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Article

Isolation and Characterization of Lactobacillus gasseri Strains from Women for Potential Vaginal Health Applications

by
Eui-Chun Chung
1,
Jong Seo Lee
1,
Hye Ji Lim
1,
Seok-Jin Kim
1,
Youn-Jee Chung
2,* and
Kum-Joo Shin
1,*
1
R&D Center, Hecto Healthcare Co., Ltd., Seoul 06142, Republic of Korea
2
Departments of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(1), 12; https://doi.org/10.3390/microbiolres16010012
Submission received: 3 December 2024 / Revised: 24 December 2024 / Accepted: 8 January 2025 / Published: 10 January 2025
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Abstract

:
Lactobacillus, a genus of lactic acid bacteria, is known to coexist symbiotically in the female vaginal microbiota and has gained attention as a potential probiotic with benefits for female reproductive health. This study aimed to evaluate the probiotic potential of Lactobacillus gasseri BELG74(BELG74), isolated from the vaginal microbiota of Korean women, in promoting vaginal health through growth ability, pH reduction, lactic acid production, and antimicrobial activity. Among 36 Lactobacillus gasseri strains, BELG74 demonstrated the highest growth capacity at 1.84 × 109 CFU/mL and the lowest pH of 3.84. BELG74 produced the most lactic acid at a concentration of 20.12 g/L, which correlated with anti-pathogenic activity against Gardnerella vaginalis, Fannyhessea vaginae, and Candida albicans of more than 90%. It also showed high acid resistance (92.2%) and bile resistance (25.3%), ensuring its survival through the gastrointestinal tract. Furthermore, BELG74 exhibited strong biofilm formation and adhesion capacity of 28.7% to HeLa cells, making it effective in colonizing the vaginal environment and suppressing pathogenic bacteria. The reduction of IL-1β by 63% suggested anti-inflammatory effects. Additionally, BELG74 effectively neutralized trimethylamine and ammonia by over 99.9%, suggesting its ability to reduce unpleasant vaginal odors. These findings indicate that BELG74 could be a promising probiotic for improving vaginal health, with further clinical studies needed to confirm these benefits.

1. Introduction

Lactic acid bacteria (LAB), anaerobic microorganisms capable of producing various metabolic substances, are well suited to inhabit specific tissues or organs of their host [1]. Consequently, LAB are commonly found in plants, fermented foods, and the mucosa of humans and animals. Within humans and animals, LAB naturally reside in various ecosystems, including the gastrointestinal and urogenital tracts, as part of normal microbial communities [2].
The vagina, located at the entrance of the uterus and in close proximity to the urethra and anus, is susceptible to exposure to colonic microbiota or uropathogenic bacteria [3]. A healthy vaginal microbiome plays a crucial role in reproductive health. The vaginal microbiome comprises more than about 580 species of microorganisms [4]. In healthy women, various types of LAB primarily inhabit the vagina, protecting it from pathophysiological and structural risks [5]. Research on the vaginal microbiome shows that, in healthy women, approximately 80% of the total vaginal microbiome is composed of Lactobacillus species. Among these, about 60% belong to Lactobacillus crispatus, 20% to Lactobacillus iners, and Lactobacillus gasseri and Lactobacillus jensenii are each present at a similar level of 5%, as dominant Lactobacilli in the female vagina [6]. These species help maintain a low vaginal pH (below 4.5) by producing lactic acid, which protects against pathogens. However, Lactobacillus iners has been found to be less suitable for vaginal health due to its tendency to increase vaginal pH, creating a more pathogen-susceptible environment [7,8]. The composition of Lactobacillus species in the vagina varies among individuals due to factors such as environmental influences (e.g., diet), hormonal levels (e.g., estrogen), and health status (e.g., sexually transmitted infections). Additionally, this composition can change in response to various factors, including infections, alterations in pH levels, or pregnancy, all of which significantly influence the vaginal microbiota [5].
Bacterial vaginosis (BV) is a condition characterized by a decline in beneficial LAB and an overgrowth of anaerobic and other bacterial species, such as Gardnerella vaginalis, Fannyhessea, Mobiluncus, Prevotella, and Bacteroides [9,10]. Among these, Gardnerella vaginalis (G. vaginalis) has been reported as the causative agent of the clinical signs and symptoms of BV [11]. According to the change in microbial composition, lactic acid concentration rapidly decreases from about 110 mM to less than 20 mM, and vaginal pH increases above 4.5 along with vaginosis symptoms such as itching and grayish-white discharge [12,13]. Antibiotics are commonly used to treat BV and are effective in the short term. However, repeated use can lead to increased recurrence rates, the emergence of antibiotic-resistant strains, and a reduction in microbial diversity in both the gut and vagina [14]. Additionally, disruptions in the vaginal microbiota can trigger conditions such as vulvovaginal candidiasis caused by Candida albicans [15]. LAB, particularly Lactobacillus species, play a key role in preventing such disruptions by maintaining a low vaginal pH and producing antimicrobial compounds [16]. The association between high lactic acid concentration and a Lactobacilli-dominant vaginal environment suggests beneficial properties attributed to Lactobacilli, indicating that Lactobacillus probiotics may improve vaginal health.
Probiotics can colonize the gastrointestinal tract, providing long-term beneficial effects, and thus have been used for over a century in the treatment of various mucosal surface infections (e.g., intestinal and vaginal infections) [17]. Probiotics lower pH by producing organic acids in the mucosal lumen, inhibit the adhesion of pathogenic strains to epithelial cells, and produce antimicrobial compounds such as bacteriocins, defensins, and H2O2 to block the colonization of pathogenic bacteria. Furthermore, Lactobacillus gasseri (L. gasseri), one of the dominant species in the vaginal microbiota of healthy women, has been known to enhance mucosal immune responses through interactions with epithelial and lymphoid cells, making them a promising alternative or adjunct to antibiotics for treating vaginal infections [18,19,20]. These characteristics may play an important role in supporting women’s reproductive health. Additionally, L. gasseri is classified as a species with proven safety and commercial viability by Korea’s Ministry of Food and Drug Safety (MFDS).
In this study, we isolated L. gasseri strains from the vaginal microbiota of healthy Korean women and evaluated their lactic acid production capacity and antimicrobial activity to identify effective strains for improving vaginal health. Additionally, we assessed various physiological properties of the selected strains to confirm their potential as probiotics, as a promising alternative to antibiotics for treating BV and related conditions.
As far as we know, extensive research has been conducted globally on the use of lactic acid bacteria isolated from the female vagina to promote vaginal health. However, such studies have not been carried out in Korea. Therefore, there is an opportunity for expanding our understanding of the vaginal microbiota and its potential application in women’s health.

2. Materials and Methods

2.1. Sample Collection and Isolation of LAB

A total of 21 healthy Korean women of reproductive age (20–45 years), enrolled at Seoul St. Mary’s Hospital, The Catholic University of Korea, provided vaginal swab samples. Samples were combined with sterile 0.85% NaCl saline, stored at 4 °C, and used for isolation of LAB within 2 h. This study was conducted after obtaining informed consent from the participants under approval of the Institutional Review Board of Seoul St. Mary’s Hospital (KC22TISI0524).
Each vaginal swab sample was diluted tenfold in phosphate-buffered saline (PBS), and 0.1 mL of the diluted sample was plated on MRS agar (Difco, Sparks, NV, USA) and MRS agar supplemented with mucin (Sigma Aldrich, St. Louis, MO, USA) to confirm pure isolation. The isolated strains were stored in 15% glycerol solution at −80 °C. For strain identification, 16S rRNA sequencing was performed by Bionics (Seoul, Republic of Korea). DNA was extracted from a single colony using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA). The extracted DNA served as a template for PCR amplification and the 16S rRNA gene region was amplified using the primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′), 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′), 518F (5′-CCA GCA GCC GCG GTA ATA CG-3′), and 800R (5′-TAC CAG GGT ATC TAA TCC-3′). The sequences were compared for homology with other standard strains in the EzBioCloud 16S rRNA database (http://www.ezbiocloud.net).

2.2. Measurement of Growth Ability

The isolated LAB were anaerobically cultured in an incubator (Don Whitley scientific, Bingley, UK) at 37 °C for 18 h in MRS broth. The culture was then diluted 1000-fold in MRS broth and dispensed into a 96-well plate (SPL, Pocheon, Republic of Korea). Optical density (OD) values were measured at 600 nm using a microplate reader (Molecular Devices, San Jose, CA, USA) at 0, 4, 8, and 24 h, and after 24 h, colony forming units (CFU/mL) were counted.

2.3. Measurement of pH in Culture Medium

The isolated LAB were anaerobically cultured at 37 °C for 20 h in MRS broth. The culture was centrifuged at 3500 rpm for 15 min (LABOGENE, Lillerød, Denmark) to obtain the cell-free supernatant, which was then filtered using a 0.22 µm syringe filter (Merck Millipore, Burlington, VT, USA). The pH was measured using a pH meter (Mettler Toledo, Greifensee, Switzerland).

2.4. Measurement of Lactic Acid in Culture Medium

The LAB were anaerobically cultured at 37 °C for 20 h in MRS broth. The culture was centrifuged (3600 rpm for 15 min) to obtain the cell-free supernatant, which was filtered through a 0.22 µm syringe filter. Total lactic acid was measured using the D/L-Lactic acid (Rapid) assay kit (Megazyme, Bray, Ireland), following the protocol provided in the kit.

2.5. Inhibition of Pathogenic Bacteria and Fungi

The LAB were inoculated into MRS broth and anaerobically cultured at 37 °C for 20 h, followed by centrifugation to obtain the cell-free supernatant (CFS). G. vaginalis KCTC 5096T and Fannyhessea vaginae (F. vaginae) KCTC 15240 T strains, obtained from the Korea Collection for Type Cultures (KCTC, Jeongeup, Republic of Korea), were cultured anaerobically at 37 °C for 24 h in Columbia blood broth (MB cell, Seoul, Republic of Korea) containing 5% sheep blood. The pathogenic bacteria culture was adjusted to a final concentration of 1 × 105 CFU/mL. The LAB CFS was mixed with the cultures of G. vaginalis and F. vaginae at ratios of 1:4 and 1:8, respectively, and the mixtures were incubated anaerobically at 37 °C for 24 h. The CFU of the mixtures was measured using Columbia blood agar (MB cell, Seoul, Republic of Korea).
C. albicans SC5314 obtained from the Korea Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea) was cultured in yeast malt (YM) broth (MB cell, Seoul, Republic of Korea) at 37 °C for 24 h under aerobic conditions. The C. albicans culture was adjusted to a final concentration of 1 × 103 CFU/mL, and then, the LAB CFS and C. albicans culture were mixed in a 1:2 ratio. The mixture was incubated aerobically at 37 °C for 48 h (SCI FIENTECH, Seoul, Republic of Korea). The CFU of the mixtures was measured using YM agar.

2.6. Measurement of Acid and Bile Salt Tolerance and Biofilm Formation

Lactobacillus gasseri BELG74 (BELG74) was anaerobically cultured in MRS broth at 37 °C for 20 h. The culture was centrifuged at 3600 rpm for 15 min to remove the supernatant, and the bacterial pellet was washed with PBS. To test acid tolerance, the pellet was exposed to 1000 units/mL pepsin solution (pH 2.5) (Roche, Rotkreuz, Switzerland) for 2 h at 37 °C. The culture was then diluted in 0.85% NaCl to measure the bacterial count, and survival rates were calculated based on counts before treatment with the pepsin solution. For bile salt tolerance, the bacterial pellet was cultured in MRS broth containing 0.3% oxgall (Difco, Sparks, NV, USA) for 5 h at 37 °C. The bacterial count was measured after dilution in 0.85% NaCl, and survival rates were calculated based on counts before treatment with the bile salts.
Biofilm formation ability was assessed by transferring 200 μL of the subcultured BELG74 to 5 mL of MRS medium without Tween 80, stirring the mixture, and then inoculating it into a 96-well plate. The microplate was incubated anaerobically for 72 h. After incubation, the supernatant was removed, and the cells were washed once with PBS. The cells were fixed at 60 °C for 1 h, followed by staining with 0.1% crystal violet for 30 min. After staining, the cells were washed twice with distilled water and decolorized with 200 μL of 96% ethanol. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices, San Jose, CA, USA). Additionally, a sterile culture medium was used as negative control (ODc). The strain was considered as nonbiofilm (OD ≤ ODc); weak biofilm producers (ODc ≤ OD ≤ 2 × ODc); moderate biofilm producers (2 × ODc ≤ OD ≤ 4 × ODc); and strong biofilm producers (4 × ODc ≤ OD ≤ 8 × ODc).

2.7. Measurement of Adhesion Indicators

Auto-aggregation was assessed by culturing BELG74 in MRS broth at 37 °C for 20 h, followed by centrifugation at 3600 rpm for 15 min. The supernatant was discarded, and the pellet was washed twice with PBS. The pellet was resuspended in 10 mL of PBS to adjust the OD to 0.55–0.6 (A0). This suspension was placed in a 15 mL conical tube, incubated anaerobically at 37 °C for 24 h, and 0.2 mL of upper suspension was transferred to a 96-well plate to measure OD at 600 nm (A1). The auto-aggregation was evaluated by measuring the residual turbidity in the supernatant, which was calculated using the following formula: %Auto-aggregation = (1−A1/A0) × 100.
Hydrophobicity was measured by culturing BELG74 in MRS broth at 37 °C for 20 h, followed by centrifugation at 3600 rpm for 15 min. After washing the bacterial pellet twice with PBS, the pellet was resuspended in 10 mL of PBS to adjust the absorbance to 0.55–0.6 in 10 mL of PBS. (H0). A volume of 3 mL of this suspension was mixed with 0.6 mL of hexane and vortexed for 1 min, then allowed to stabilize at room temperature for 10 min. A 0.2 mL aliquot of the aqueous phase was transferred to a 96-well plate to measure OD at 600 nm (H1). Hydrophobicity was calculated using the following formula: %Hydrophobicity = (H0−H1) H0 × 100.
To evaluate adhesion to cervical cells, HeLa cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in EMEM media (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 mg/mL) in an incubator (Thermo Fisher, Waltham, MA, US) with 5% CO2 at 37 °C. BELG74 was cultured in MRS broth at 37 °C for 20 h, then washed twice with PBS and resuspended with EMEM media. For the adhesion assay, HeLa cells were seeded at a density of 1.5 × 106 cells in a 60 mm cell culture dish. After 24 h, when a monolayer was formed, the prepared BELG74 of 1.5 × 108 CFU (MOI 100) was inoculated and co-cultured for 2 h at 37 °C in an incubator with 5% CO2. Afterward, the supernatant was carefully removed, and the cells along with attached bacteria were detached using 200 µL of 0.25% trypsin-EDTA. An additional 800 µL of PBS was added to recover the bacteria, and the CFU of the recovered bacteria was measured.

2.8. Measurement of Cytokine Production in Macrophages

To assess the anti-inflammatory effect of BELG74 in preventing or improving vaginal inflammation, the ability of the strain to regulate IL-1β and IL-6 secretion in RAW 264.7 cells and THP-1 cells was evaluated, respectively.
RAW 264.7 cells (obtained from Manassas, VA, USA) were cultured in DMEM (ATCC, Manassas, VA, USA) supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 mg/mL). Cells were plated at a density of 2.5 × 105 cells in a 24-well plate (Sigma Aldrich, St. Louis, MO, USA) and incubated for 4 h in an incubator with 5% CO2 at 37 °C. Then, 1.25 × 107 CFU (MOI 50) of BELG74 was inoculated into wells containing RAW 264.7 cells, followed by treatment with 1 µL of lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 1 mg/mL. The cells were incubated for 24 h in an incubator with 5% CO2 at 37 °C. After incubation, the media were carefully collected and centrifuged. Then, the supernatant was analyzed for IL-1β and IL-6 levels using an ELISA Kit (Cusabio, Houston, TX, USA). The negative control group was treated with neither the strain nor LPS. The positive control group was treated with only LPS at a concentration of 1 mg/mL.
THP-1 cells obtained from ATCC (Manassas, VA, USA) were cultured in RPMI supplemented with 10% FBS, 50 mM ß-mercaptoethanol, penicillin (100 IU/mL), and streptomycin (100 mg/mL). Cells were plated at a density of 2 × 105 cells in a 96-well plate with phorbol 12-myristate 13-acetate (10 μg/mL) and incubated for 24 h in an incubator with 5% CO2 at 37 °C. BELG74 was cultured in MRS broth at 37 °C for 20 h. Then, 1 × 107 CFU (MOI 50) of BELG74 was inoculated into wells containing THP-1 cells, followed by treatment with 1 µL of LPS (1 mg/mL). The cells were incubated for 24 h in an incubator with 5% CO2 at 37 °C.

2.9. Measurement of Odor Removal Capacity for Ammonia and Trimethylamine

The odor removal capacity of BELG74 against trimethylamine (TMA) and ammonia was conducted by the Korea Standard Testing and Research Institute (KSTR) using the standard testing method KSI 2218, with a detector tube gas measurement system. One milliliter of bacterial culture supernatant was placed in a 5 L chamber with 100 mg/kg TMA or 28 mg/kg ammonia gas, and the deodorization rate was measured after 2 h.

2.10. Statistical Analysis

All experiments were performed in triplicate. Results are expressed as mean ±standard deviation. To ascertain whether mean differences between variables were statistically significant (p ≤ 0.05), statistical analysis was performed using Student’s t-test.

3. Results and Discussion

3.1. Growth and pH of L. gasseri Strains

The healthy female vagina is mildly acidic due to lactic-acid-producing Lactobacillus species fermenting glycogen to generate lactic acid and maintain this optimal environment [21]. L. gasseri, one of the key species found in the vaginal microbiota, is known for various health benefits, including protection against pathogens and enhancement of mucosal immune responses [22,23]. Among 36 L. gasseri strains isolated from healthy Korean women, the strains with superior growth and low pH-inducing ability were selected by measuring the viable cell counts in the culture and pH of the supernatants. As a result, four strains, BCC-LG-49, BCC-LG-50, BCC-LG70, and BELG74, were selected with high viable cell counts of over 1 × 109 CFU/mL and relatively low pH, which is represented in a box in Figure 1.

3.2. Lactic Acid Production of L. gasseri Strains

Lactic acid production was measured for four strains with high growth activity. In the human body, lactic acid predominantly exists in the form of L-lactate and is one of the main organic acids produced by LAB, known to effectively improve the vaginal environment [10]. We measured the D/L-lactate content in the culture supernatants of the four selected L. gasseri strains, and the results are depicted in Figure 2. The lactic acid concentrations ranged from 13.94 to 20.12 g/L, with BELG74 showing the highest content at 20.12 g/L, confirming its superior lactic acid production capability among the four strains.
The average vaginal pH in healthy women is reported to be around 3.5 to 4.5, and high lactate production can support this environment [24]. Previous studies have shown that the lactic acid produced by L. gasseri can exhibit antimicrobial activity. For instance, growth of pathogenic gram-negative bacteria was inhibited when exposed to 9 g/L lactic acid produced by L. gasseri [25]. In the present study, the lactic acid production capacity of L. gasseri BELG74 suggests its potential not only to improve the vaginal environment but also to exhibit antimicrobial activity.

3.3. Antimicrobial Activity Against Vaginal Pathogens

The vagina hosts a variety of microbial species, and when harmful bacteria and fungi such as anaerobic microorganisms increase in number beyond the dominant species, gynecological conditions such as BV and vaginitis can occur. L. gasseri is a dominant LAB species present in the female vagina, and previous studies have reported that the L. gasseri VHProbio E09 strain can inhibit the growth of G. vaginalis and C. albicans under co-culture conditions [26]. Since there is a need to evaluate the potential impact of antimicrobial substances and organic acids produced by L. gasseri, we assessed the antimicrobial activity of supernatants from vagina-derived L. gasseri strains. The antimicrobial activity of four selected L. gasseri strains was assessed against three representative vaginal pathogenic species, G. vaginalis, F. vaginae, and C. albicans. All four L. gasseri strains exhibited over 50% antibacterial activity against G. vaginalis and F. vaginae, with BELG74 showing the highest antibacterial activities of 99.99% and 95.04% against G. vaginalis and F. vaginae, respectively (Figure 3a,b). Moreover, all four strains exhibited >80% inhibition against C. albicans, with BELG74 demonstrating the highest inhibition rate of approximately 96.4% (Figure 3c). Both BELG74 and L. gasseri BCC-LG-50 strains exhibited over 95% inhibition against G. vaginalis. However, BELG74 demonstrated a broader antimicrobial spectrum, showing over 95% inhibition against three species, including F. vaginae and C. albicans.
The antimicrobial mechanisms of Lactobacillus spp. can vary among strains, depending on the types and quantities of antimicrobial substances produced, such as organic acids, especially lactic acid, bacteriocins, and hydrogen peroxide, as well as their specific modes of action against particular pathogens [27]. Therefore, the similar antimicrobial activity of BCC-LG-50 and BELG74 against G. vaginalis, as well as the higher antimicrobial activity of BELG74 against F. vaginae and C. albicans, could be mediated by the specific types and quantities of antimicrobial substances produced and their modes of action against these pathogens. Further investigation is needed to elucidate these mechanisms in the future. Thus, based on the growth, lactic acid production, and antimicrobial activity, BELG74 was selected for further evaluation of its physiological activities beneficial to vaginal health.

3.4. Genetic Identification of L. gasseri BELG74

Among 36 vaginal-derived L. gasseri strains, four strains with good growth performance were selected, and BELG74 was ultimately chosen as the best strain based on its lactic acid production capability and antimicrobial activity. As a predominant species of the Lactobacillus genus, L. gasseri is a well-documented safe probiotic bacterium. L. gasseri is a species known for its various functional benefits and is naturally found in environments such as fermented products, the gastrointestinal tract, oral cavity, and vaginal ecosystem. It has been reported to possess multiple probiotic properties, including regulating the gut microbiota, exhibiting anti-inflammatory and antimicrobial activities, and maintaining the vaginal microbiota in women [28]. The genetic identification of the selected BELG74 revealed a sequence that showed 99.86% homology with Lactobacillus gasseri ATCC3323. Using the Mega X program, we analyzed the evolutionary distance among Lactobacillus species, and the phylogenetic tree is illustrated in Figure 4.

3.5. Acid and Bile Resistance, and Biofilm Formation of L. gasseri BELG74

Probiotics must survive passage through the gastrointestinal tract to provide potential benefits; they need to remain viable in sufficient numbers to interact with the host in the small or large intestine. However, they can be affected by the high acidity and digestive enzymes in the stomach [29]. Various studies have indicated that probiotics administered orally can transition through the gastrointestinal tract to the vaginal area, potentially improving conditions such as BV [30]. The biofilm formed by probiotic bacteria, such as Lactobacillus spp., is a beneficial trait as it can enhance colonization, promote long-term persistence at host gastrointestinal mucosal surfaces, and inhibit the colonization of pathogenic strains [31]. To confirm the strain’s survival capabilities in the gastrointestinal tract, we assessed its acid and bile resistance, as well as biofilm formation ability, as shown in Table 1. BELG74 exhibited high acid resistance of approximately 92.2% after exposure to a pepsin solution at pH 2.5 for 2 h, and about 25.3% resistance to 0.3% oxgall.
Lacticaseibacillus rhamnosus GG (LGG) and Lactiplantibacillus plantarum WCFS1, widely used probiotics, showed survival rates of 23.26% and 19.89%, respectively, after being exposed to artificial gastric fluid at pH 2.5 for 2 h [32]. In contrast, LGG showed a high bile resistance of 89.1% [33]. Therefore, BELG74 demonstrated better acid resistance and less bile resistance than known probiotics. Considering probiotics should pass through the stomach and intestine, BELG74 has a competitive characteristic in terms of acid and bile resistance.
Additionally, BELG74 demonstrated strong biofilm production, as evidenced by an OD more than four times higher compared to the negative control (Table 1). These characteristics indicate its excellent capabilities as a probiotic strain suited for gastrointestinal passage. Oral ingestion of Lactobacillus allows the bacteria to reach the vagina via perineal skin contact, after passing through the oral cavity, stomach, and intestines [30]. BELG74 demonstrates high acid and bile resistance and biofilm formation, suggesting its potential to reach the vagina and exert its beneficial effects.

3.6. Adhesion Properties of L. gasseri BELG74

Adhesion ability is essential for LAB to function as probiotics, but it is also a key property for pathogenic bacteria such as G. vaginalis, C. albicans, and Escherichia coli, which attach to epithelial cells and form biofilms, leading to diseases [34]. Therefore, LAB can inhibit the growth of pathogens by colonizing vaginal epithelial cells and preventing the pathogens from adhering to the host cells [35]. The auto-aggregation and hydrophobicity characteristics of LAB are related to their ability to adhere to epithelial barriers [36]. High auto-aggregation capacity in LAB strains can influence their ability to remain longer in the host [37], and the hydrophobicity of bacterial cell surfaces provides an advantage for adhering to epithelial barriers [38]. Therefore, the auto-aggregation ability and cell surface hydrophobicity of BELG74 were measured as indicators of adhesion ability, and its adhesion capacity was further evaluated using HeLa cells.
BELG74 exhibited an auto-aggregation ability of 52.3% (Table 2). In a previous report by Rajab S et al., auto-aggregation levels of LAB ranged from approximately 11.3% to 48.5%, therefore, BELG74 may possess superior aggregation ability among LAB strains. Regarding cell surface hydrophobicity, previous studies reported a range of 6% to 78% hydrophobicity for LAB [39]. In this study, BELG74 demonstrated a hydrophobicity of 92.1% (Table 2), indicating an exceptional level compared to previous studies. The adhesion ability of BELG74 to HeLa cervical cells was evaluated, and the viable cell count after co-incubating BELG74 with HeLa cells for 2 h is presented in Table 2. The result showed that BELG74 exhibited a relatively high adhesion capacity with about 28.7% of the initially applied bacteria adhering to the HeLa cells. In a previous study of the adhesion ability of probiotics to HeLa cells, the UREX (GR-1, RC14) strains, which are known as probiotics for vaginal health, showed an adhesion ability of approximately 1 × 107 or less after co-incubation with 1 × 109 CFU/mL for 1 h, which was calculated to be less than 1% [40]. In contrast, BELG74 in this study demonstrated 28.8% adhesion ability, which is superior to the commercial UREX strains.
These results related to adhesion properties suggest that BELG74 may be effective in adhering to vaginal epithelial cells, which is supported by previous studies showing that vaginally derived Lactobacillus spp. strains can effectively adhere to vaginal epithelial cells [41]. It has been reported that L. gasseri isolated in the female vagina produces extracellular polysaccharides, which can enhance adhesion ability, leading to an increase in biofilm formation [42]. While our study confirmed the adhesion-related bioactivity of BELG74, further research is needed to elucidate the underlying mechanisms. Considering the adhesion property is a critical factor in inhibiting BV, clinical studies are needed to further evaluate whether BELG74 can colonize the vagina, suppress pathogenic microorganisms, and alleviate BV.

3.7. Anti-Inflammatory Cytokine Modulation by L. gasseri BELG74

In inflammation, macrophages play a significant role in regulating inflammatory responses and maintaining homeostasis [43]. LPS is a bacteria-derived material widely used to induce inflammatory responses in research [44]. In this study, the anti-inflammatory potential of BELG74 was assessed using RAW 264.7 murine macrophage and THP-1 human macrophage cell lines stimulated with LPS. After 24 h of co-incubation of BELG74 with RAW 264.7 or THP-1 cells in the presence of LPS, IL-1β levels in RAW 264.7 cells and IL-6 levels in THP-1 cells were measured, resulting in significant reduction by 63% and 21%, respectively (Figure 5).
BV is the most common condition affecting the vaginal microbiota and triggers inflammatory immune responses in the cervical and vaginal mucosa. In inflammatory responses, IL-1β is induced in response to infection and tissue damage [45]. Previous studies indicate increased cytokine levels in the cervico-vaginal fluid during BV, particularly with elevated IL-1β levels, correlating with higher levels of G. vaginalis and F. vaginae. Additionally, in vitro studies using cell lines have demonstrated that G. vaginalis, F. vaginae, and other BV-associated bacterial species induce IL-6 expression [46]. In this study, BELG74 inhibited the pro-inflammatory cytokines IL-1β by 63% and IL-6 by 21% in macrophages. The observed anti-inflammatory effects suggest its potential to improve vaginal inflammation induced by BV. Given that BELG74 is a strain isolated from the vaginal microbiota, it is likely to exert these anti-inflammatory effects within the vaginal environment, supporting its potential use as a beneficial strain for women’s vaginal health. Research has shown that L. gasseri LGV03, isolated from the cervical-vaginal area, can modulate immune responses by altering neutrophil levels in zebrafish [47]. Additionally, a study has reported that L. gasseri can reduce pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages infected with Helicobacter pylori [48]. These findings show similar trends to our BELG74 results. Although the anti-inflammatory mechanism of L. gasseri strains is not yet fully understood, various components of L. gasseri can be hypothesized to show anti-inflammatory activity. The cell wall of Lactobacillus contains components like lipoteichoic acid, peptidoglycan, wall teichoic acid, extracellular polysaccharides, and lipoic acid, which regulate immune responses via Toll-like receptor 4 [49]. Metabolites from Lactobacillus culture medium have been shown to inhibit IL-1β in mouse macrophages [50]. Moreover, a study demonstrated that extracellular vesicles from Lactobacillus effectively reduced the expression of LPS-induced pro-inflammatory cytokines, including IL-1α, IL-1β, IL-2, and TNFα, while enhancing the expression of anti-inflammatory cytokines, such as IL-10 and TGFβ, in vitro [51]. The molecular mechanisms underlying the anti-inflammatory effects of BELG74, whether they are due to cellular components, metabolites, extracellular vesicles, or other substances, need to be elucidated in future studies. Moreover, examination of the anti-inflammatory effects of BELG74 was conducted using both murine and human-derived macrophages, and further investigation is required to confirm the anti-inflammatory effect of BELG74 in clinical settings.

3.8. Odor Neutralization Ability of L. gasseri BELG74

BV is clinically characterized by a “fishy odor”. This odor originates from biogenic amines (BAs), which are organic compounds containing one or more amine (NH2) groups. These compounds, including polyamines such as putrescine, cadaverine, and trimethylamine, are associated with an increase in vaginal BAs [52]. The unpleasant odor associated with vaginal discharge is primarily attributed to trimethylamine (TMA). TMA is a key indicator for the clinical diagnosis of BV, characterized by a depletion of lactobacilli and an overgrowth of anaerobic bacteria. Studies have shown that up to 27% of women experience a depletion of Lactobacillus spp. in their vagina [10]. This microbial dysbiosis, with reduced Lactobacillus, leads to increased production of TMA due to the breakdown of coline compounds by outgrown pathogenic vaginal bacteria [52]. To assess the odor neutralization capability of BELG74, we evaluated its effectiveness against TMA and ammonia, and the results are summarized in Table 3. BELG74 culture effectively reduced ammonia and TMA levels by 99.9%. Taken together, the deodorizing capability of BELG74 against TMA and ammonia may help mitigate unpleasant odors associated with vaginal microbial imbalances.
Previous studies have reported the presence of BA-degrading amino oxidase genes, including monoamine oxidase, diamine oxidase, and multicopper oxidase, in Lactobacillus species [53,54]. Additionally, novel enzymes from LAB such as laccase and glyceraldehyde-3-phosphate dehydrogenase have been identified as capable of reducing BAs [55,56]. Studies on specific enzymatic mechanisms by which BELG74 reduces BAs such as ammonia and trimethylamine are needed in the future.

4. Conclusions

In conclusion, this in vitro study demonstrated that BELG74 effectively inhibited the vaginal pathogens, G. vaginalis and C. albicans, and exhibited bioactive properties that could function effectively in the vaginal environment such as epithelial cell adhesion, anti-inflammation and odor neutralization. These findings suggest that BELG74 has potential as a probiotic for improving vaginal health.
The composition of the vaginal microbiota varies by ethnicity, with differences reported between countries in terms of species diversity and environmental factors such as pH [57]. These differences suggest that the required efficacy of probiotics may vary depending on ethnicity. Currently, no studies have examined the application of vaginal probiotics across different ethnic groups. However, since commercial probiotic products are used worldwide, it is likely that their applicability will extend across populations, which could apply to the BELG74 identified and characterized in this study, although there is still a need to investigate this further.
In the future, it is necessary to investigate the mechanisms underlying the antibacterial and anti-inflammatory effects of BELG74, such as its metabolites and cell wall components. Additionally, the in vivo efficacy of BELG74 should be evaluated using a mouse model infected with G. vaginalis, which will involve assessing the inhibition of vaginal infection and examining both local and systemic immune responses. Furthermore, clinical studies need to be conducted with patients diagnosed with BV to evaluate the efficacy through various clinical parameters, microbiological evaluations, and patient’s quality of life.

Author Contributions

Conceptualization, K.-J.S.; methodology, E.-C.C. and J.S.L.; software, E.-C.C.; validation, J.S.L.; formal analysis, H.J.L. and E.-C.C.; resources, Y.-J.C. and H.J.L.; data curation, H.J.L. and E.-C.C.; writing—original draft preparation, E.-C.C.; writing—review and editing, J.S.L. and K.-J.S.; visualization, E.-C.C.; supervision, J.S.L.; project administration, S.-J.K. and K.-J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2019R1G1A1100229) and the Catholic Medical Center Research Foundation in the program year of 2019.

Institutional Review Board Statement

The study was approved by the Public Institutional Review Board designated by the Ministry of Health and Welfare (IRB number: KC22TISI0524).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement

The dataset used and/or analyzed in the current study is available from the corresponding author upon reasonable request.

Conflicts of Interest

Authors E.-C.C., J.S.L., H.J.L., K.-J.S. and S.-J.K. were employed by the company Hectohealthcare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Microbiolres 16 00012 g001
Figure 1. Viable cell counts and pH of 36 strains of Lactobacillus gasseri (L. gasseri) isolated from Korean female vagina.
Figure 1. Viable cell counts and pH of 36 strains of Lactobacillus gasseri (L. gasseri) isolated from Korean female vagina.
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Figure 2. Lactic acid production capacity of four strains of L. gasseri with high growth activity. Each column represents the mean of three independent experiments; the bars represent the standard deviation.
Figure 2. Lactic acid production capacity of four strains of L. gasseri with high growth activity. Each column represents the mean of three independent experiments; the bars represent the standard deviation.
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Figure 3. Antimicrobial activity of L. gasseri strains against female vaginal pathogens: (a) antimicrobial activity against G. vaginalis, (b) antimicrobial activity against F. vaginae, (c) antifungal activity against C. albicans. Each column represents the mean of three independent experiments, each performed in duplicate; the bars represent the standard deviation.
Figure 3. Antimicrobial activity of L. gasseri strains against female vaginal pathogens: (a) antimicrobial activity against G. vaginalis, (b) antimicrobial activity against F. vaginae, (c) antifungal activity against C. albicans. Each column represents the mean of three independent experiments, each performed in duplicate; the bars represent the standard deviation.
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Figure 4. Phylogenetic tree of L. gasseri BELG74 based on 16S rRNA sequences.
Figure 4. Phylogenetic tree of L. gasseri BELG74 based on 16S rRNA sequences.
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Figure 5. Anti-inflammatory activities of L. gasseri BELG74: (a) inhibition of pro-inflammatory cytokine (IL-1β) release in RAW 264.7 cells, (b) inhibition of inflammatory cytokine (IL-6) release in THP-1 cells. Statistical significance is indicated by * p < 0.05 and ** p < 0.01 as compared to the untreated group. NC, negative control.
Figure 5. Anti-inflammatory activities of L. gasseri BELG74: (a) inhibition of pro-inflammatory cytokine (IL-1β) release in RAW 264.7 cells, (b) inhibition of inflammatory cytokine (IL-6) release in THP-1 cells. Statistical significance is indicated by * p < 0.05 and ** p < 0.01 as compared to the untreated group. NC, negative control.
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Table 1. Acid and bile resistance and biofilm formation of L. gasseri BELG74.
Table 1. Acid and bile resistance and biofilm formation of L. gasseri BELG74.
Acid Resistance
(%)		Bile Resistance
(%)		Biofilm Formation
(Negative Control:0.1)
92.2 ± 7.0725.3 ± 3.100.44 ± 0.03
Each column represents the mean of three independent experiments.
Table 2. Adhesion ability of L. gasseri BELG74.
Table 2. Adhesion ability of L. gasseri BELG74.
Auto-Aggregation
(%)		Hydrophobicity
(%)		Adhesion Activity
(%)
52.3.2 ± 7.9492.1 ± 0.2428.8 ± 1.80
Each column represents the mean of three independent experiments.
Table 3. Trimethylamine and Ammonia Reduction by L. gasseri BELG74.
Table 3. Trimethylamine and Ammonia Reduction by L. gasseri BELG74.
MaterialTest GroupVolatile Nitrogen Compounds (mg/kg)Inhibition (%)
NH3
(Ammonia)		Negative Control1000
BELG74099.9
C3H9N
(Trimethylamine)		Negative Control280
BELG74099.9
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Chung, E.-C.; Lee, J.S.; Lim, H.J.; Kim, S.-J.; Chung, Y.-J.; Shin, K.-J. Isolation and Characterization of Lactobacillus gasseri Strains from Women for Potential Vaginal Health Applications. Microbiol. Res. 2025, 16, 12. https://doi.org/10.3390/microbiolres16010012

AMA Style

Chung E-C, Lee JS, Lim HJ, Kim S-J, Chung Y-J, Shin K-J. Isolation and Characterization of Lactobacillus gasseri Strains from Women for Potential Vaginal Health Applications. Microbiology Research. 2025; 16(1):12. https://doi.org/10.3390/microbiolres16010012

Chicago/Turabian Style

Chung, Eui-Chun, Jong Seo Lee, Hye Ji Lim, Seok-Jin Kim, Youn-Jee Chung, and Kum-Joo Shin. 2025. "Isolation and Characterization of Lactobacillus gasseri Strains from Women for Potential Vaginal Health Applications" Microbiology Research 16, no. 1: 12. https://doi.org/10.3390/microbiolres16010012

APA Style

Chung, E.-C., Lee, J. S., Lim, H. J., Kim, S.-J., Chung, Y.-J., & Shin, K.-J. (2025). Isolation and Characterization of Lactobacillus gasseri Strains from Women for Potential Vaginal Health Applications. Microbiology Research, 16(1), 12. https://doi.org/10.3390/microbiolres16010012

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