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 α-defensins human neutrophil peptides 1 through 3 (HNP1-3) that are released from the neutrophils, and the β-defensins human β-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 –20°C 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: © 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/125280 doi:10.1067/mob.2002.125280

ActaMed® Library - This service is offered by an educational grant of AVENTIS PHARMA S.P.A.

Lactate assay.5 GT buffer and used for the CFU assay. pH 4.5 mL of a 50% slurry of Macroprep CM resin (Bio-Rad.1% acetic acid to obtain proteins that had not been extracted by the first water extraction. and the fraction was centrifuged into a new tube (as per the manufacturer’s instructions) then brought to its original 250-µL volume with pH 4. resuspended in loading buffer. pH 4. Bacterial growth inhibition assay. followed by a second extraction with an equal volume of 0. Fluid that passed through the membrane (the <3-kd fraction) contained no protein. The resin was allowed to settle. 1.1 The >3-kd fraction was re- covered by the filter unit was turned over.562 Valore et al September 2002 Am J Obstet Gynecol the fluid retained in the tampon was squeezed out into the same centrifuge tube by compression in a large syringe. followed by two washes with genital tract (GT) buffer (20 mmol/L potassium phosphate. Calif) in 25 mmol/L ammonium acetate pH 7. Mo]. lyophilized to dryness. 25258. A portion of the water extract (250 µL) was centrifuged through a filter spin unit (Microcon 3K YM. Millipore Corporation. Sparks. then proteins were eluted from the resin. Bedford. For antimicrobial and physiologic studies.This service is offered by an educational grant of AVENTIS PHARMA S. Exogenous bacteria were represented by the gram-negative Escherichia coli ML-35p and a gram-positive clinical isolate of β-hemolysing Streptococcus group B. The pH was determined by a micro pH electrode (Corning.01 TSB) and incubated for 3 hours to allow diffusion of protein bands into the bacteria/agarose layer. ϫ0. GT buffer. Mass) that had been prewashed with water to remove membrane stabilizers. Epithelial cells and tampon debris were removed by centrifugation.5) and then placed on a plate that contained bacteria which were embedded in agarose (4 ϫ 105 bacteria/mL in 1% low electroendo osmosis (EEO) agarose [Sigma Chemical Company. For studies of the activity of the protein fraction. L crispatus. This method recovered 96% of the extraction fluid. and the pK of lactate is 3. Total lactate in the vaginal fluid samples was determined with a microtiter plate diagnostic kit (catalog No.1 Lactobacillus MRS broth (Difco. 250 µL of a second tampon extract (with acetic acid. and No.1% acetic acid. . The resin was washed several times in ammonium acetate buffer. Culture medium was prepared in the following manner: ActaMed® Library . The gel was then removed. and the supernatant was decanted off. Md). 33197. The other Lactobacillus species were grown under aerobic condition in MRS broth. CFU assay. as determined by silver stained AU-PAGE (Fig 1) and was used for the colonyforming unit (CFU) assay.1% or 5% acetic acid. The water extract was lyophilized and resuspended in sterile water to the original volume of vaginal fluid. No. The extraction fluid for protein analysis was 0. 735. as determined by weight (1 g = 1 mL). The gel overlay assay was accomplished as previously described.7 Briefly. under conditions that minimized evaporation and condensation. Microbial suspensions (3 µL in nutrient solution) were added to either vaginal fluid or GT buffer to a final volume of 30 µL.5 was added and incubated on a rotator for 3 to 4 hours at room temperature. the second extract was combined with the first extract. with one resin volume equivalent of 10% acetic acid followed by two of the same volume elutions with 5% acetic acid. and subjected to AU-PAGE. Becton Dickinson. NY) in a tube that contained a 100-µL aliquot of water-extracted vaginal fluid.5. To increase protein recovery from the tampon. 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).P. Test strains that represented the endogenous flora of the vagina6 included gram-positive Lactobacillus jensenii. The high molecular weight fraction was then washed on the filter with equal volumes of water. The final nutrient concentration corresponded to either ϫ0. The water and acid extracts were separately resuspended in water to the original volume by weight. which generated an anaerobic carbon dioxide–enriched environment. The eluates were pooled. 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. and the fluid was further processed for protein analysis or antimicrobial testing.A. Areas that corresponded to antimicrobial peptides that were transferred from the AU-PAGE gel were visualized as zones of clearance in the microbial lawn. St Louis. Vaginal fluid fractionation. The amount of lactic acid present in the vaginal fluid was calculated by the Henderson-Hasselbach formula: [HA] = [HA]0 · 10pK/(10pH + 10pK).01 trypticase soy broth (TSB) or ϫ0. and dissolved in 0. New York. Processing for antimicrobial assays. After neutralization. Hercules. where [HA]0 is the molar concentration of lactate plus lactic acid. the tampons were first extracted with water. the remaining mixture was incubated in an environmental shaker at 37°C for the times indicated. which was designed to mimic the electrolyte composition and pH of vaginal fluid. and L vaginalis (ATCC No. nutrient agarose (ϫ2 TSB. Cationic protein extraction from vaginal fluid. Sigma Chemical Company). and the surviving bacteria were allowed to grow overnight. 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. 35 µL of vaginal fluid was lyophilized. The influence of pH and lactic acid on the growth of E coli ML-35p was assessed. The gel was washed three times for 4 minutes each in 100 mL of 10 mmol/L sodium phosphate (pH 7. 49540. respectively) and the yeast Candida albicans. The extract was first neutralized to pH 7 with ammonium hydroxide. L crispatus was grown under anaerobic conditions with the use of a BBL GasPak pouch (Becton Dickinson). Gel overlay assay. 60 mmol/L sodium chloride. 1% agarose) was poured over the top of the lawn.5). [HA] is the molar concentration of lactic acid.83.

Denmark). 5.This service is offered by an educational grant of AVENTIS PHARMA S.1 TSB.1 0.1% bovine serum albumin. Ill). in which case a single sample that was collected approximately on day 20 was used from each donor.3 4. Number 3 Am J Obstet Gynecol Valore et al 563 Table I.01% CETAB. pH 4.1 0. 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. Second antibody conjugated to alkaline phosphatase was purchased from Pierce (Rockford. Roskilde.4 0. Wash) in coating buffer (BupH ready mix buffers. anti–HBD-2. Sunnyvale. Sunnyvale.3 4. analyzed by AU-PAGE.8 0 –3 0 –3 –3 +1 –3 +1 0 –3 Samples were collected during two different menstrual cycles (sets 1 and 2).01 11. 10.7 36 11 21 41 0.2 4.8 0. adjusted to the same pH as its lactate-containing counterpart.9 3.04 ± 0. Denmark) were coated with a 100-µL volume of a 1:5000 dilution of HBD-1 mouse ascites fluid (IB5A. anti-lysozyme and anti-lactoferrin were purchased from DAKO (Glostrup. University of Washington. 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. 7. as needed.A.35 ± 0.8 Antibodies that were used for Western blot analysis were as follows: rabbit anti–HBD-1. as previously described. The lyophilate of the acid extract from tampons was resuspended in loading buffer. lactic acid was dissolved in GT (pH 6.9 4. 5 donors. Minn).2 3. Total lactate and pH were determined. Plates were washed and incubated with 100 µL rabbit ActaMed® Library .Volume 187. DC). and the pH was adjusted with sodium hydroxide or hydrochloric acid.57 ± 0.5 hour with mixing. with the exception of the histone and LL-37 Western blots. to a final concentration of 2 ϫ 106 bacteria/well.1 TSB to concentrations of 5. The optical density at 620 nm (OD620) of the plate was read on a Spectra Max enzyme immunoassay (EIA) reader (Molecular Devices. Semiquantitative Western blot densitometry. with the use of a densitometer and the ImageQuant program (both from Molecular Dynamics Inc. and was added to each well with a calibrated pipette.4 0. respectively. The control medium contained GT buffer (20 mmol/L KHPO4.4 4. Seattle.7 37 33 27 18 0. anti-LL-37 was obtained from Dr Robert Lehrer (UCLA Department of Medicine). Calif). antiSLPI was purchased from R&D Systems (Minneapolis.9 5. and anti–HNP-1 through 3 were generated in our laboratory8-11. The corresponding antimicrobial activity is presented as a change in the log CFU of E coli after 3-hour incubation in vaginal fluid.2 5. Dale.3 5.13 0. then samples in 100 µL volume were added to the appropriate well and serially diluted in the wells of the plate and allowed to incubate for 1.02 10. The plate was then incubated in an environmental shaker at 37°C.5 4. produced in collaboration with Dr Beverly A. 0. as determined by the Henderson-Hasselbach formula. and transferred to Immobilon-P (Millipore Corporation).05% Tween 20.P. . Pierce) and incubated overnight. and 20 hours to determine bacterial growth. and 2 µL of ϫ100 concentrated bacteria was washed in GT buffer. The 96-well Maxisorp plates (Nunc. 20. Culture medium (198 µL) was placed in triplicate wells.5) buffer that contained ϫ0. 3. incubated with 150 µL blocking buffer (1% bovine serum albumin in BupH–phosphate-buffered saline solution. anti–HD-5. 0.3 10.07 0.7 ± 0. 2.02 protein. 5. Vaginal fluid Western blots were analyzed and compared with standard Western blots that contained known amounts of the appropriate Table II.9 ± 0. and 40 mmol/L. and anti-histone H2B was purchased from Serotec (Washington. 60 mmol/L NaCl) with ϫ0. HBD-1 quantification by enzyme-linked immunosorbent assay. and the OD620 was read at 3. standards and samples were analyzed in triplicate and duplicate. Antimicrobial polypeptides in vaginal fluid of the donor panel (n = 20. to attain the desired concentration of lactic acid (1. All steps of the protocol were performed at room temperature. Calif) to determine the initial value.01% thimerosal) for 1 hour.7 4.5. 4 samples each) Protein Calprotectin Lysozyme Lactoferrin SLPI HBD-2 HNP1-3 HBD-1 Mean concentration ± SE (µg/mL) 34 ± 7 13 ± 2 0. Wells were washed (0. and the lactic acid concentration was calculated. in phosphate-buffered saline solution). rabbit anti-calprotectin (MRP8 and MRP10) was a gift from Dr Kenneth Miyasaki (UCLA Department of Dentistry). 0.2 0. and 7 mmol/L).

. Bacteria were added to fluid that was supplemented with ϫ0. 4. the CFUs were determined after overnight incubation. A silverstained AU-PAGE. and 24 hours. when a CFU assay was done in the whole and fractionated fluid. 15. donor 4. To determine whether the antimicrobial activity of vaginal fluid samples was due to small or large molecules. days 9. Aliquots were taken at 0. and 1. 5 µL of the <3-kd fraction. virtually all of the antimicrobial activity against E coli could be attributed to the low molecular weight fraction (Fig 1).This service is offered by an educational grant of AVENTIS PHARMA S. Antimicrobial activity of vaginal fluid. For the analysis of proteins during the menstrual cycle. <3-kd fraction (open circles). 1.3 µg lysozyme. and E coli ML-35p) were added to vaginal fluid and incubated at 37°C.2. the protein-containing fraction was permissive for bacterial growth that was similar to the buffer control. D. the fluid was separated into two fractions by passing through a 3-kd molecular weight cutoff membrane.A. Vaginal fluid was collected from five healthy volunteers. Aliquots were plated onto TSB plates. donor 3 (open triangles).564 Valore et al September 2002 Am J Obstet Gynecol Fig 1. 5 µL of the protein fraction (>3 kd). donor 2. and 0. 12. and donor 5.and low-molecular-weight fractions.P. and 1. donors 2 and 5 exhibited strong antimicrobial activity against L jensenii (3 log decrease after 2 hours). The vaginal fluid from all of the donors was permissive for the growth of L crispatus. 14. and 23 of a 27-day cycle (respective weights: 1. the plate was developed with orrthophenylenediamine (OPD) solution (Sigma Chemical Company). L crispatus. 2. similar to growth in the GT buffer control. with the exception of a sample from donor 2 that exerted a bacteriostatic effect on E coli.01 TSB for a nutrient source and incubated at 37°C for 0.5. donor 1 (open circles). 19.5. 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]). For all but one of the donors. Samples included GT buffer control (closed circles). The antimicrobial activity of whole vaginal fluid (closed circles). Interestingly.2. L vaginalis. 19. and C albicans. 3. the reaction was stopped with 2. 1. 17. days 7. 0. Calif). organisms that are considered to be endogenous flora of the vagina. C. 4.3. 20. and 25 of a 35-day cycle (respective weights: 1. days 7. L vaginalis. Molecular Devices.7 g). donor 5 (open inverted triangles). days 6. 1. and 0. E. and 25 of a 29-day cycle (respective weights: 2. The same three donors (2.2.4. 1. donor 3. and 8 hours and were plated in triplicate on TSB agar plates. Microbes (L jensenii. Vaginal fluid was fractionated into two parts by passage through a 3-kd molecular weight cutoff membrane. The antimicrobial activity of vaginal fluid.2.5 µg HBD-2. Samples from donors 1 and 3 allowed moderate growth of all the organisms that were tested. and 26 of a 26day cycle (respective weights: 0. After being washed with water. and GT buffer control (open inverted triangles) from each donor is shown. whereas the fraction that passed through the membrane contained no protein (as visualized by silver stained AU-PAGE [lane B]). 1. 1. who ranged in age from 25 to 44 years and had regular menstrual cycles of approximately 28 days. 0. ActaMed® Library . 1. and CFUs were determined after overnight incubation.8.0. anti–HBD-1 polyclonal antibody (diluted 1:2000 in blocking buffer) for 45 minutes. 14. and the OD620 was read on a spectrophotometer (Spectra-Max. B. donor 4 exhibited moderate activity (3 log decrease after 8 hours). the five donors collected samples on the following days: donor 1.6 g). 1.1. 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]). single samples were collected at day 20 through 25 of the cycle. and 1. 1. 1. and 24 of a 25-day cycle (respective weights: 1. Effects of high. donor 4 (open diamonds). The antimicrobial activity of vaginal fluid fractions against E coli ML35p.2.5N hydrogen sulfate.9 g). donor 2 (open squares). Results Fluid collections.5 g). Fig 2.6. and donor samples 1 and 3 allowed growth.7. 5 µL vaginal fluid. and C albicans (Fig 2). 21. protein-rich >3-kd fraction (closed inverted triangles). group B Streptococcus. followed by washing and 1hour incubation in 100 µL of a 1:2000 dilution of horseradish peroxidase that was conjugated to goat antirabbit antibody (Pierce). and 5) whose fluids killed L jensenii also exhibited strong/moderate killing capabilities against group B Streptococcus and E coli.8 g). 12. A. For antimicrobial assays. 1.9. days 8. Sunnyvale.3 However.

In previous studies. and 24 hours and plated in triplicate on TSB agar plates. The vaginal fraction retained by a 3-kd molecular weight cutoff membrane was dissolved to ϫ1. Antimicrobial effects of the high molecular weight fraction. a gel overlay assay was performed. To determine whether antimicrobial activity increases with the concentration of the high molecular weight solutes.and 5-fold and then tested in the CFU assay. It is likely that the high molecular weight components (eg. and the amount of lactic acid that was present in each sample was calculated from the measured pH. Similar results were also seen for group B streptococci (data not shown). As can be seen by the zones of clearance. In each graph. To determine the separate effects of pH and lactic acid on the growth of E coli.This service is offered by an educational grant of AVENTIS PHARMA S. The unconcentrated high molecular weight fraction (ϫ1) initially decreased the viability of bacteria (Fig 4). total lactate of the water-extracted vaginal fluid was determined. The antimicrobial effect of the protein-rich fraction of vaginal fluid. 20.01 TSB). and the plate was placed in an environmental shaker at 37°C. vaginal fluid contains many proteins that kill E coli (Fig 5).002. A volume of 198 µL culture medium was placed in triplicate wells of a 96-well plate and 2 µL bacteria (2 ϫ 109 bacteria/mL) were added to each well. 60 mmol/L sodium chloride) with ϫ0. The symbols represent bacterial CFU in GT or protein-rich vaginal fluid fraction. 5. At 1 and 2 mmol/L lactic acid. with recovery and even enhanced growth at 20 hours likely to be due to the opposing effects of lactic acid (inhibition) and lactate (enhancement). . The bacterial concentration represented by the OD620 reading is shown at 0. The high molecular weight fraction that was concentrated 2. and the concentration of lactic acid ranged from 0.5.0 mmol/L (Table I). ϫ0. Protein composition of vaginal fluid.P.13 To analyze the role of lactic acid in antimicrobial activity. 3. n = 10 samples from 5 donors) and between the pH and the log change in CFUs (R = 0. Fig 3. To estimate whether one or more antimicrobial proteins in vaginal fluid exert predominant activity.941. 3. proteins) of vaginal fluid are more concentrated on and in the epithelial surface than in the fluid layer. we performed a growth inhibition assay. but by 24 hours the CFU rose to match the buffer control.1. 5.5. GT buffer (20 mmol/L potassium phosphate. the growth of E coli was significantly inhibited.Volume 187.2 to 5. among which lactic acid predominated. bacterial growth in the lactate-con- taining medium is compared with growth in lactate-free medium at the same pH (Fig 3). The controls at the matched pH contained no lactate (open circles). vaginal fluid was reported to contain a mixture of organic acids. aliquots were taken at 0. The antibacterial effect of lactic acid. concentrated as indicated ϫ1-ϫ5 . and 20 hours. Gel overlay antimicrobial assay.12.839. the protein-containing high molecular weight fraction (donor 5) was concentrated 2. 6.1 TSB that contained 5. there was inhibition of growth at the early time points.00005). there is a marked additional growth-suppressive effect of lactic acid concentrations of ≥3 mmol/L. P = . 10. P = . 1.or 5-fold exerted a microbicidal effect throughout the assay. ϫ2. 7.7 to 11. The pH of the samples ranged from 4. and CFUs were determined after overnight incubation. The effects of pH and lactate/lactic acid on the growth of E coli. 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. E coli ML35p was added to each sample. Proteins from samples that were collected throughout the menstrual cycle from five donors were analyzed by semiquantitative West- ActaMed® Library .1 (regardless of lactic acid concentration). 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. At a pH of ≤4. Number 3 Am J Obstet Gynecol Valore et al 565 Fig 4. 2. At >pH 4.A. or 7 mmol/L (closed circles). and ϫ5 original concentration in GT buffer (pH 4. 3. R = –0.

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. L jensenii. the other proteins were detected at concentrations of <1 µg/mL. The “permissive” vaginal fluids contained ≤3. . and defensins HNP1-3. 20 ng/mL). Overall. In contrast. a slight increase in pH of vaginal fluid (4. additional antimicrobial peptides were sought in donor panel samples that were collected in mid menstrual cycle. with an average of 500 ng/mL (n = 5).12 Hydrogen peroxide is known to be antimicrobial. It has been widely assumed that lactic acid is an antimicrobial factor in vaginal secretions. acetic. there were only minor changes in protein concentrations during the menstrual cycle (Fig 6). The human cathelicidin peptide LL-3714 was detected in the donor panel. In this donor. and lactic acid. butyric. The microcolonies were absent from the zones of clearance that indicate multiple active antimicrobial polypeptides in each sample. In contrast. Histone15 was detected in only one of the five vaginal fluid samples (donor 1) and was present at approximately 2 µg/mL (detection limit. The corresponding pattern of AU-PAGE Coomassie blue–stained polypeptides is shown in the lower panel. the predominant acid in normal vaginal fluid.12 is nonvolatile and is retained during the extraction procedure. and E coli were killed by three of the five samples. 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. propionic. this is the first study to test this hypothesis. Subsequently.2 mmol/L lactic acid and had a higher pH than the more potently antimicrobial vaginal fluid samples. the ability of vaginal fluid from five different donors to exert an antimicrobial effect on several different microbes was assessed.2 mmol/L in the second sample compared with 5.3 vs 4. succinic. ern blot for previously described antimicrobial components of epithelial secretions (calprotectin. in our model of lactic acid–dependent antimicrobial activity. and HBD-2) and by enzyme-linked immunosorbent assay for HBD-1. lane M1 contains the human protein markers lactoferrin. lactic acid. The antimicrobial polypeptide “fingerprint” of vaginal fluids. Most antimicrobial activity could be attributed to the low-molecular-weight (protein-free) fraction of vaginal fluid. As with vaginal fluids. This may explain the paradoxic difference in activity of 2 samples that were collected from donor 4. two of the three lactobacillus species (L crispatus.566 Valore et al September 2002 Am J Obstet Gynecol Comment In this study. and lysozyme. 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. Of the endogenous vaginal microbes. 1. and the data were pooled therefore to calculate the mean concentration of each protein (n = 20. However. HD-5 was not detected in the vaginal fluid by this method (detection limit.7) resulted in a significant decrease in calculated lactic acid concentration (3. group B Streptococcus.8-11 mmol/L. ranging from 65 to 1000 ng/mL. With further incubation of the plate.3 µg/mL). approximately 3 mmol/L lactic acid represented a transition zone below which we observed no lactic acid–dependent antimicrobial activity and above which there was complete killing of E coli (Fig 3). we found that the vaginal fluid with the highest levels of antimicrobial activity also contained the highest levels of lactic acid (3. surviving bacteria formed microcolonies. Table I). which straddled the transition zone between killing and no Fig 5. Resident vaginal bacteria can produce hydrogen peroxide and convert host-derived nutrients into organic acids that include formic. 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.This service is offered by an educational grant of AVENTIS PHARMA S. ActaMed® Library . In this study. SLPI. 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. and lane M2 contains histone H2B and SLPI.A. lysozyme. Arrowheads indicate marker bands that exhibit antimicrobial activity in this assay.P. HNP-1.3 mmol/L in the first sample). 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. From top to bottom.16 However. however. Table II). L vaginalis) and C albicans were able to grow in vaginal fluids from all of the donors. to our knowledge. Calprotectin and lysozyme were present at concentrations of >10 µg/mL.

The solid line indicates the log-linear regression (log trend) of all of the points in each graph. the former because of neutrophil influx and the latter by the induction of ep- ithelial synthesis. some studies have found that many of these antimicrobial peptides can act synergistically. in vaginal fluid.This service is offered by an educational grant of AVENTIS PHARMA S. .19 SLPI. the acid form of total lactate may be a major contributor to antimicrobial activity.A. there was little change in polypeptide concentration during the cycle (Fig 6). Antimicrobial polypeptide levels in vaginal fluid collected throughout the menstrual cycle of the donor panel: donor 1 (closed squares). calprotectin has also been found to inhibit the adherence of exogenous microbes to cultured epithelial cells. calprotectin and lysozyme are present at concentrations previously found to be antimicrobial. killing of E coli. Thus. and donor 5 (closed inverted triangles). they did find synergism with the following combinations: lysozyme/lactoferrin. In addition.P. even though individually many of the proteins are present in vaginal fluid at concentra- ActaMed® Library .18 In addition to its role as an antimicrobial polypeptide. like in the model system.17. 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. lactoferrin/SLPI. Thus. Number 3 Am J Obstet Gynecol Valore et al 567 Fig 6. lysozyme/SLPI.18 although the other peptides and proteins could participate in synergistic activity.Volume 187. donor 4 (closed circles). Thus. the concentrations of these defensins would be expected to increase in response those infections that induce an inflammatory response. Of the polypeptides that were detected in the vaginal fluid. In addition. Although Singh et al23 found only an additive effect between HBD-2 and lysozyme. 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. which inhibits serine proteases such as elastase and cathepsin G. Surprisingly. also has been found to have antibacterial and antifungal capabilities.2022 Both HNP1-3 and HBD-2 concentrations increase in response to inflammatory stimuli. we found that the protein component of the vaginal fluid was able to exert a significant antimicrobial effect when concentrated 2. donor 3 (open circles). and lysozyme/ 5-fold (Fig 4). donor 2 (open inverted triangles). This study marks the first time antimicrobial polypeptides have been assessed in vaginal fluid and observed during the course of the menstrual cycle.

J Biol Chem 1997.A. Ganz T. Donders GG. Scand J Urol Nephrol Suppl 1984. Amsterdam: Elsevier/North-Holland Biomedical Press. Dale I. Anton PA. Thwin SS. Larsen B. Gain RE. Harder J. Kistner RW. 20. Chan WC. In our study. Vol 2. Defensins: natural peptide antibiotics of human neutrophils. Ahangari G. it is possible that other vaginal fluid samples also contain antimicrobial concentrations of histone. J Exp Med 1958. such as bacterial vaginosis and candidiasis. Welsh MJ. Cohen MS. Hiemstra PS.24 In our study. Porter for her critical review of this manuscript and her insightful scientific discussions and Dr Rose Linzmeier for her helpful contributions to this project.182:872-8. Agerberth B. 5.67:3267-75. Dekeersmaecker A. Hillier SL. ActaMed® Library . the role of histones in vaginal fluid must be more carefully examined in future studies. was the role of lymphoid populations in the vagina and immunoglobulin A and G in the vaginal fluid. Amsterdam: Elsevier/North-Holland Biomedical Press. Innate antimicrobial activity of nasal secretions.568 Valore et al September 2002 Am J Obstet Gynecol tions below their active range. Gabrielsen TO. J Clin Invest 1985. Streit V. J Infect Dis 1991. 24. Conway BA. The human vagina. et al. Infect Immun 1999. Ganz T. J Infect Dis 1999. Agnew K. 8.65:2389-95. Bauman JE. Wigzell H. Fagerhol MK. . 109-20. J Clin Invest 1998. Quayle AJ.76:1427-35. Wang L. REFERENCES 1. Vaginal organic acids and hormonal changes in the menstrual cycle. J Immunol Methods 1991. 13. Travis SM. et al. Heinzel-Wieland R. Vereecken A. Bailey K. Keyte JW. Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Antibacterial activity of antileukoprotease. Stanek R. Potential role of epithelial cell-derived histone H1 proteins in innate antimicrobial defense in the human gastrointestinal tract. Bainton DF. Liden S. 12. Klebanoff SJ. McCray PB Jr. Infect Immun 2001. Valore EV. 17. contribute to the resistance of the normal vagina to colonization by exogenous microbes represented in our experiments by E coli. Ganz T.176:740-7. Smith JJ. Host defences and the vaginal mucosa: a re-evaluation. Bosmans E. Biomed Chromatogr 1992. 15. editors. 6. Fertil Steril 1982. In: Hafez ES. 9. Steinbakk M. Singh PK. Harwig SS. Christophers E. 1978. Maassen RJ. Am J Obstet Gynecol 2000. The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. Patton DL. Eschenbach DA. Oren A. Structure and mapping of the human β-defensin HBD-2 gene and its expression at sites of inflammation. 4. Greenwood D. Jia HP. Schutte BC.272:15258-63. Control of the microbial flora of the vagina by H2O2-generating lactobacilli. Dijkman JH. Brandtzaeg P. Vaginal fluid. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. Liu L.This service is offered by an educational grant of AVENTIS PHARMA S. 19.248:904-9. Infect Immun 1997. Heinzel-Wieland R. Cole AM. Stapleton AE. Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes.P.164:94-100. editors. Clearly. Webster SK. McCray PB. p. Infect Immun 1996. Ganz T. 11. The human vagina.86:13-22. Evans TN. Lehrer RI. Stahle-Backdahl M. et al. Influence of the normal menstrual cycle on vaginal tissue. and the fluid is sampled from most of the vaginal surface. Clin Infect Dis 2000. Nisapakultorn K. Proctor RA. Activity of abundant antimicrobials of the human airway. Hiemstra PS. Porter EM. Evans TN.30:901-10. Hawes SE. 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. they could inhibit the attachment of microbes to the epithelia. Liu L. 18. Gene 1998. Antimicrobial polypeptides could have a greater role in vaginal host defense of women who have low concentrations of lactate and high vaginal pH. to a lesser extent antimicrobial peptides and proteins. Levin RJ. Sparling PF.20:872-9. and together they may constitute an effective barrier to microbial invasion of the epithelial surface. Hirsch JG. et al. Harwig SS.6:231-5.15. Wiedow O. 7. Eisenhauer P.137:167-73. Mahida YR. This method has the advantage that quantitation is precise. 22. Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi.279:L799-805. 121-37. Steffens GJ. Ross KF. 2.108:925-44. but this mixture is physiologically relevant. Wiles KR. Dewan P. Am J Respir Cell Mol Biol 1999. Herzberg MC. Bartels J. 21. Hillier SL. The limitations imposed by the available antibody enabled only a relatively high detection limit. 10. High performance ion exclusion chromatographic characterization of the vaginal organic acids in women with bacterial vaginosis. Szklarek D. 3. Heng HHQ. We thank Dr Edith M. J Infect Dis 1997. 23. discharge. In: Hafez ES. Ultrasensitive assays for endogenous antimicrobial polypeptides. Infect Immun 1998. Jackson R. Van Bulck B. Daher K. Tack BF. Calprotectin expression inhibits bacterial binding to mucosal epithelial cells.66:3255-63. Wagner G. Spitz B. the sampled fluid is a mixture that represents both vaginal and cervical sources. Zabner J. Although such antibodies are not directly microbicidal. A potentially important factor that was not considered in this study. Rose FR. Singh PK. Histone and fragments of histone are cationic polypeptides with antimicrobial activity. 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. Glover DD. Localization of human intestinal defensin 5 in Paneth cell granules. Therefore. Antonio MA. Kolodny RC. 1978. Zhao C. Stolk J. Adv Exp Med Biol 1995. Am J Physiol Lung Cell Mol Physiol 2000. 16. Kauffman HF. 14. p.371:201-6. Physiology of the vagina. Hooton TM. Eschenbach DA.69:3692-6. Pathogenesis of abnormal vaginal bacterial flora. Rosenman M.64:4520-4. histone was found in one vaginal fluid sample at a concentration of 2 µg/mL. We presented evidence that vaginal fluid is selectively antimicrobial and that lactic acid and. The likely contribution of antimicrobial products that originate from vaginal bacteria also was not specifically addressed in this study.38:572-9. in or on the epithelium. Epithelial cells in normal vaginal fluid frequently appear lysed25 and may release nuclear histone into the vaginal fluid milieu. Biochem Biophys Res Commun 1998. Black JR. because of our focus on innate immunity.222:237-44. et al. Anderson NN. Tomee JF. Frohm M. Waltersdorph AM. and microflora. Muller F. Bactericidal action of histone.101:1633-42. Selsted ME. 25. Park CH.180:1950-6. The leucocyte protein L1 (calprotectin): a putative nonspecific defence factor at epithelial surfaces.