Antimicrobial components of vaginal fluid

Erika V. Valore, MS, Christina H. Park, BS, Sorina L. Igreti, BS, and Tomas Ganz, PhD, MD Los Angeles, Calif
OBJECTIVE: We examined the antimicrobial activity and composition of vaginal fluid. STUDY DESIGN: Vaginal fluid from preweighed tampons was assayed for pH, lactic acid, and antimicrobial polypeptides. The fluid was also fractionated by molecular filtration. Antimicrobial activity of whole fluid was determined against representative resident and exogenous microbes, and its fractions were tested against Escherichia coli. RESULTS: Vaginal fluids (5/5 donors) were permissive for Lactobacillus crispatus and vaginalis and Candida albicans, but not for Escherichia coli, Streptococcus group B, and Lactobacillus jensenii in three of five donors. The antimicrobial activity against E coli was predominantly in a <3-kd fraction and correlated with both low pH and high lactic acid content. Compared with a matched pH buffer, lactic acid markedly suppressed the growth of E coli. Concentrated 2- or 5-fold, the protein-rich fraction was active against E coli. CONCLUSION: Vaginal fluid exerts selective antimicrobial activity against nonresident bacterial species. The activity is mediated by lactic acid, low pH, and antimicrobial polypeptides. (Am J Obstet Gynecol 2002;187:561-8.)

Key words: Lactic acid, antimicrobial polypeptides, vaginal microflora

The human vagina supports certain commensal microbes but, at the same time, resists colonization by exogenous microbes and presents a barrier to microbial entry into deeper tissues. The vaginal surface is lined by a moist noncornified stratified squamous epithelium and is kept moist by a fluid that is, in part, secreted as a plasma transudate through the vaginal wall,1,2 with additional contributions from the cervical and vestibular glands. The volume of the fluid in the sexually unstimulated vagina3 has been reported to be between 1 and 4 mL. Several mechanisms of vaginal defense against colonization by exogenous microbes have been proposed. The squamous epithelial layer continuously sloughs off, removing attached microbes. The commensal microbes, among which lactobacilli normally predominate, compete with exogenous microbes by using nutrients, establishing and maintaining a low pH,2 and generating antimicrobial bacteriocins, peroxides, and organic acids. The presence of epithelia-derived antimicrobial proteins in the vaginal fluid was also noted.4 However, detailed analysis of its antimicrobial components has not been re-

ported. Antimicrobial polypeptides that are abundant in other epithelial fluids5 include lysozyme, lactoferrin, secretory leukoprotease inhibitor (SLPI), calprotectin, human -defensins human neutrophil peptides 1 through 3 (HNP1-3) that are released from the neutrophils, and the -defensins human -defensins 1 and 2 (HBD-1, HBD-2) from epithelial cells. The objective of this study was to identify antimicrobial polypeptides and other antimicrobial components that are present in vaginal fluid, to determine whether these components are regulated during the menstrual cycle, and to determine the effect of vaginal fluid on the growth of selected resident and exogenous microorganisms. Material and methods Sample collection. The study protocol was approved by the UCLA institutional review board. Preweighed tampons (rayon fiber with cotton cord; Tampax, Proctor and Gamble, Cincinnati, Ohio) were inserted in the vagina for 8 to 10 hours and then were reweighed to determine the amount of fluid that had collected. The tampons were processed on the day of collection or, in some cases, stored at 20C until extraction. Freezing did not appear to affect the integrity and recoverability of the proteins that were present in the sample, as determined by protein profiles that were observed after separation by acid urea polyacrylamide gel electrophoresis (AU-PAGE) before and after freezing (data not shown). Vaginal fluid extraction. Fifty milliliters of extraction fluid was added to each tampon and incubated, with rotation at room temperature for 3 hours. The bulk of the extraction fluid was then transferred to a centrifuge tube; 561

From the Department of Medicine and Pathology, School of Medicine, University of California, Los Angeles. Supported by National Institutes of Health grants No. AI 37945 and AI46514. Received for publication November 15, 2001; revised February 26, 2002; accepted March 28, 2002. Reprint requests: Tomas Ganz, PhD, MD, UCLA Department of Medicine, CHS 37-055, Los Angeles, CA 90095-1690. E-mail: tganz@mednet.ucla.edu 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/125280 doi:10.1067/mob.2002.125280

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the fluid retained in the tampon was squeezed out into the same centrifuge tube by compression in a large syringe. This method recovered 96% of the extraction fluid, as determined by comparing the weight of a tampon prewetted with 1 mL of water (to mimic vaginal fluid volume) before and after extraction with 50 mL of water. Epithelial cells and tampon debris were removed by centrifugation, and the fluid was further processed for protein analysis or antimicrobial testing. The extraction fluid for protein analysis was 0.1% or 5% acetic acid. For antimicrobial and physiologic studies, the tampons were first extracted with water, followed by a second extraction with an equal volume of 0.1% acetic acid to obtain proteins that had not been extracted by the first water extraction. For studies of the activity of the protein fraction, the second extract was combined with the first extract. Cationic protein extraction from vaginal fluid. The extract was first neutralized to pH 7 with ammonium hydroxide. After neutralization, 1.5 mL of a 50% slurry of Macroprep CM resin (Bio-Rad, Hercules, Calif) in 25 mmol/L ammonium acetate pH 7.5 was added and incubated on a rotator for 3 to 4 hours at room temperature. The resin was allowed to settle, and the supernatant was decanted off. The resin was washed several times in ammonium acetate buffer, then proteins were eluted from the resin, with one resin volume equivalent of 10% acetic acid followed by two of the same volume elutions with 5% acetic acid. The eluates were pooled, lyophilized to dryness, and dissolved in 0.1% acetic acid. Processing for antimicrobial assays. The water extract was lyophilized and resuspended in sterile water to the original volume of vaginal fluid, as determined by weight (1 g = 1 mL). The pH was determined by a micro pH electrode (Corning, New York, NY) in a tube that contained a 100-L aliquot of water-extracted vaginal fluid. Vaginal fluid fractionation. The water and acid extracts were separately resuspended in water to the original volume by weight. A portion of the water extract (250 L) was centrifuged through a filter spin unit (Microcon 3K YM; Millipore Corporation, Bedford, Mass) that had been prewashed with water to remove membrane stabilizers. Fluid that passed through the membrane (the <3-kd fraction) contained no protein, as determined by silver stained AU-PAGE (Fig 1) and was used for the colonyforming unit (CFU) assay. To increase protein recovery from the tampon, 250 L of a second tampon extract (with acetic acid, as described earlier) was added to the >3-kd fraction and then subjected to centrifugation on the 3K membrane to remove the acid and residual small molecules. The high molecular weight fraction was then washed on the filter with equal volumes of water, followed by two washes with genital tract (GT) buffer (20 mmol/L potassium phosphate, 60 mmol/L sodium chloride, pH 4.5), which was designed to mimic the electrolyte composition and pH of vaginal fluid.1 The >3-kd fraction was re-

covered by the filter unit was turned over, and the fraction was centrifuged into a new tube (as per the manufacturers instructions) then brought to its original 250-L volume with pH 4.5 GT buffer and used for the CFU assay. CFU assay. Microbial suspensions (3 L in nutrient solution) were added to either vaginal fluid or GT buffer to a final volume of 30 L. The final nutrient concentration corresponded to either 0.01 trypticase soy broth (TSB) or 0.1 Lactobacillus MRS broth (Difco, Becton Dickinson, Sparks, Md). An aliquot was removed and immediately plated in triplicate on TSB plates (or MRS plates for Lactobacillus) to determine the initial microbe concentration (T = 0); the remaining mixture was incubated in an environmental shaker at 37C for the times indicated, under conditions that minimized evaporation and condensation. Test strains that represented the endogenous flora of the vagina6 included gram-positive Lactobacillus jensenii, L crispatus, and L vaginalis (ATCC No. 25258, No. 33197, and No. 49540, respectively) and the yeast Candida albicans. Exogenous bacteria were represented by the gram-negative Escherichia coli ML-35p and a gram-positive clinical isolate of -hemolysing Streptococcus group B. L crispatus was grown under anaerobic conditions with the use of a BBL GasPak pouch (Becton Dickinson), which generated an anaerobic carbon dioxideenriched environment. The other Lactobacillus species were grown under aerobic condition in MRS broth. Gel overlay assay. The gel overlay assay was accomplished as previously described.7 Briefly, 35 L of vaginal fluid was lyophilized, resuspended in loading buffer, and subjected to AU-PAGE. The gel was washed three times for 4 minutes each in 100 mL of 10 mmol/L sodium phosphate (pH 7.5) and then placed on a plate that contained bacteria which were embedded in agarose (4 105 bacteria/mL in 1% low electroendo osmosis (EEO) agarose [Sigma Chemical Company, St Louis, Mo], GT buffer, pH 4.5, 0.01 TSB) and incubated for 3 hours to allow diffusion of protein bands into the bacteria/agarose layer. The gel was then removed; nutrient agarose (2 TSB; 1% agarose) was poured over the top of the lawn, and the surviving bacteria were allowed to grow overnight. Areas that corresponded to antimicrobial peptides that were transferred from the AU-PAGE gel were visualized as zones of clearance in the microbial lawn. Lactate assay. Total lactate in the vaginal fluid samples was determined with a microtiter plate diagnostic kit (catalog No. 735; Sigma Chemical Company). The amount of lactic acid present in the vaginal fluid was calculated by the Henderson-Hasselbach formula: [HA] = [HA]0 10pK/(10pH + 10pK), where [HA]0 is the molar concentration of lactate plus lactic acid, [HA] is the molar concentration of lactic acid, and the pK of lactate is 3.83. Bacterial growth inhibition assay. The influence of pH and lactic acid on the growth of E coli ML-35p was assessed. Culture medium was prepared in the following manner:

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Table I. Lactate concentration and pH of vaginal fluid of the donor panel

Donor panel Set 1 1 2 3 4 5 Set 2 1 2 3 4 5 pH Total lactate (mmol/L) Calculated concentration of lactic acid (mmol/L) Log change in E coli CFU

5.3 4.2 4.9 4.3 4.3 5.5 4.2 5.4 4.7 4.4

0.7 36 11 21 41 0.7 37 33 27 18

0.02 10.8 0.9 5.3 10.4 0.01 11.1 0.9 3.2 3.8

0 3 0 3 3 +1 3 +1 0 3

Samples were collected during two different menstrual cycles (sets 1 and 2). Total lactate and pH were determined, and the lactic acid concentration was calculated. The corresponding antimicrobial activity is presented as a change in the log CFU of E coli after 3-hour incubation in vaginal fluid.

lactic acid was dissolved in GT (pH 6.5) buffer that contained 0.1 TSB to concentrations of 5, 10, 20, and 40 mmol/L, and the pH was adjusted with sodium hydroxide or hydrochloric acid, as needed, to attain the desired concentration of lactic acid (1, 2, 3, 5, and 7 mmol/L), as determined by the Henderson-Hasselbach formula. Culture medium (198 L) was placed in triplicate wells, and 2 L of 100 concentrated bacteria was washed in GT buffer, pH 4.5, and was added to each well with a calibrated pipette, to a final concentration of 2 106 bacteria/well. The optical density at 620 nm (OD620) of the plate was read on a Spectra Max enzyme immunoassay (EIA) reader (Molecular Devices, Sunnyvale, Calif) to determine the initial value. The plate was then incubated in an environmental shaker at 37C, and the OD620 was read at 3, 5, 7, and 20 hours to determine bacterial growth. The control medium contained GT buffer (20 mmol/L KHPO4, 60 mmol/L NaCl) with 0.1 TSB, adjusted to the same pH as its lactate-containing counterpart. Semiquantitative Western blot densitometry. The lyophilate of the acid extract from tampons was resuspended in loading buffer, analyzed by AU-PAGE, and transferred to Immobilon-P (Millipore Corporation), as previously described.8 Antibodies that were used for Western blot analysis were as follows: rabbit antiHBD-1, antiHBD-2, antiHD-5, and antiHNP-1 through 3 were generated in our laboratory8-11; rabbit anti-calprotectin (MRP8 and MRP10) was a gift from Dr Kenneth Miyasaki (UCLA Department of Dentistry), anti-lysozyme and anti-lactoferrin were purchased from DAKO (Glostrup, Denmark); antiSLPI was purchased from R&D Systems (Minneapolis, Minn); anti-LL-37 was obtained from Dr Robert Lehrer (UCLA Department of Medicine), and anti-histone H2B was purchased from Serotec (Washington, DC). Second antibody conjugated to alkaline phosphatase was purchased from Pierce (Rockford, Ill). Vaginal fluid Western blots were analyzed and compared with standard Western blots that contained known amounts of the appropriate

Table II. Antimicrobial polypeptides in vaginal fluid of the donor panel (n = 20; 5 donors, 4 samples each)
Protein Calprotectin Lysozyme Lactoferrin SLPI HBD-2 HNP1-3 HBD-1 Mean concentration SE (g/mL) 34 7 13 2 0.9 0.2 0.7 0.1 0.57 0.13 0.35 0.07 0.04 0.02

protein, with the use of a densitometer and the ImageQuant program (both from Molecular Dynamics Inc, Sunnyvale, Calif). Donor panel samples that were used for Western blot analysis consisted of 4 samples that were collected approximately 7 days apart during one menstrual cycle, with the exception of the histone and LL-37 Western blots, in which case a single sample that was collected approximately on day 20 was used from each donor. HBD-1 quantification by enzyme-linked immunosorbent assay. The 96-well Maxisorp plates (Nunc, Roskilde, Denmark) were coated with a 100-L volume of a 1:5000 dilution of HBD-1 mouse ascites fluid (IB5A; produced in collaboration with Dr Beverly A. Dale, University of Washington, Seattle, Wash) in coating buffer (BupH ready mix buffers; Pierce) and incubated overnight. All steps of the protocol were performed at room temperature; standards and samples were analyzed in triplicate and duplicate, respectively. Wells were washed (0.1% bovine serum albumin, 0.05% Tween 20, in phosphate-buffered saline solution), incubated with 150 L blocking buffer (1% bovine serum albumin in BupHphosphate-buffered saline solution, 0.01% CETAB, 0.01% thimerosal) for 1 hour; then samples in 100 L volume were added to the appropriate well and serially diluted in the wells of the plate and allowed to incubate for 1.5 hour with mixing. Plates were washed and incubated with 100 L rabbit

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Fig 1. The antimicrobial activity of vaginal fluid fractions against E coli ML35p. Vaginal fluid was fractionated into two parts by passage through a 3-kd molecular weight cutoff membrane. A silverstained AU-PAGE. A, 5 L vaginal fluid. B, 5 L of the <3-kd fraction. C, 5 L of the protein fraction (>3 kd). D, 0.3 g lysozyme. E, 0.5 g HBD-2. The antimicrobial activity of whole vaginal fluid (closed circles), protein-rich >3-kd fraction (closed inverted triangles), <3-kd fraction (open circles), and GT buffer control (open inverted triangles) from each donor is shown. Bacteria were added to fluid that was supplemented with 0.01 TSB for a nutrient source and incubated at 37C for 0, 1, 3, and 24 hours. Aliquots were plated onto TSB plates, and CFUs were determined after overnight incubation.

Fig 2. The antimicrobial activity of vaginal fluid. Microbes (L jensenii, L crispatus, L vaginalis, and C albicans, group B Streptococcus, and E coli ML-35p) were added to vaginal fluid and incubated at 37C. Aliquots were taken at 0, 1, 4, and 8 hours and were plated in triplicate on TSB agar plates; the CFUs were determined after overnight incubation. Samples included GT buffer control (closed circles), donor 1 (open circles), donor 2 (open squares), donor 3 (open triangles), donor 4 (open diamonds), donor 5 (open inverted triangles).

antiHBD-1 polyclonal antibody (diluted 1:2000 in blocking buffer) for 45 minutes, followed by washing and 1hour incubation in 100 L of a 1:2000 dilution of horseradish peroxidase that was conjugated to goat antirabbit antibody (Pierce). After being washed with water, the plate was developed with orrthophenylenediamine (OPD) solution (Sigma Chemical Company); the reaction was stopped with 2.5N hydrogen sulfate, and the OD620 was read on a spectrophotometer (Spectra-Max; Molecular Devices, Sunnyvale, Calif). Results Fluid collections. Vaginal fluid was collected from five healthy volunteers, who ranged in age from 25 to 44 years and had regular menstrual cycles of approximately 28 days. For the analysis of proteins during the menstrual cycle, the five donors collected samples on the following days: donor 1, days 6, 12, 17, and 24 of a 25-day cycle (respective weights: 1.1, 1.2, 1.6, and 0.9 g); donor 2, days 8, 12, 19, and 23 of a 27-day cycle (respective weights: 1.9, 1.5, 2.4, and 1.5 g); donor 3, days 7, 14, 20, and 26 of a 26day cycle (respective weights: 0.7, 1.2, 1.2, and 0.8 g); donor 4, days 7, 14, 21, and 25 of a 35-day cycle (respective weights: 1.2, 1.8, 1.2, and 1.7 g); and donor 5, days 9, 15, 19, and 25 of a 29-day cycle (respective weights: 2.0, 1.5, 1.3, and 1.6 g). For antimicrobial assays, single samples were collected at day 20 through 25 of the cycle. Antimicrobial activity of vaginal fluid. The vaginal fluid from all of the donors was permissive for the growth of L

crispatus, L vaginalis, and C albicans (Fig 2), organisms that are considered to be endogenous flora of the vagina.3 However, donors 2 and 5 exhibited strong antimicrobial activity against L jensenii (3 log decrease after 2 hours), donor 4 exhibited moderate activity (3 log decrease after 8 hours), and donor samples 1 and 3 allowed growth. The same three donors (2, 4, and 5) whose fluids killed L jensenii also exhibited strong/moderate killing capabilities against group B Streptococcus and E coli. Samples from donors 1 and 3 allowed moderate growth of all the organisms that were tested, similar to growth in the GT buffer control. Effects of high- and low-molecular-weight fractions. To determine whether the antimicrobial activity of vaginal fluid samples was due to small or large molecules, the fluid was separated into two fractions by passing through a 3-kd molecular weight cutoff membrane. The fraction that was retained by the membrane contained virtually all of the protein (as can be seen in the lower right panel of Fig 1 [lane C]), whereas the fraction that passed through the membrane contained no protein (as visualized by silver stained AU-PAGE [lane B]). Nearly all the protein in the vaginal fluid was recovered from the membrane (as can be seen by comparing lane A [whole fluid] to lane C [>3 kd protein fraction]). Interestingly, when a CFU assay was done in the whole and fractionated fluid, virtually all of the antimicrobial activity against E coli could be attributed to the low molecular weight fraction (Fig 1). For all but one of the donors, the protein-containing fraction was permissive for bacterial growth that was similar to the buffer control, with the exception of a sample from donor 2 that exerted a bacteriostatic effect on E coli.

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Fig 4. The antimicrobial effect of the protein-rich fraction of vaginal fluid. The vaginal fraction retained by a 3-kd molecular weight cutoff membrane was dissolved to 1, 2, and 5 original concentration in GT buffer (pH 4.5; 0.01 TSB). E coli ML35p was added to each sample; aliquots were taken at 0, 1, 3, and 24 hours and plated in triplicate on TSB agar plates, and CFUs were determined after overnight incubation. The symbols represent bacterial CFU in GT or protein-rich vaginal fluid fraction, concentrated as indicated 1-5 . Fig 3. The effects of pH and lactate/lactic acid on the growth of E coli. GT buffer (20 mmol/L potassium phosphate, 60 mmol/L sodium chloride) with 0.1 TSB that contained 5, 10, 20, or 40 mmol/L lactate was adjusted to the appropriate pH with sodium hydroxide or hydrochloric acid so that the calculated amount of lactic acid form was 1, 2, 3, 5, or 7 mmol/L (closed circles). The controls at the matched pH contained no lactate (open circles). A volume of 198 L culture medium was placed in triplicate wells of a 96-well plate and 2 L bacteria (2 109 bacteria/mL) were added to each well, and the plate was placed in an environmental shaker at 37C. The bacterial concentration represented by the OD620 reading is shown at 0, 3, 5, 6, 7, and 20 hours.

The antibacterial effect of lactic acid. In previous studies, vaginal fluid was reported to contain a mixture of organic acids, among which lactic acid predominated.12,13 To analyze the role of lactic acid in antimicrobial activity, total lactate of the water-extracted vaginal fluid was determined, and the amount of lactic acid that was present in each sample was calculated from the measured pH. The pH of the samples ranged from 4.2 to 5.5, and the concentration of lactic acid ranged from 0.7 to 11.0 mmol/L (Table I). The corresponding antimicrobial activity against E coli is presented as a change in the CFU (expressed in log) from the inoculum after incubation in the vaginal fluid and shows a significant correlation between the lactic acid concentration and the log change of CFU (Pearson product moment correlation coefficient, R = 0.839; P = .002; n = 10 samples from 5 donors) and between the pH and the log change in CFUs (R = 0.941; P = .00005). To determine the separate effects of pH and lactic acid on the growth of E coli, we performed a growth inhibition assay. In each graph, bacterial growth in the lactate-con-

taining medium is compared with growth in lactate-free medium at the same pH (Fig 3). At a pH of 4.1 (regardless of lactic acid concentration), the growth of E coli was significantly inhibited. At >pH 4.1, there is a marked additional growth-suppressive effect of lactic acid concentrations of 3 mmol/L. At 1 and 2 mmol/L lactic acid, there was inhibition of growth at the early time points, with recovery and even enhanced growth at 20 hours likely to be due to the opposing effects of lactic acid (inhibition) and lactate (enhancement). Antimicrobial effects of the high molecular weight fraction. It is likely that the high molecular weight components (eg, proteins) of vaginal fluid are more concentrated on and in the epithelial surface than in the fluid layer. To determine whether antimicrobial activity increases with the concentration of the high molecular weight solutes, the protein-containing high molecular weight fraction (donor 5) was concentrated 2- and 5-fold and then tested in the CFU assay. The unconcentrated high molecular weight fraction (1) initially decreased the viability of bacteria (Fig 4), but by 24 hours the CFU rose to match the buffer control. The high molecular weight fraction that was concentrated 2- or 5-fold exerted a microbicidal effect throughout the assay. Gel overlay antimicrobial assay. To estimate whether one or more antimicrobial proteins in vaginal fluid exert predominant activity, a gel overlay assay was performed. As can be seen by the zones of clearance, vaginal fluid contains many proteins that kill E coli (Fig 5). Similar results were also seen for group B streptococci (data not shown). Protein composition of vaginal fluid. Proteins from samples that were collected throughout the menstrual cycle from five donors were analyzed by semiquantitative West-

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Comment In this study, the ability of vaginal fluid from five different donors to exert an antimicrobial effect on several different microbes was assessed. Of the endogenous vaginal microbes, two of the three lactobacillus species (L crispatus, L vaginalis) and C albicans were able to grow in vaginal fluids from all of the donors. In contrast, L jensenii, group B Streptococcus, and E coli were killed by three of the five samples. Most antimicrobial activity could be attributed to the low-molecular-weight (protein-free) fraction of vaginal fluid. However, it is possible that the antimicrobial activity of the polypeptide fraction may have been underestimated because of potential losses during tampon extraction or extract storage. Resident vaginal bacteria can produce hydrogen peroxide and convert host-derived nutrients into organic acids that include formic, succinic, acetic, propionic, butyric, and lactic acid.12 Hydrogen peroxide is known to be antimicrobial, and several studies have shown that women in whom colonies of hydrogen peroxide-producing lactobacilli have been found are much less prone to sexually transmitted diseases and bacterial vaginosis.16 However, hydrogen peroxide and several of the organic acids are volatile and would likely be removed or greatly reduced by the extraction procedure used in this study. In contrast, lactic acid, the predominant acid in normal vaginal fluid,12 is nonvolatile and is retained during the extraction procedure. In this study, we found that the vaginal fluid with the highest levels of antimicrobial activity also contained the highest levels of lactic acid (3.8-11 mmol/L; Table I). The permissive vaginal fluids contained 3.2 mmol/L lactic acid and had a higher pH than the more potently antimicrobial vaginal fluid samples. It has been widely assumed that lactic acid is an antimicrobial factor in vaginal secretions; however, to our knowledge, this is the first study to test this hypothesis. We performed a checkerboard study of lactate and pH effects on the growth of E coli and surmised that the acid form of total lactate is the major determinant of antimicrobial activity. As with vaginal fluids, in our model of lactic aciddependent antimicrobial activity, approximately 3 mmol/L lactic acid represented a transition zone below which we observed no lactic aciddependent antimicrobial activity and above which there was complete killing of E coli (Fig 3). This may explain the paradoxic difference in activity of 2 samples that were collected from donor 4, in which (despite an increase in total lactate from 21 to 27 mmol/L between the first and second samples) there was a pronounced decrease of antimicrobial activity. In this donor, a slight increase in pH of vaginal fluid (4.3 vs 4.7) resulted in a significant decrease in calculated lactic acid concentration (3.2 mmol/L in the second sample compared with 5.3 mmol/L in the first sample), which straddled the transition zone between killing and no

Fig 5. The antimicrobial polypeptide fingerprint of vaginal fluids. Vaginal fluids (donors 1-5) were analyzed by AU-PAGE and overlaid for 3 hours onto a plate of E coli ML-35p which were embedded in agarose. With further incubation of the plate, surviving bacteria formed microcolonies. The microcolonies were absent from the zones of clearance that indicate multiple active antimicrobial polypeptides in each sample. The corresponding pattern of AU-PAGE Coomassie bluestained polypeptides is shown in the lower panel. From top to bottom, lane M1 contains the human protein markers lactoferrin, HNP-1, and lysozyme, and lane M2 contains histone H2B and SLPI. Arrowheads indicate marker bands that exhibit antimicrobial activity in this assay.

ern blot for previously described antimicrobial components of epithelial secretions (calprotectin, lysozyme, SLPI, and defensins HNP1-3, and HBD-2) and by enzyme-linked immunosorbent assay for HBD-1. Overall, there were only minor changes in protein concentrations during the menstrual cycle (Fig 6), and the data were pooled therefore to calculate the mean concentration of each protein (n = 20; Table II). Calprotectin and lysozyme were present at concentrations of >10 g/mL; the other proteins were detected at concentrations of <1 g/mL. HD-5 was not detected in the vaginal fluid by this method (detection limit, 20 ng/mL). Subsequently, additional antimicrobial peptides were sought in donor panel samples that were collected in mid menstrual cycle. The human cathelicidin peptide LL-3714 was detected in the donor panel, ranging from 65 to 1000 ng/mL, with an average of 500 ng/mL (n = 5). Histone15 was detected in only one of the five vaginal fluid samples (donor 1) and was present at approximately 2 g/mL (detection limit, 1.3 g/mL).

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Fig 6. Antimicrobial polypeptide levels in vaginal fluid collected throughout the menstrual cycle of the donor panel: donor 1 (closed squares), donor 2 (open inverted triangles), donor 3 (open circles), donor 4 (closed circles), and donor 5 (closed inverted triangles). The solid line indicates the log-linear regression (log trend) of all of the points in each graph.

killing of E coli. Thus, in vaginal fluid, like in the model system, the acid form of total lactate may be a major contributor to antimicrobial activity. This study marks the first time antimicrobial polypeptides have been assessed in vaginal fluid and observed during the course of the menstrual cycle. Surprisingly, there was little change in polypeptide concentration during the cycle (Fig 6). Of the polypeptides that were detected in the vaginal fluid, calprotectin and lysozyme are present at concentrations previously found to be antimicrobial,17,18 although the other peptides and proteins could participate in synergistic activity.18 In addition to its role as an antimicrobial polypeptide, calprotectin has also been found to inhibit the adherence of exogenous microbes to cultured epithelial cells.19 SLPI, which inhibits serine proteases such as elastase and cathepsin G, also has been found to have antibacterial and antifungal capabilities.2022 Both HNP1-3 and HBD-2 concentrations increase in response to inflammatory stimuli, the former because of neutrophil influx and the latter by the induction of ep-

ithelial synthesis. Thus, the concentrations of these defensins would be expected to increase in response those infections that induce an inflammatory response. The gel overlay assay (Fig 5) shows that the proteins and peptides of the vaginal fluid are antimicrobial when placed directly on a lawn of bacteria. In addition, we found that the protein component of the vaginal fluid was able to exert a significant antimicrobial effect when concentrated 2- to 5-fold (Fig 4). This observation could be biologically significant because some of the proteins in the vaginal fluid are produced within the epithelium and are likely to be present at much higher levels at the surface and in the interstices of the epithelial cell layers. In addition, some studies have found that many of these antimicrobial peptides can act synergistically. Although Singh et al23 found only an additive effect between HBD-2 and lysozyme, they did find synergism with the following combinations: lysozyme/lactoferrin, lysozyme/SLPI, lactoferrin/SLPI, and lysozyme/LL-37. Thus, even though individually many of the proteins are present in vaginal fluid at concentra-

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tions below their active range, in or on the epithelium, and together they may constitute an effective barrier to microbial invasion of the epithelial surface. Histone and fragments of histone are cationic polypeptides with antimicrobial activity.15,24 In our study, histone was found in one vaginal fluid sample at a concentration of 2 g/mL. The limitations imposed by the available antibody enabled only a relatively high detection limit; it is possible that other vaginal fluid samples also contain antimicrobial concentrations of histone. Epithelial cells in normal vaginal fluid frequently appear lysed25 and may release nuclear histone into the vaginal fluid milieu. Therefore, the role of histones in vaginal fluid must be more carefully examined in future studies. A potentially important factor that was not considered in this study, because of our focus on innate immunity, was the role of lymphoid populations in the vagina and immunoglobulin A and G in the vaginal fluid. Although such antibodies are not directly microbicidal, they could inhibit the attachment of microbes to the epithelia. The likely contribution of antimicrobial products that originate from vaginal bacteria also was not specifically addressed in this study. In our study, vaginal fluid was collected on tampons that were inserted in the vagina for 8 to 10 hours then removed and weighed to determine the volume absorbed over this time period. This method has the advantage that quantitation is precise, and the fluid is sampled from most of the vaginal surface. Clearly, the sampled fluid is a mixture that represents both vaginal and cervical sources, but this mixture is physiologically relevant. We presented evidence that vaginal fluid is selectively antimicrobial and that lactic acid and, to a lesser extent antimicrobial peptides and proteins, contribute to the resistance of the normal vagina to colonization by exogenous microbes represented in our experiments by E coli. Antimicrobial polypeptides could have a greater role in vaginal host defense of women who have low concentrations of lactate and high vaginal pH. Future studies will be necessary to determine whether there are differences in the concentration of antimicrobial polypeptides in the vaginal fluid of women who are normal compared with women who have recurrent problems with abnormal levels of microbial colonization, such as bacterial vaginosis and candidiasis. We thank Dr Edith M. Porter for her critical review of this manuscript and her insightful scientific discussions and Dr Rose Linzmeier for her helpful contributions to this project.
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