 1086V were coisolated from 4 women, and both L. jensenii and L. 1086V were coisolated from 6 women. Other isolated pairs included L. jensenii and L. gasseri, L. crispatus and L. fermentum, L. 1086V and L. gasseri, and L. jensenii with an isolate not homologous to any of the probes used.

Nearly all of the L. crispatus and L. jensenii strains produced H2O2 (95% and 94%, respectively; table 1). Only 14 L. gasseri strains were identified, and 71% of them produced H2O2. In contrast, only 9% of L. 1086V isolates produced H2O2.

The P values in tables 2 and 3 were obtained by comparing women with L. crispatus or L. jensenii with women not colonized by either species, including women lacking lactobacilli. This comparison was chosen because nearly all of the L. crispatus and L. jensenii strains produced H2O2. Either L. crispatus or L. jensenii colonized a total of 154 women (51%). There were 61 lactobacilli-colonized women (20%) who did not have either species. In some instances, data were not available for some of the women, resulting in a different denominator.

Table 2

Demographic characteristics and birth control usage of women colonized or not colonized by Lactobacillus species, stratified by species of lactobacilli.

Table 3

Microbiologic characteristics of women colonized or not colonized by lactobacilli.

Demographic characteristics and birth control usage stratified by species of lactobacilli are shown in table 2. Marital status, oral contraception, or douching did not appear to have any significant effect on the colonization of L. crispatus or L. jensenii. However, women with L. crispatus or L. jensenii were less likely to be <20 years old (37% vs. 49%; P = .05) and more likely to be white (72% vs. 50%; P ⩽ .001) and users of barrier contraceptives (41% vs. 27%; P = .008).

The microbiologic findings for the 302 women are shown in table 3. Of the sexually transmitted pathogens, N. gonorrhoeae and C. trachomatis, only N. gonorrhoeae infection was significantly decreased in women colonized by L. crispatus or L. jensenii (1% vs. 5%; P = .03). Women with L. crispatus or L. jensenii were also less likely to have BV (9% vs. 69%; P ⩽.001) and to have decreased colonization of BV-related species: Gardnerella vaginalis (38% vs. 84%; P ⩽ .001); anaerobic non-pigmented gram-negative rods (38% vs. 77%; P ⩽ .001); anaerobic black-pigmented gram-negative rods (11% vs. 33%; P ⩽ .001); and Mycoplasma hominis (22% vs. 61%; P ⩽ .001).

Except for Escherichia coli, there was no significant decrease in colonization of other vaginal bacterial species, such as Ureaplasma urealyticum, Group B Streptococcus and Enterococcus, and Staphylococcus species. E. coli was less likely to be recovered from women with L. crispatus or L. jensenii (15% vs. 27%; P =.01).

Discussion

L. crispatus and L. jensenii are the predominant vaginal Lactobacillus species colonizing women of reproductive age. L. acidophilus was not recovered from any of the women in this study. This study confirms the DNA homology study of Giorgi et al. [14], who did not identify L. acidophilus in a group of 27 asymptomatic women but did find strains homologous to L. gasseri, L. jensenii, and L. crispatus. In light of the differences in species identification by use of phenotypic and DNA-based methods, we encourage the use of genomic-based methods for identifying lactobacilli to the species level. The identification of L. crispatus and L. jensenii allows us to focus on the potential benefits of these species in the vagina.

To our knowledge, this is the first published report to support the species specificity of H2O2 production by lactobacilli. Most clinical isolates of L. crispatus and L. jensenii produce H2O2, compared with only a few isolates of L. 1086V. McGroarty et al. [26] and Eschenbach et al. [21] both reported that all of their identified L. jensenii isolates were H2O2 producers. Nagy et al. [8], however, reported H2O2 production by only 56% of L. jensenii strains. Although some Lactobacillus species may be biochemically identifiable, the discrepancy of the latter result may be due to the unreliability and irreproducibility of the classic methods of identification between laboratories. In addition, the percentage of L. acidophilus isolates producing H2O2 was reported to be 77% by McGroarty et al. [26], 43% by Eschenbach et al. [21], and 71% by Nagy et al. [8]. This disparity may result from the inability of biochemical assays to differentiate between the species formerly belonging to the L. acidophilus group.

In this study, an increased frequency of L. crispatus or L. jensenii was significantly linked to a lower prevalence of BV. L. crispatus or L. jensenii colonized three-fourths of women without BV, whereas only 12% of women with BV were colonized by either species (table 3). Similarly, Eschenbach et al. [21] reported that L. acidophilus or L. jensenii colonized 64% of 28 uninfected women and only 7% of 67 BV-positive women. This differs from the study by Nagy et al. [8], which suggested that there were no differences in the Lactobacillus species distribution between asymptomatic women without BV and women with BV.

The clinical relevance of H2O2-producing isolates of lactobacilli was suggested by Hawes et al. [1] in a longitudinal study of nonpregnant women. After an adjustment was made for douching and having multiple sex partners, it was shown that nonpregnant women lacking H2O2-producing vaginal lactobacilli were twice as likely to develop BV than were women colonized by H2O2-producing lactobacilli. As the primary species of lactobacilli that colonize the vagina and produce H2O2, L. crispatus and L. jensenii may protect a woman from developing BV by inhibiting the growth of BV-related microorganisms. G. vaginalis, anaerobic gram-negative rods and M. hominis were less frequently recovered from women colonized by these H2O2-producing lactobacilli, compared with women not colonized by these species. Klebanoff et al. [27] demonstrated in vitro that H2O2 produced by vaginal lactobacilli was the source of the cidal activity against G vaginalis and Prevotella bivia (formerly Bacteroides bivius). This activity was observed either in the presence of a halide-peroxidase complex or alone.

An association between H2O2-producing lactobacilli and decreased diagnosis of BV also has been reported in pregnant women [28]. This is noteworthy, since BV has been linked to pregnancy complications, such as preterm birth [29] and histologic chorioamnionitis [30]. The vaginal colonization of L. crispatus or L. jensenii during pregnancy may offer protection against these complications. Concentrations of ⩾ 107 cfu of vaginal lactobacilli per gram of vaginal fluid have been linked to a decreased incidence of preterm delivery [31].

We do not know whether L. crispatus and L. jensenii complement one another. However, it is beneficial to know that none of the women colonized by both of these species were diagnosed with BV. In this study, 8% of the women were colonized by >1 Lactobacillus species. The L. crispatus and L. jensenii combination accounted for nearly half of the Lactobacillus combinations found. In addition, Lactobacillus 1086V was coisolated with L. crispatus or L. jensenii in 40% of the women colonized by a species combination. This prevalence is unexpected, contradicting the claim that no 2 women are colonized by the same Lactobacillus species combination [32].

There were significantly fewer diagnoses of gonorrheal infection in women colonized by L. crispatus or L. jensenii, compared with women not colonized by either species or lacking lactobacilli. Previously, Saigh et al. [33], who were studying the inhibition of N. gonorrhoeae by lactobacilli, reported that the presence of inhibitory lactobacilli may be protective for women with gonorrhea-infected partners, since fewer of these women subsequently developed gonorrhea [33]. Unfortunately, it is unknown whether the presence of vaginal lactobacilli benefits a woman with a gonorrhea-infected partner by decreasing the efficiency of transmission. Hawes et al. [1] found that women colonized by H2O2-producing lactobacilli were less frequently infected by N. gonorrhoeae than were women lacking H2O2-producing lactobacilli. Zheng et al. [34] discovered that production of lactic acid and a catalase inhibitor by lactobacilli inhibits N. gonorrhoeae growth. It is unknown whether the production of catalase inhibitor is universal among lactobacilli.

The absence of lactobacilli, as determined by Gram stain, is positively associated with an increased prevalence of HIV [2–5]. Some authors have suggested that treatment of BV may reduce HIV transmission, because women with abnormal Gram stain scores had an increased risk of HIV, compared with women with predominant vaginal lactobacilli [3]. One recent study of women in Zimbabwe reported an increased rate of HIV seroconversion among pregnant women with BV [35], compared with those without clinical evidence of BV. Since women colonized by L. crispatus and L. jensenii are less likely to have BV, further studies are needed to evaluate whether these 2 Lactobacillus species may be protective against HIV. Klebanoff et al. [36] implicated H2O2, either alone or in combination with a halide and a peroxidase, in the cidal activity against HIV. They suggested that women lacking H2O2-producing lactobacilli may be at greater risk of being infected with HIV.

The identification of L. crispatus and L. jensenii as the predominant vaginal lactobacilli narrows the list of potential probiotic strains and focuses research to either species or to both. These 2 species may offer protection against some vaginal infections, such as BV and gonorrhea. These species inherently meet 2 requirements for use in successful probiotic therapy: They are endogenous vaginal lactobacilli, and they adhere to vaginal epithelial cells [37]. In addition, these 2 species are known to produce H2O2, a well-accepted mode of host defense. Vaginal colonization of women with these species may be advantageous in the maintenance of a normal microflora and the prevention of sexually transmitted diseases. Randomized, controlled trials will be needed to test this hypothesis.

Acknowledgments

We thank Alan G. Barbour, Staffan Normark, Hans Wolf-Watz, Jana Jass, and Dominic McCafferty for critically reading the manuscript; Kjell Grankvist for evaluating the hematological results; and Elisabeth Dahlberg, Birgitta Ekblom, and Sigrid Kilter for their skilful technical assistance.

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Presented in part: International Society for Sexually Transmitted Disease Research Meeting, New Orleans, August 1995 (abstract 207).

Informed consent was obtained from each woman in a protocol approved by the Human Subjects Committee at the University of Washington. In the conduct of clinical research, human experimentation guidelines of the US Department of Health and Human Services were followed.

Grant support: NIH (AI-31448, AI-38513).

© 1999 by the Infectious Diseases Society of America

Topic:

• chromosomes
• demography
• hydrogen peroxide
• lactobacillus
• vagina
• microbial colonization

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