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. Author manuscript; available in PMC: 2022 Jan 27.
Published in final edited form as: Sex Transm Dis. 2021 Jan;48(1):63–70. doi: 10.1097/OLQ.0000000000001272

Clinical and personal lubricants impact growth of vaginal Lactobacillus species and colonization of vaginal epithelial cells: an in vitro study

Paweł Łaniewski 1, Kimberley A Owen 1,2, Michael Khnanisho 1,3, Rebecca M Brotman 4, Melissa M Herbst-Kralovetz 1,#
PMCID: PMC8793461  NIHMSID: NIHMS1771385  PMID: 32842049

Abstract

Background:

Vaginal lubricants are commonly used during gynecological exams, sexual activities or to alleviate vaginal dryness. Many lubricants contain potentially bacteriostatic or bactericidal agents (parabens, chlorhexidine gluconate, nonoxynol-9). Our objective was to evaluate the impact of lubricants that vary in formulation, on the growth and viability of vaginal Lactobacillus species and vaginal epithelial cell (VEC) colonization in an in vitro model.

Methods:

Growth curve, disk diffusion and minimal inhibitory assays were used to determine impact of lubricants or excipients on the growth of L. crispatus, L. gasseri, L. jensenii and L. iners. Two L. crispatus strains were utilized in VEC colonization assays. Statistical differences were determined by ANOVA.

Results:

Lubricants containing chlorhexidine gluconate or nonoxynol-9 (Conceptrol®, K-Y Jelly, and Surgilube®) significantly inhibited Lactobacillus spp. growth (P<0.05). In contrast, other clinical lubricants (E-Z Lubricating Jelly, McKesson Lubricating) and personal lubricants (Astroglide® Liquid, Good Clean Love Almost Naked, K-Y Warming Jelly) did not exhibit this effect. Chlorhexidine gluconate had a detrimental effect on Lactobacillus growth and exhibited stronger antimicrobial activity compared to methylparaben and propylparaben (P<0.0001). There were lubricants that did not induce cytotoxicity in VEC (Good Clean Love Almost Naked, E-Z Lubricating Jelly, McKesson Lubricating Jelly), but these products did substantially decrease attachment of Lactobacillus to VEC, particularly when VEC were pre-exposed to lubricants prior to inoculation with bacteria (P<0.0001).

Conclusions:

This in vitro model indicates that select vaginal lubricants, particularly those with chlorhexidine gluconate, have potentially adverse effects on women’s health by reducing growth and re-colonization of vaginal Lactobacillus species.

Keywords: chlorhexidine gluconate, host–microbe interactions, paraben, vaginal microbiota, women’s health

Short summary

An in vitro study found that vaginal lubricants containing chlorhexidine gluconate can deleteriously affect protective Lactobacillus species, which may lead to increased risk for vaginitis or other adverse gynecologic outcomes.

Introduction

Women commonly report use of personal lubricants in sexual practices and to help alleviate vaginal dryness and the genitourinary syndrome of menopause.1 In addition, clinicians frequently use vaginal lubricants in the conduct of gynecologic exams and recommend them to patients with gynecologic cancer to help mitigate adverse effects of treatments, which cause vulvovaginal atrophy and an overall reduction in quality of life.2 Yet, the effect of lubricants on the cervicovaginal microenvironment, including local microbiota, has not been comprehensively studied. In the majority of healthy, reproductive-age women, the vaginal microbiota is dominated by one or few Lactobacillus species, such as L. crispatus, L. gasseri, L. jensenii or L. iners.3 However, in postmenopausal women, as a result of hormonal changes, the vaginal microbiota frequently lacks Lactobacillus dominance.3 Colonization of the vagina with lactic-acid producing Lactobacillus species has broadly been associated with vaginal health, since the acidic microenvironment consequently protects the host from invading pathogens, including sexually transmitted infections (STIs).3

Studies investigating the impact of lubricants on the human mucous membranes are limited. Two reports, utilizing in vitro biomimetic models, demonstrated that lubricants with high osmolality reduce epithelial barrier integrity, cause cellular damage and alter inflammatory responses.4,5 Relative to other body sites, hyperosmolar lubricants have been shown to cause rectal epithelial cell damage or denudation.6 Furthermore, consistent use of hyperosmolar lubricants during anal intercourse has been associated with higher prevalence of STIs among men who have sex with men.7 The World Health Organization (WHO) guidelines published in 2012 state that lubricants’ osmolality should not exceed 1200 mOsm/kg and by comparison, but the majority of lubricants on the market exceed this osmolality recommendation and the osmolality of vaginal secretions is 260–290 mOsm/kg.8

Most clinical and personal lubricants also contain excipients with antimicrobial properties, such as parabens and chlorhexidine gluconate (CHG). Parabens are commonly used preservatives with a broad spectrum activity against fungi and bacteria.9 CHG is a broad spectrum antiseptic effective against gram-positive and gram-negative bacteria, as well as fungi.9 The mechanism of parabens’ and CHG’s action relates to the damage of cell membrane and wall integrity.9 Intriguingly, CHG has been banned by the United States Food and Drug Administration (FDA) from use in over-the-counter healthcare antiseptics and hand sanitizers in 2018 and 2020, respectively.10,11 Ultimately, the effect of lubricants containing these excipients on the healthy constituents of the vaginal microbiota is unknown. In this study, we aimed to determine the effects of a broad range of personal and clinical lubricants on the growth of vaginal Lactobacillus species and investigated the impact of lubricants on Lactobacillus colonization of vaginal epithelial cells (VEC).

Materials and Methods

Lubricants and bacterial strains

Personal lubricants were obtained from a local drugstore and clinical lubricants were obtained from clinics located in Phoenix, AZ and Baltimore, MD (Table 1). All lubricants were used prior to the expiration date. Bacterial strains were obtained from the American Type Culture Collection (ATCC) or the Biodefense and Emerging Infections (BEI) Research Resources Repository and included five vaginal Lactobacillus strains: L. crispatus JV-V01, L. gasseri JV-V03, L. jensenii JV-V16 and L. iners AB107, as well as L. crispatus type strain VPI 3199 (the latter isolated from eye). L. crispatus, L. gasseri, and L. jensenii were grown on de Man, Rogosa and Sharpe agar or in MRS broth at 37 °C under 5% CO2. L. iners was grown on tryptic soy agar (TSA) supplemented with 5% defibrinated sheep blood (Quad Five, Ryegate, MT) or in tryptic soy broth (TSB) supplemented with 5% horse serum at 37 °C under anaerobic conditions, generated with a GasPak EZ Anaerobe Container System. All bacterial culture media and supplements were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ).

Table 1. Formulations of tested clinical and personal lubricants.

Excipients exhibiting potential antimicrobial properties against vaginal Lactobacillus spp. are indicated in bold.

Lubricant Osmolality [mOsm/kg] Ingredients Manufacturer
Clinical lubricants
E-Z Lubricating Jelly 2243 Water, glycerin, carbomer, sodium hydroxide, PEG-150, methylparaben, propylparaben Athena Medical Products
McKesson Lubricating Jelly 2125 Water, glycerin, sodium hydroxide, carbomer 140g, polyethylene glycol, propylparaben, methylparaben McKesson Medical-Surgical Inc.
Surgilube® Surgical Lubricant Not tested Water, hydroxypropylmethylcellulose, propylene glycol, chlorhexidine gluconate HR Pharmaceuticals Inc.
Personal lubricants
Astroglide® Liquid 6100 Purified water, glycerin, propylene glycol, polyquaternium 15, methylparaben, propylparaben Biofilm Inc.
Conceptrol® 1257 Nonoxynol-9 (4%), lactic acid, methylparaben, povidone, propylene glycol, purified water, sodium carboxymethycellulose, sorbic acid, sorbitol solution Caldwell Consumer Health LCC
Good Clean Love Almost Naked 270 Organic aloe barbadensis leaf juice, xanthan gum, agar, potassium sorbate, sodium benzoate, sodium lactate, lactic acid, natural flavors Good Clean Love Inc.
K-Y Jelly 2500 Water, glycerin, hydroxyethylcellulose, chlorhexidine gluconate, gluconolactone, methylparaben, sodium hydroxide Reckitt Benckiser Group plc
K-Y Warming Jelly 10300 Propylene glycol, PEG-8, hydroxypropylcellulose, tocopherol Reckitt Benckiser Group plc

Growth curve assay.

To determine the bacteriostatic and/or bactericidal effect of lubricants on vaginal Lactobacillus spp., the growth of bacteria in liquid media with or without lubricants, was analyzed. Bacteria were inoculated into MRS or supplemented TSB liquid media and cultured overnight at 37 °C under 5% CO2 or anaerobic conditions. The broths containing 10% (v/v) lubricants were inoculated with Lactobacillus spp. at the final optical density at 600 nm (OD600) of 0.5 and cultured as described earlier. The broth without any lubricant was used as a positive control. The growth of bacteria with or without lubricants was determined after 4 and 24 h by measuring the OD600 of bacterial cultures and the standard plating assay. For the standard plating assay, bacterial cultures were serially diluted in phosphate-buffered saline (PBS) and spotted on MRS or supplemented TSA plates. The agar plates were incubated at 37 °C for 48–72 hours for enumeration of colony-forming units (CFU). The concentration of viable bacterial cells in each culture was calculated and presented as CFU/mL.

Disk diffusion assay.

The disk diffusion assay was used to determine antimicrobial properties of select excipients, such as parabens and chlorhexidine gluconate, on vaginal Lactobacillus spp. MRS or supplemented TSA plates were inoculated with 1 × 107 CFU of bacteria. Under aseptic conditions, 6 mm Whatman filter disks (GE Healthcare, Chicago, IL) were impregnated with 20 μl of 20% (w/v) solution of chlorhexidine gluconate in water (Tokyo Chemical Industry, Tokyo, Japan) or 20% (w/v) solutions of methylparaben or propylparaben in ethanol (Tokyo Chemical Industry). Bleach and ethanol were used as positive and negative controls, respectively. Disks were placed onto inoculated agar plates and incubated at 37 °C under 5% CO2 (for L. crispatus, L. gasseri and L. jensenii) or anaerobic conditions (for L. iners). Following 24 h incubation, zones of inhibitions were measured in millimeters.

Minimal inhibitory concentration assay.

Minimal inhibitory concentrations (MIC) of parabens and chlorhexidine gluconate were assessed using the broth microdilution method. Lactobacillus spp. were grown overnight on MRS or TSA plates and resuspended in sterile PBS. Bacterial suspensions were adjusted to the OD600 of 1.0 and diluted in MRS or supplemented TSB liquid media. Two-fold serial dilutions of excipients (methylparaben, propylparaben and chlorhexidine gluconate) in appropriate broths (50 μl) were aliquoted into respective wells in a sterile 96-well microtiter plate. 50 μl aliquots of bacterial inoculum containing 5 × 105 CFU were added to wells with excipient dilutions. Broth without added excipients or bacterial inoculum were used as a growth and sterility controls, respectively. The inoculated microtiter plate was incubated at 37 °C under 5% CO2 or anaerobic conditions. Following 24 h incubation, the OD600 was recorded using a Safire II Multi-Mode Microplate Reader (Tecan, Männedorf, Switzerland). The MIC was defined as the lowest concentration of the excipient that inhibits the visible growth of the tested bacteria.

Colonization assays.

Human vaginal epithelial (V19I) cells were cultured as monolayers in 1:1 (v/v) mixture of EpiLife and Keratinocyte Serum-Free Media (Life Technologies, Carlsbad, CA) at 37 °C under 5% CO2. For experimental manipulations, cells were quantified using trypan blue (0.25%; v/v) exclusion staining and seeded into 24-well tissue culture-treated plates at a density of 2 × 105 cells/mL. L. crispatus strain JV-V01 was used for colonization assays. Bacteria were grown for 16–18 h on MRS agar plates, resuspended in sterile Dulbecco’s PBS and used for in vitro colonization of VEC at a multiplicity of infection (MOI) of 10. The impact of lubricants on L. crispatus colonization was tested using pre- and post-exposure approaches. For pre-exposure assays, VEC were pre-exposed to 10% (v/v) solution of select lubricants in cell culture media following immediate inoculation with bacteria. After adding bacterial inoculum to the cells, plates were centrifuged for 10 min at 900 × g to ensure bacterial inoculum reached the epithelial monolayers and incubated under standard conditions. For post-exposure assays, VEC were pre-colonized with bacteria prior to exposure to lubricants. VEC were infected for 2 h, washed three times with Dulbecco’s PBS to remove bacteria not attached to the cells and then treated with 10% (v/v) solution of lubricants. Dulbecco’s PBS or 10% (v/v) solution of glycerol was used as a negative treatment control. Following 4 or 24 h incubation, VEC were washed three times with Dulbecco’s PBS, trypsinized and resuspended in Dulbecco’s PBS. Suspensions of bacteria and VEC were vortexed for 5 min and serially diluted, plated on MRS agar and incubated for 48–72 h for bacterial quantification. All incubations were performed at 37 °C under 5% CO2.

Statistical analysis

All experiments were performed at least in triplicate. The statistical differences were determined using an analysis of variance (ANOVA) with Dunnett or Bonferroni adjustments for multiple comparisons. P values <0.05 were considered significant.

Results

Herein, we tested three clinical lubricants and five commercially available personal lubricants, which differed in osmolality and formulation. Six out of eight lubricants contained an antimicrobial, such as methylparaben, propylparaben, polyquaternium 15 or CHG (Table 1). The impact of each lubricant on the growth of the four most predominant vaginal Lactobacillus spp. (L. crispatus, L. gasseri, L. jensenii and L. iners) was evaluated. Initially, we assessed the bacterial growth by measuring optical densities of bacterial cultures containing lubricants. The presence of Conceptrol®, K-Y Jelly or Surgilube® in liquid media significantly inhibited the growth of L. crispatus, L. gasseri, L. jensenii and L. iners following 24 h exposure when compared to controls (P ranging from <0.05 to <0.0001) (Fig. 1). Then, we quantified viable bacteria following 4 and 24 h exposure to each lubricant using standard plating assay. In the presence of K-Y Jelly or Surgilube®, concentrations of viable Lactobacillus spp. were significantly lower compared to cultures without lubricant (P ranging from <0.05 to <0.001) (Fig. 2). However, these numbers did not significantly decline relative to the numbers of viable bacteria at the time of inoculation, suggesting a bacteriostatic effect of lubricants on the Lactobacillus growth. In contrast, GCL Almost Naked, McKesson Lubricating Jelly and Astroglide® Liquid had minor or no effect on the viability of tested vaginal Lactobacillus spp. (Fig. 2). Notably, the lubricants that significantly inhibited the growth of Lactobacillus spp., i.e. Conceptrol®, K-Y Jelly and Surgilube®, were not distinguished by the highest osmolality; however, all contained antimicrobial agents, such as parabens or CHG.

Figure 1. Select lubricants (K-Y Jelly, Surgilube® and Conceptrol®) inhibit the growth of vaginal Lactobacillus spp.

Figure 1.

L. crispatus strain VPI 3199, L. crispatus strain JV-V01, L. gasseri strain JV-V03, L. jensenii strain JV-V16 and L. iners strain AB107 were grown in MRS or supplemented TSB broth with 10% (v/v) lubricants at 37°C under 5% CO2 or anaerobic conditions. Bacterial growth was assessed by measuring optical densities at 600 nm (OD600) at 4 h and 24 h post inoculation. The broth without any lubricant was used as a positive control. OD600 measurements of each culture with lubricants were compared to cultures without lubricants at respective time points. Data are shown as means ± SE from at least three independent experiments. P values were calculated using one-way ANOVA with Dunnett post-test (* P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001).

Figure 2. Select lubricants (K-Y Jelly, Surgilube® and Conceptrol®) exhibit bacteriostatic effect on vaginal Lactobacillus spp.

Figure 2.

L. crispatus strain VPI 3199 (A), L. crispatus strain JV-V01 (B), L. gasseri strain JV-V03 (C), L. jensenii strain JV-V16 (D) and L. iners strain AB107 (E) were grown in MRS or supplemented TSB broth with 10% (v/v) lubricants at 37°C under 5% CO2 or anaerobic conditions. Bacterial growth kinetics were assessed by measuring number of colony forming units (CFU) representing viable bacteria in the cultures in appropriate liquid media containing 10% solutions of lubricants at 4 and 24 h following the exposure to 10% (v/v) lubricants using standard plating assay. The broth without any lubricant was used as a positive control. Concentrations of viable bacteria in each culture were calculated as CFU/mL and compared to culture without lubricants at respective time points. Data are shown as means ± SE from three independent experiments. P values were calculated using one-way ANOVA with Dunnett post-test (* P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001).

To test the antimicrobial properties of excipients listed as key components of lubricants (Table 1) against vaginal Lactobacillus spp., we performed disk diffusion assays. We did not test polyquaternium 15 since this compound is not commercially available. In addition, lack of bacterial growth inhibition following exposure to Astroglide® Liquid, containing polyquaternium 15, indicates that this compound does not impact the growth of Lactobacillus spp. (at least at the concentration present in the tested lubricant). The growth of L. crispatus, L. gasseri and L. jensenii on agar plates had significant zones of inhibition from all excipients tested when compared to the negative control (P ranging from 0.0007 to <0.0001) (Fig. 3A). For L. iners, CHG caused significant (P<0.001) growth inhibition, whereas zones of inhibition from parabens did not reach statistical significance. Furthermore, CHG inhibited Lactobacillus spp. at a significantly higher magnitude (19–24 mm) compared to parabens (8–12 mm) (P<0.0001) (Fig. 3A).

Figure 3. Chlorhexidine gluconate exhibit stronger antimicrobial properties than parabens across all tested vaginal Lactobacillus spp.

Figure 3.

A. The impact of each excipient on the growth of Lactobacillus spp. was tested using disk diffusion assay. Bacteria were grown on appropriate agar plates with 6-mm disks impregnated with 20% solutions (w/w) of methylparaben, propylparaben and chlorhexidine gluconate (CHG). Ethanol (solvent for parabens) and bleach was used as negative and positive controls, respectively. Zones of inhibition were recorded 24 h post inoculation and compared to a negative control. Data are shown as means ± SE from three independent experiments. P values were calculated using one-way ANOVA with Bonferroni post-test (** P<0.01; *** P<0.001; **** P<0.0001). B. Minimal inhibitory concentrations (MIC) of methylparaben, propylparaben and CHG were determined using the broth microdilution method. The MIC was defined as the lowest concentration of the excipient that inhibits the visible growth of the tested Lactobacillus spp.

To better delineate the differences in antimicrobial potentials of parabens and CHG against vaginal Lactobacillus spp., we determined the minimal inhibitory concentrations (MIC) for these compounds. Both parabens inhibited bacterial growth at 8,000 mg/L for all tested Lactobacillus spp. except L. iners, which was inhibited at 2,000 mg/L and 4,000 mg/L of methylparaben and ethylparaben, respectively (Fig. 3B). In contrast, CHG inhibited growth of Lactobacillus spp. in a species-specific manner at a concentration ranging from 1.25 to 10 mg/L, which are 200–6400 times (2.3–3.8 log) lower than parabens.

To assess the effect of lubricants on the colonization of the vaginal epithelium with Lactobacillus associated with optimal vaginal health, we exposed human vaginal epithelial cells (VEC), grown as monolayers, to lubricants and infected the in vitro VEC cultures with L. crispatus. We were able to test only three lubricants: GCL Almost Naked, E-Z Lubricating Jelly and McKesson Lubricating Jelly. The other lubricants used in this study induce substantial cytotoxicity, including condensation of chromatin and a loss of intercellular connections in the in vitro VEC model5, which precluded testing the colonization. Glycerol was used as an additional control to mimic the viscosity of lubricants in the event that this property impacted colonization alone. We also used two different approaches to determine the impact of lubricants on bacterial colonization. First, we tested the effect of lubricants on VEC that were pre-colonized with L. crispatus prior to exposure lubricants. Four-hour exposure to lubricants did not significantly impact the colonization levels; however, 24 h exposure to E-Z Lubricating Jelly or McKesson Lubricating Jelly significantly decreased L. crispatus colonization levels by 0.9 log (P<0.05) (Fig. 4A). In contrast, GCL Almost Naked did not impact the colonization at any tested time points compared to untreated or glycerol controls (Fig. 4A). Second, we tested the effect of lubricants on L. crispatus colonization levels when VEC were pre-exposed to lubricants following infection with L. crispatus. Pre-exposure to all tested lubricants for 4 h or 24 h significantly impacted the VEC colonization by L. crispatus compared to negative controls (VEC cultures without lubricants or with glycerol) (P<0.0001 and <0.001, respectively). The colonization levels were reduced by 1.7–2.3 logs at 4 h and 1.3–1.4 logs at 24 h when compared to untreated controls (Fig. 4B).

Figure 4. Lubricants reduce the colonization of vaginal epithelial cells (VEC) with L. crispatus particularly when VEC are colonized with bacteria following the lubricant exposure.

Figure 4.

Three non-cytotoxic lubricants: Good Clean Love (GCL) Almost Naked, E-Z Lubricating Jelly, McKesson Lubricating Jelly) were tested to determine their impact on colonization of in vitro VEC model with L. crispatus strain JV-V01. A. VEC were pre-colonized with bacteria at the multiplicity of infection (MOI) 10 for 2 h prior to exposure to 10% (v/v) lubricants B. VEC were exposed to 10% (v/v) lubricants and immediately colonized with L. crispatus for 4 h or 24 h as described above. Colonization levels were reported as number of viable bacteria (CFU) attached to VEC per well. Data are shown as means ± SE from at least three independent experiments. P values were calculated using two-way ANOVA with Bonferroni post-test (** P<0.01; **** P<0.0001).

Discussion

The vaginal microbiota play a critical role in women’s health and disease.3,12 Particularly, Lactobacillus-dominant communities contribute to homeostasis and protect the cervicovaginal microenvironment from invading pathogens.3 In contrast, the depletion of Lactobacillus spp. and the overgrowth of diverse anaerobes (characteristic of bacterial vaginosis (BV)) can lead to numerous gynecologic and obstetric sequalae, including increased risk of STIs, preterm birth, spontaneous miscarriages, pelvic inflammatory disease and gynecologic cancer.12,13 Multiple factors have been shown to affect the vaginal microbiome, including sexual practices and use of lubricants, sex toys, and feminine hygiene products.14 Indeed, lubricants, which frequently contain antimicrobial preservatives, may directly impact bacterial communities in the cervicovaginal microenvironment.

The available epidemiological data suggest that the use of lubricants is associated with increased risk of vaginal dysbiosis or BV.1517 However, mechanistic in vitro studies investigating vaginal lubricants or feminine hygiene products are still very limited. Previously, we demonstrated the detrimental effect of hyperosmolar lubricants on the vaginal epithelium using in vitro models.5 Herein, we sought to determine the impact of personal and clinical lubricants, which varied in osmolality and formulations, on health-associated vaginal Lactobacillus spp. We examined lubricants routinely used in clinics for vaginal ultrasounds and pelvic exams (E-Z Lubricating Jelly, McKesson Lubricating Jelly, K-Y Jelly, Surgilube®), as well as, over-the-counter (OTC) personal lubricants used for sexual practices, alleviating vaginal dryness (Astroglide® Liquid, GCL Almost Naked, K-Y Jelly, K-Y Warming Jelly) or prevention of pregnancy (Conceptrol®) (Table 1).

In this study, we demonstrated that certain lubricants, such as K-Y Jelly, and Conceptrol®, exhibited antimicrobial properties against vaginal Lactobacillus spp. in vitro, despite a lack of association to high osmolality, which relates to the concentration of glycols and glycerin (Table 1). These antimicrobial properties could potentially be due to their excipients i.e. CHG, parabens, polyquaternium-15 or a known spermicide, nonoxynol-9 (N-9). Intriguingly, lubricants containing CHG (K-Y Jelly, Surgilube®) or N-9 (Conceptrol®) inhibited the most bacterial growth, whereas lubricants containing parabens or polyquartenium-15 (Astroglide® Liquid, E-Z Lubricating Jelly) did not have this effect (Fig. 1, 2) in our in vitro systems. These findings strongly suggest that CHG or N-9 in these lubricants are responsible for the bacterial growth inhibition. The disk diffusion and MIC assays performed in this study confirmed that CHG exhibit stronger antimicrobial properties against vaginal Lactobacillus spp. compared to parabens (Fig. 3).

CHG is a broad-spectrum microbicide, which can be found in a variety of products, including OTC antiseptic mouthwashes, creams, wipes, toothpastes, deodorants, sunscreens, eye drops, hair conditioners and more.9 In the context of the oral cavity, previous clinical studies demonstrated that use of mouthwash containing CHG has been linked to major shifts in the microbiota composition.18 Studies on the impact of CHG on skin microbiota have shown conflicting findings. On one hand, a minimal effect of CHG on the skin microbiota has been demonstrated, suggesting high stability and resilience of bacterial communities,19 whereas other studies showed decreased bacterial density on CHG-treated skin.20

Data presented in this in vitro study suggests that intravaginal use of products containing CHG may also have a detrimental effect on the vaginal microbiota by decreasing the overall bacterial load, including health-associated Lactobacillus spp. This might allow BV-associated bacteria to colonize the vagina, leading to vaginal dysbiosis or BV. Our findings are in accordance with a previous report showing that CHG inhibit growth not only of genital pathogens (such as Neisseria gonorrhoeae or Trichomonas vaginalis), but also vaginal Lactobacillus spp.21 A 2010 study also demonstrated that use of CHG-based Surgilube® during pelvic examination decreased the detection of group B Streptococcus (GBS), a common vaginal opportunistic pathogen.22 Intriguingly, despite the links to anaphylaxis and recent FDA ban of CHG in healthcare antiseptics and classification as ‘not generally recognized as safe and effective for use’10, the American College of Obstetricians and Gynecologists (ACOG) approved the off-label use of CHG for surgical vaginal preparation, as there is no other FDA-approved alternative to povidone-iodine.23 This approval comes from reports demonstrating that CHG is more effective than povidone-iodine at killing vaginal bacteria24 and thus far CHG has not been linked to signs of vaginal irritation.25 The discordance in recommendations from different bodies highlights the urgent need for better safety screening of female hygiene products, for example, utilizing in vitro 3-D biomimetic models and larger epidemiologic studies and trials.5

The other antimicrobial excipient, N-9, is a surfactant spermicide and an active ingredient in Conceptrol®. It has been shown in clinical and in vitro studies to cause genital inflammation and barrier breach,26 and consequently has been associated with increased HIV-1 transmission in high-risk women.27 Furthermore, the adverse effect of N-9 on vaginal Lactobacillus spp. were well characterized by several studies in the 1990s.28,29 Adverse findings on N-9 were also validated in a 2012 study, utilizing 16S rRNA sequencing, which demonstrated shifts in composition from Lactobacillus-dominant communities to communities dominated by anaerobes associated with BV, as well as Streptococcus, Enterococcus, and Escherichia, following twice-daily vaginal application of 4% N-9.30 In accordance to previous reports, Conceptrol®, containing 4% N-9, also reduced growth of vaginal Lactobacillus species in our study.

Our in vitro evidence suggest that lubricants might impact attachment of Lactobacillus to the vaginal epithelium (Fig. 4). A limited number of lubricants were tested for bacterial colonization, since the majority of products induce substantial damage to human epithelial cells due to high osmolality.5 The viscosity of the tested lubricants could have been a limitation in experiments. However, the viscosity of the lubricants does not explain the effect of tested lubricants on colonization of VEC with bacteria. This was confirmed by the use of glycerol as a viscosity control, which did not elicit any substantial decrease in Lactobacillus colonization of epithelial cells. Therefore, the reason for the reduction of L. crispatus colonization on VEC was potentially due to specific ingredients or other physical properties of lubricants than the viscosity.

Overall, this in vitro study suggests that personal and clinical lubricants containing N-9 or CHG might exhibit adverse effects on the growth of vaginal microbiota species and highlights the need for consumers and clinicians to utilize these lubricants with caution. Future clinical studies, particularly with longitudinal study designs, are needed to show whether the use of these vaginally applied products have a long-lasting effect on the microbiota in vivo and to confirm the clinical relevance of our in vitro findings. Notably, the WHO warned consumers to avoid certain ingredients that are commonly found in OTC lubricants (e.g., N-9, polyquaternium, etc.) as these may increase the risk of HIV infection.6,8 However, currently, they have made no comment of CHG regarding consumer products. Our data suggest that feminine hygiene products containing CHG could impact vaginal Lactobacillus growth and potentially re-colonization of the vagina. Ultimately, additional clinical studies and mechanistic in vitro studies, are required to investigate the impact of vaginal lubricants and other feminine products, including moisturizers, washes, wipes, creams, sprays, powders, douches, as well as probiotics/pharmaceutical vehicles and other intravaginal practices, on vaginal Lactobacillus species and cervicovaginal epithelium.

Acknowledgments

The following reagents were obtained through BEI Resources, NIAID, NIH as part of Human Microbiome Project: L. crispatus JV-V01 (bacteria, HM-103), L. gasseri JV-V03 (bacteria, HM-104), and L. jensenii JV-V16 (bacteria, HM-105). The authors would like to acknowledge the University of Bath Placement Program.

Funding

This work was supported by the National Institutes of Health NIAID Grant R01-AI119012 (to R.M.B.).

Footnotes

Potential conflicts of interest

The authors declare no conflict of interest.

References

  • 1.Herbenick D, Reece M, Schick V, Sanders SA, Fortenberry JD. Women’s use and perceptions of commercial lubricants: prevalence and characteristics in a nationally representative sample of American adults. J Sex Med. 2014; 11(3):642–652. [DOI] [PubMed] [Google Scholar]
  • 2.Crean-Tate KK, Faubion SS, Pederson HJ, Vencill JA, Batur P. Management of genitourinary syndrome of menopause in female cancer patients: a focus on vaginal hormonal therapy. Am J Obstet Gynecol. 2020; 222(2):103–113. [DOI] [PubMed] [Google Scholar]
  • 3.Martin DH, Marrazzo JM. The vaginal microbiome: Current understanding and future directions. J Infect Dis. 2016; 214 Suppl 1:S36–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ayehunie S, Wang YY, Landry T, Bogojevic S, Cone RA. Hyperosmolal vaginal lubricants markedly reduce epithelial barrier properties in a three-dimensional vaginal epithelium model. Toxicol Rep. 2018; 5:134–140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wilkinson EM, Łaniewski P, Herbst-Kralovetz MM, Brotman RM. Personal and clinical vaginal lubricants: Impact on local vaginal microenvironment and implications for epithelial cell host response and barrier function. J Infect Dis. 2019; 220(12):2009–2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Fuchs EJ, Lee LA, Torbenson MS, et al. Hyperosmolar sexual lubricant causes epithelial damage in the distal colon: potential implication for HIV transmission. J Infect Dis. 2007; 195(5):703–710. [DOI] [PubMed] [Google Scholar]
  • 7.Gorbach PM, Weiss RE, Fuchs E, et al. The slippery slope: lubricant use and rectal sexually transmitted infections: A newly identified risk. Sex Transm Dis. 2012; 39(1):59–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.World Health Organization. Use and procurement of additional lubricants for male and female condoms: WHO/UNFPA/FHI360. Advisory note. 2012.
  • 9.Halla N, Fernandes IP, Heleno SA, et al. Cosmetics preservation: A review on present strategies. Molecules. 2018; 23(7). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Food and Drug Administration. Safety and effectiveness of health care antiseptics; topical antimicrobial drug products for over-the-counter human use. Federal Register. 2017; 82(243):60474–60503. [PubMed] [Google Scholar]
  • 11.Food and Drug Administration. Safety and effectiveness of consumer antiseptic rubs; topical antimicrobial drug products for over-the-counter human use. Federal Register. 2019; 84(71):14847–14864. [PubMed] [Google Scholar]
  • 12.Hillier SL, Marrazzo J, Holmes KK. Bacterial Vaginosis. In: Holmes KK, Sparling PF, Stamm WE, et al. , eds. Sexually Transmitted Diseases, Fourth Edition. McGraw-Hill Education; 2007:737–768. [Google Scholar]
  • 13.Łaniewski P, Ilhan ZE, Herbst-Kralovetz MM. The microbiome and gynaecological cancer development, prevention and therapy. Nat Rev Urol. 2020; 17(4):232–250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Plummer EL, Vodstrcil LA, Fairley CK, et al. Sexual practices have a significant impact on the vaginal microbiota of women who have sex with women. Sci Rep. 2019; 9(1):19749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Brotman RM, Ravel J, Cone RA, Zenilman JM. Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis. Sex Transm Infect. 2010; 86(4):297–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Marrazzo JM, Thomas KK, Agnew K, Ringwood K. Prevalence and risks for bacterial vaginosis in women who have sex with women. Sex Transm Dis. 2010; 37(5):335–339. [PMC free article] [PubMed] [Google Scholar]
  • 17.Mitchell C, Manhart LE, Thomas KK, Agnew K, Marrazzo JM. Effect of sexual activity on vaginal colonization with hydrogen peroxide-producing lactobacilli and Gardnerella vaginalis. Sex Transm Dis. 2011; 38(12):1137–1144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bescos R, Ashworth A, Cutler C, et al. Effects of chlorhexidine mouthwash on the oral microbiome. Sci Rep. 2020; 10(1):5254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.SanMiguel AJ, Meisel JS, Horwinski J, Zheng Q, Bradley CW, Grice EA. Antiseptic Agents elicit short-term, personalized, and body site-specific shifts in resident skin bacterial communities. J Invest Dermatol. 2018; 138(10):2234–2243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Rhee Y, Palmer LJ, Okamoto K, et al. Differential effects of chlorhexidine skin cleansing methods on residual chlorhexidine skin concentrations and bacterial recovery. Infect Control Hosp Epidemiol. 2018; 39(4):405–411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rabe LK, Hillier SL. Effect of chlorhexidine on genital microflora, Neisseria gonorrhoeae, and Trichomonas vaginalis in vitro. Sex Transm Dis. 2000; 27(2):74–78. [DOI] [PubMed] [Google Scholar]
  • 22.Schwope OI, Chen KT, Mehta I, Re M, Rand L. The effect of a chlorhexidine-based surgical lubricant during pelvic examination on the detection of group B Streptococcus. Am J Obstet Gynecol. 2010; 202(3):276 e271–273. [DOI] [PubMed] [Google Scholar]
  • 23.Lee ASD. Conversion to Chlorhexidine Gluconate for Perioperative Vaginal Preparation: An Evidence-Based Process Improvement Project. AORN J. 2019; 110(2):145–152. [DOI] [PubMed] [Google Scholar]
  • 24.Vorherr H, Vorherr UF, Mehta P, Ulrich JA, Messer RH. Antimicrobial effect of chlorhexidine and povidone-iodine on vaginal bacteria. J Infect. 1984; 8(3):195–199. [DOI] [PubMed] [Google Scholar]
  • 25.Al-Niaimi A, Rice LW, Shitanshu U, et al. Safety and tolerability of chlorhexidine gluconate (2%) as a vaginal operative preparation in patients undergoing gynecologic surgery. Am J Infect Control. 2016; 44(9):996–998. [DOI] [PubMed] [Google Scholar]
  • 26.Hjelm BE, Berta AN, Nickerson CA, Arntzen CJ, Herbst-Kralovetz MM. Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. Biol Reprod. 2010; 82(3):617–627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Van Damme L, Ramjee G, Alary M, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet. 2002; 360(9338):971–977. [DOI] [PubMed] [Google Scholar]
  • 28.Hooton TM, Fennell CL, Clark AM, Stamm WE. Nonoxynol-9: differential antibacterial activity and enhancement of bacterial adherence to vaginal epithelial cells. J Infect Dis. 1991; 164(6):1216–1219. [DOI] [PubMed] [Google Scholar]
  • 29.Watts DH, Rabe L, Krohn MA, Aura J, Hillier SL. The effects of three nonoxynol-9 preparations on vaginal flora and epithelium. J Infect Dis. 1999; 180(2):426–437. [DOI] [PubMed] [Google Scholar]
  • 30.Ravel J, Gajer P, Fu L, et al. Twice-daily application of HIV microbicides alter the vaginal microbiota. mBio. 2012; 3(6). [DOI] [PMC free article] [PubMed] [Google Scholar]

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