Characterization of Vaginal Lactobacilli with Potential Probiotic Properties Isolated from Healthy Women in Northern Iran

Abstract

Vaginal lactobacilli protect against bacterial vaginosis and vaginal candidiasis. They may have probiotic properties and help maintain the balance and health of the vaginal ecosystem while the loss of these bacteria predisposes females to urinary and genital infections. The aim of this study was to investigate the probiotic potential of vaginal Lactobacillus among healthy females in northern Iran. The Lactobacillus strains were isolated from vaginal samples and were identified by sequencing of the 16S rRNA fragment. Functional properties such as tolerance to low pH, H₂O₂ production, adherence ability to Hela cells and antagonistic activity against Candida albicans was examined. A total of 38 vaginal lactobacilli strains from five species, including Lactobacillus crispatus (n = 13), Lactobacillus gasseri (n = 10), Lactobacillus acidophilus (n = 6), Lactobacillus jensenii (n = 5) and Lactobacillus johnsonii (n = 4), were identified. All of the species showed significant tolerance to low pH over 24 h (p < 0.001). The best adherence ability to Hela cells was seen in Lactobacillus gasseri strains. Nearly 17 of the strains had higher anti-candida activity compared to the other strains. According to the findings, four lactobacilli strains isolated in the vaginal samples of healthy Iranian women had the best probiotic potential.

Introduction

Over 50 different microbial species have been identified in the human vagina that promote female health by protecting against pathogens, modulating immunity, preventing infection, and maintaining the pH below 4.7 [1]. A variety of Lactobacillus species have been identified as the predominant biota in the vagina, and over 70% of all bacteria in the vagina of healthy females are identified as lactobacilli [2, 3]. The most common species in the vagina include L. jensenii, L. johnsonii, L. gasseri, L. iners, and L. crispatus, which protect the vaginal mucosa against pathogenic microorganisms, sexually transmitted infections (STIs), bacterial vaginitis, vulvovaginal candidiasis and Neisseria gonorrhoeae [4]. This protection is mainly due to the adherence of lactobacilli to vaginal epithelial cells, thus preventing pathogenic growth. Lactobacillus species use various mechanisms to achieve this property, such as immunomodulation, production of antimicrobial substances such as organic acids, hydrogen peroxide and bacteriocins, and competition with pathogens for food supplies [5, 6]. Also, Lactobacillus species inhibit the adhesion of pathogens to the receptors of epithelial cells through the removal or production of biofilm [7].

Probiotics are known as living microorganisms that, if consumed sufficiently, create positive effects on the health of the host. In the last 15 years, research on probiotic bacteria has increased, and attention has been paid to their use as medicine or in nutrition [8].

Vaginal lactobacilli may have probiotic properties and are known to be responsible for maintaining the balance and health of the vaginal ecosystem, and the loss of these bacteria makes females prone to urinary and genital infections [6]. Therefore, the use of selected lactobacilli may be effective in improving the vaginal microbiota and preventing infection [9]. Researchers have also discussed the effective role of probiotic bacteria in the prevention and treatment of cancer, including uterine cancer, and are further investigating the subject [10]. The ability to inhibit the growth of Hela cells, which originate from the cervical epithelial cells, is one of the anti-cancer properties of Lactobacillus that has been utilized in recent years [11]. In addition, due to the increase in fungal vaginal infections, including Candida albicans in the vagina, new effective therapies are being investigated [12]. A probiotic-like product such as intravaginal lactobacilli as a medicine alone or as a supplement may inhibit Candida albicans infection [13, 14]. Therefore, this study was conducted to examine the probiotic potential of vaginal Lactobacillus, their ability for adherence to Hela cells and their antagonistic activity against Candida albicans among healthy females in northern Iran.

Materials and Methods

Study Groups and Sampling

One hundred healthy females referred to gynecology clinics for routine gynecological consultations in Amol, Iran, were recruited for this study. The inclusion criteria were: Age 22–50 years (premenopausal), no history of sexually transmitted diseases, diabetes, immunodeficiency, vaginitis or vaginal candidiasis infection, and not being menopausal or having vaginal bleeding. The exclusion criteria were: Pregnancy, use of antibiotics or antifungal compounds, vaginal medications or suppositories or contraceptive spermicides over the past 30 days, smoking, high blood pressure and alcohol consumption. A questionnaire was designed to collect the subjects’ demographic information, such as age, history of pregnancy, history of abortion, number of births, type of delivery, and abstinence from antibiotics and antifungals over the 2 months preceding sampling, history of diabetes, and blood pressure. Sterile swabs were used for sampling the exocervix and the side of the vagina; then, the swab was put into MRS broth medium (Liofilchem, Italy) and immediately incubated at 37 °C for 24 h.

Purification of Lactobacillus Isolates

After incubation, the samples were cultured on MRS agar (Liofilchem, Italy) and incubated at 37 °C in an anaerobic jar using Anaerocult C (Merck, Germany) for 24–48 h. Then, gram reaction, morphology, and catalase test were used to identify and select Lactobacillus single colonies grown on MRS agar (Liofilchem, Italy). Total gram-positive bacilli and negative catalase test results were identified as lactobacilli and stored in MRS broth containing 20% (w/v) glycerol at − 20 °C for further investigations. The phenotypic characteristic of some vaginal samples was yeast or cocci, and Lactobacillus did not grow after 48–72 h on MRS agar incubation at 37 °C; therefore, they were excluded from the study.

Molecular Identification of Lactobacillus Isolates

Bacterial DNA was extracted from all the isolated strains using High Pure Template PCR extraction kit (Roche, Germany). The quality and quantity of the extracted DNA were checked with a nanodrop device and electrophoresis on 0.8% agarose gel.

The sequences of the 16S ribosomal RNA intergenic spacer region of phylogenetically related species were retrieved from GenBank (www.ncbi.nlm.nih.gov). DNA fragments encoding 16S rRNA were amplified using specific primer sequences; forward (5′-CTCAAAACTAAACAAAGTTTC-3′) and reverse (5′-CTTGTACACACCGCCCGTCA-3′) primers, which confirmed Lactobacillus genus [15]. The PCR reaction mix with a total volume of 25 µl consisted of distilled water) 17 µl(, buffer )2.5 µl(, Mgcl₂) 1.25 µl(, dNTP) 0.4 µl (, 0.25 µl TaqDNA polymerase, 0.8 µl forward, 0.8 µl reverse primer and 2 µl of DNA. A DNA-free vial was used as a negative control. PCR was performed by thermocycler SimpliAmp-ABI (USA) using the following schedule: Initial denaturation at 95 °C for 5 min followed by 35 cycles, each consisting of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and a final elongation step at 72 °C for 5 min. The PCR product was electrophoresed in a 1% (W/V) agarose gel in TBE buffer at 100 V for 45 min and stained with ethidium bromide, and the gel was visualized with a UV transilluminator (UVitec, UK). A 100-bp molecular mass marker (SinaClon, Iran) was used for assessing the size of the PCR products.

Sequencing

The PCR product was sent to Niagen Noor Company (Tehran, Iran) along with the 16S rRNA primer for sequencing. The chromatograms were edited using Chromas version 3.1 software. After sequencing the amplified fragment of the 16S rRNA gene, the isolated strains were compared with other sequences in GenBank on NCBI website using BLAST software.

Bile Salt Tolerance Assay

For this test, MRS broth medium (Liofilchem, Italy) supplemented with 0.3% Deoxycholate bile salt (Merck, Germany) was used and 100 µl of the full-grown culture isolates were added to 10 ml of this medium. Then, the cells were incubated at 37 °C and optical densities (OD 600 nm) were measured at 1, 2 h, 4 h, and 24 h by a spectrophotometer. The culture medium was used without oxgall as the control. All measurements were performed two times and the average of two replicates was calculated [16, 17].

Tolerance to Low pH

In order to investigate the resistance of lactobacilli, 1 M HCL was added to MRS broth before autoclaving and the sterile MRS broth medium was prepared with pH of 2, 2.5, 3, 4 and 6; then, (1% v/v) full-grown culture isolates were inoculated in these mediums and incubated at 37 °C. Their optical densities (OD 620 nm) were measured at 1, 2, 4, and 24 h by a spectrophotometer. The experiment was repeated twice and each reading represents the average of two replicates [18].

Hemolysis Activity Test

For the hemolysis test, all the Lactobacillus isolates were cultured (in MRS agar medium for 48 h at 37 °C in anaerobic conditions) and then a single colony isolated from Lactobacillus was cultured on blood agar medium (Merck, Germany) containing sheep blood 5% and incubated in anaerobic conditions using Anaerocult C for 24–48 h at 37 °C. The hemolytic activity was visually detected and distinguished as β-hemolysis, α-hemolysis and γ-hemolysis based on the appearance of a clear zone, green halo zone and no zone around the colonies. Streptococcus pyogenes (ATCC 19615) was used as the positive control. After incubation, hemolysis around the isolates was recorded [19].

H₂O₂ Production

For this test, the strains are cultured on MRS agar containing 0.25 mg/mL of 3,3′,5,5′-tetramethylbenzidine (Merck, Germany) and 0.01 mg/mL of horseradish peroxidase (Sigma-Aldrich, USA) in anaerobic conditions for 48–72 h. The plates were exposed to air for 30 min and hydrogen peroxide was evaluated based on the time required for a blue coloration appearance. The strains were scored as weak (score of 1, time ≥ 20 min), medium (score of 2, time 10–20 min) and strong (score of 3, time ≤ 10 min) H₂O₂ producer and a score of 0 meant not producing the blue coloration [20, 21].

Evaluation of Antagonistic Activity

Antagonistic Activity of Lactobacilli Against Urinary Pathogens by Spot Agar Assay

To investigate the antimicrobial activity of Lactobacillus isolates, Lactobacillus isolates were first cultured in spot form on MRS agar medium by sterile loop, and then incubated anaerobically at 37 °C for 48 h. Afterwards, E. coli ATCC 25922 and two strains of E. coli isolated from females with urinary tract infections and identified by phenotypic tests were used for this exam. Later, overnight-cultured E. coli strains (approximately 7 × 10⁸ CFU) were mixed in the nutrient agar medium (Merck, Germany) and poured on MRS agar medium which Lactobacillus isolates that had been previously cultured. Afterwards, the plates were incubated in anaerobic conditions at 37 °C for 24 h. Finally, the zone of inhibition of the growth of E. coli around the Lactobacillus isolates was measured [22].

Antagonistic Activity of Lactobacilli Against Candida albicans by Agar Overlay Method

Anticandidal activity was assessed using the modified agar overlay technique; briefly, 0.5 McFarland turbidity of the Candida albicans (ATCC 10231) that had grown on Sabouraud agar medium (Merck, Germany) was prepared, and the isolated lactobacilli were cultured in MRS broth for 24 h at 37 °C in anaerobic conditions. Afterwards, the Lactobacillus strains were cultured in the middle of MRS agar using sterile cotton swabs for culturing the bacteria and were then incubated for 48 h at 37 °C in anaerobic conditions. The Lactobacillus growth was then overlaid with melted MRS agar and the suspension of Candida albicans was streaked over the agar with solidification. After culturing, the plates were placed in the refrigerator for 4 h at a temperature of 4 °C and then in the incubator at 37 °C for 24 h. The plates were later kept at room temperature for 24 h and the antagonistic activity was measured and recorded as follows: ≥ 20 mm was considered sensitive to Lactobacillus, between 10 and 15 mm was semi-sensitive, and less than 5 mm lacked antagonistic activity to Lactobacillus strains [22].

Adherence to Hela Cells

To evaluate the adhesion of lactobacilli to Hela cells, a total of 3 × 10⁵ Hela cells were cultured in DMEM medium with fetal bovine serum 10% at 37 °C with 5% CO₂. After 24 h of incubation, the Hela cells were mixed with 1 ml of Lactobacillus (1.5 × 10⁹ cells/ml) at 37 °C for 3 h. The cells were washed twice with phosphate buffered saline and the unattached cells were removed. For the quantitative assessment of bacterial cell adhesion, the remaining cells were washed with phosphate buffered saline and lysed with 1% Triton × 100 and then centrifuged at 2200g for 5 min. The supernatant was discarded, the pellets were re-suspended, and serial dilutions were prepared in phosphate buffered saline and an appropriate dilution was cultured on MRS agar medium for 48 h at 37 °C. The grown colonies were counted and taken as the adherent bacteria. For this test, we used L. brevis ATCC 367 as the control. The following equation was used to calculate the percentage of adherent cells [23].

• N₁: The amount of adhered bacteria (log10 CFU/ml)

• N₀: The amount of applied bacteria (log10 CFU/ml)

• Adherent cells %: N₁/N₀ × 100

Statistical Analysis

Data analysis was performed in SPSS, version 21. The antagonistic activity of lactobacilli on Candida albicans and the ability of lactobacilli to bind to Hela cells were examined and the results were then compared using the one-way ANOVA. The one-way ANOVA and repeated-measures analysis were used to compare the pH tolerance and Bile salt tolerance over time and across the Lactobacillus species. In addition, a Chi-square test was used to examine the relationship between the antagonistic activity against Candida albicans and Lactobacillus species.

Results

Isolation of Lactobacillus Strains from the Vagina

The isolated Lactobacillus strains cultured on MRS agar medium were investigated and the phenotype properties, such as colony color (white to milky), were recorded. A total of 38 vaginal Lactic acid bacteria isolated from reproductive-aged women were selected. They were gram-positive, rod shaped, catalase negative. All the isolated vaginal Lactobacillus strains were finally identified by sequencing the 16S rRNA gene and compared with sequences available in GenBank database using BLAST software at NCBI and they were found to belong to five species.

Nucleotide Sequence Accession Number

The NCBI GenBank accession numbers of the spacer sequences used in this study showed in Table 1.

Table 1.

NCBI GenBank accession numbers of Lactobacillus strains sequences

Accession number	NCBI GenBank name	Name	Isolates lable	NCBI GenBank strain name	Sequence length in bp	BLAST similarity%
OR220801	Lactobacillus sp	L. crispatus	C2	C2HZA	196	99
OR220199	Lactobacillus sp	L. crispatus	C3	C3HZA	197	98
OR220200	Lactobacillus sp	L. crispatus	C4	C4HZA	215	92
OR220201	Lactobacillus sp	L. crispatus	C5	C5HZA	181	99
OR220202	Lactobacillus sp	L. crispatus	C6	C6HZA	194	99
OR220203	Lactobacillus sp	L. crispatus	C7	C7HZA	195	97
OR229087	Lactobacillus sp	L. crispatus	C8	C8HZA	190	96
OR229088	Lactobacillus sp	L. crispatus	C9	C9HZA	190	96
OR229089	Lactobacillus sp	L. crispatus	C11	C11HZA	185	94
OR229090	Lactobacillus sp	L. crispatus	C12	C12HZA	190	96
OR220204	Lactobacillus sp	L. crispatus	C13	C13HZa	221	92
OR220205	Lactobacillus sp	L. gasseri	G5	G5HZA	212	98
OR220195	Lactobacillus sp	L. gasseri	G6	G6HZA	212	88
OR220196	Lactobacillus sp	L. gasseri	G7	G7HZA	241	97
OR220197	Lactobacillus sp	L. gasseri	G8	G8HZA	238	98
OR220198	Lactobacillus sp	L. gasseri	G10	G10HZA	231	94
OR220567	Lactobacillus sp	L. acidophilus	A1	A1HZA	197	98
OR220568	Lactobacillus sp	L. jensenii	Je2	J2HZA	197	98
OR220569	Lactobacillus sp	L. jensenii	Je3	J3HZA	222	93
OR220570	Lactobacillus sp	L. jensenii	Je5	J5HZA	195	92
OR220564	Lactobacillus sp	L. johnsonii	Jh1	Jh1HZA	185	99
OR220565	Lactobacillus sp	L. johnsonii	Jh3	Jh3HZA	237	91
OR220566	Lactobacillus sp	L. johnsonii	Jh4	Jh4HZA	235	96

Demographic Data

The results of the current study showed that the females who were classified in each of these Lactobacillus strains had no significant difference in age (P = 0.618). More details are shown in (Table 2).

Table 2.

Age (mean and SD) of participate based on Lactobacillus species

Lactobacillus species	Type	Age
Species	N (%)	Mean ± SD
Total	38 (100)	37.5 ± 7.1
L. crispatus	13(32.5)	35.2 ± 7.4
L. gasseri	10(25)	39.2 ± 6.5
L. acidophilus	6(15)	36 ± 8.2
L. jensenii	5(12.5)	38.8 ± 6.7
L. johnsonii	4(10)	40 ± 8.3
F statistics	0.68
P value	0.666

A relationship was also found between the type of delivery (natural, caesarean, and both) and the frequency distribution of Lactobacillus strains (${{\chi }^2}_{df=8}$=19.12; P = 0.014). Having both delivery methods was observed only in the Lactobacillus johnsonii species group (Appendix 1).

Bile Salt Tolerance Results

The tolerance of Lactobacillus isolates to bile salt was measured based on the optical properties of the isolates at different time points by a spectrophotometer. The results showed no significant difference among the isolated lactobacilli in terms of bile salt tolerance at each time point (Fig. 1). Nonetheless, all the strains had tolerance to bile salt over time, resulting in their increased growth at the end of 24 h (P < 0.001). The results showed that Lactobacillus crispatus C9 and C10, Lactobacillus johnsonii Jh 4 and Lactobacillus gasseri G5 had the highest growth during the incubation period. By contrast, Lactobacillus jensenii Je 1 and Lactobacillus gasseri G4 showed the lowest viability after 24 h of incubation.

Fig. 1.

Bile salt tolerance according time and concentration (expressed as Mean and SD) of all strain of Lactobacillus specific species in (OD = 600 nm)

Low pH Tolerance Test

The resistance of the isolates at different pH levels was measured based on the turbidity of the culture medium. The survival of the Lactobacillus species showed a significant difference only at pH = 2 and 3 at 0 h (P = 0.038) and 24 h (P = 0.014); however, there was no significant difference between the Lactobacillus species in terms of survival in other pH levels and different hours. Overall, for all the species at all the pH levels, a significant difference was observed in terms of tolerance at different hours, such that tolerance increased significantly as 24 h approached (P < 0.001). According to the results, at the pH of 2.0, Lactobacillus gasseri G5, G7, and G10, Lactobacillus crispatus C12 and Lactobacillus johnsonii Jh2 exhibited the highest viability during 24 h of incubation. The maximum viability at pH of 3.0 was observed in Lactobacillus crispatus C10, C12, and C7 and Lactobacillus johnsonii Jh3, followed by Lactobacillus gasseri G4, which showed the minimum survival rate. More details are shown in (Fig. 2).

Fig. 2.

pH tolerance according to pH levels, time and concentration (expressed as Mean) of all strains of Lactobacillus specific species in (OD = 620nm), a pH = 2, b pH = 2.5, c pH = 3, d pH = 4, e pH = 6, L. acidophilus (n = 6), L. crispatus (n = 13), L. gasseri (n = 10), L. jensenii (n = 5), L. johnsonii (n = 4)

Hemolysis Results

After 24–48 h of incubation of the Lactobacillus strains, the hemolysis zone around the grown bacillus colonies was examined. None of the Lactobacillus strains caused lysis of sheep erythrocytes on blood agar (γ-hemolysis) while complete lysis (β-hemolysis) was observed in the case of the positive controls (Streptococcus pyogenes).

Hydrogen Peroxide Production

The qualitative analysis demonstrated that four strains of Lactobacillus crispatus (C1, C3, C7, and C13) and two strains of Lactobacillus jensenii (Je2 and Je3) showed moderate H₂O₂ production, and five strains of L. gasseri (G1, G5, G6, G9, and G10) exhibited low ability to produce hydrogen peroxide, and the other strains did not produce any H₂O₂.

Adherence to Hela Cells Results

The results of the current research revealed that all the strains had the ability to adhere to Hela cells. Moreover, Hela cell adherence showed a significant difference between different Lactobacillus strains compared to the controls (for L. brevis ATCC 367, the average adherence to Hela cells was 95%), but no significant difference was found among the Lactobacillus strains to each other (P value = 0.099), and the highest and lowest adherence percentages were observed in L. gasseri and L. jensenii species, respectively (Table 3). The strains shown below had 75% to 95% adherence ability to Hela cells: Lactobacillus gasseri G1, G2, G3, G4, G5, G6, G7, and G10, Lactobacillus crispatus C1, C3, C4, C5, C7, C12, and C13, Lactobacillus acidophilus A1, A2, A5, and A6, Lactobacillus jensenii Je1 and Lactobacillus johnsonii Jh1.

Table 3.

Adhesion percentage to Hela cells in Lactobacillus species by one way ANOVA

Lactobacillus sp	Mean ± SD
L. acidophilus (n = 6)	0.73 ± 0.11
L. crispatus (n = 13)	0.66 ± 0.25
L. gasseri (n = 10)	0.77 ± 0.20
L. jensenii (n = 5)	0.46 ± 0.22
L. johnsonii (n = 4)	0.56 ± 0.21
F statistics	2.12
P value	0.099

L. brevis ATCC 367 excluded from ANOVA test for comparing the means

Antagonistic Activity of Lactobacillus Isolates Against Candida albicans

The antagonistic activity of Lactobacillus isolates against Candida albicans was examined by measuring the range of non-growth of Candida albicans in the vicinity of Lactobacillus strains, and no significant difference was found between the Lactobacillus species with respect to their antagonistic activity on Candida albicans (P value = 0.930), (Fig. 3). The highest (≥ 20 mm) antagonistic activity against Candida albicans was observed in 17 strains, as below: Lactobacillus crispatus C1, C2, C6, C7, C8, and C12, Lactobacillus gasseri G2, G3, G5, and G6, Lactobacillus jensenii Je2, Je3, and Je4, Lactobacillus acidophilus A1, A4, and A5 and Lactobacillus johnsonii Jh3.

Fig. 3.

Antagonistic activity against Candida albicans in Lactobacillus species (χ² (df = 8) = 3.067; P value = 0.930)

Antagonistic Activity Against E. coli Strains

The antimicrobial activity of Lactobacillus against different strains of E. coli showed that there was no significant difference between the isolated strains in this regard (P > 0.05). The findings also revealed that among the Lactobacillus species, L. crispatus showed more inhibition of growth compared to the other species. More details are shown in Table 4.

Table 4.

Antagonistic activity against E. coli strains in all strains of Lactobacillus specific species (inhibition zone in mm, expressed as Mean ± SD), L. acidophilus (n = 6), L. crispatus (n = 13), L. gasseri (n = 10), L. jensenii (n = 5), L. johnsonii (n = 4)

Lactobacillus species	E. coli strain isolated from urinary infection1 (Mean ± SD)	E. coli strain isolated from urinary infection 2 (Mean ± SD)	E. coli ATCC 25922 (Mean ± SD)
L. acidophilus	7.5 ± 5.9	8.7 ± 5.3	8.2 ± 6.4
L. crispatus	9 ± 3.2	9.3 ± 3.4	10.6 ± 2.6
L. gasseri	9.9 ± 1.9	10.6 ± 1.8	10.6 ± 2.2
L. jensenii	6.6 ± 3.9	6.4 ± 3.6	7.4 ± 4.3
L. johnsonii	9.3 ± 2.5	10 ± 2.4	10 ± 2.8
Total	8.3 ± 3.9	8.8 ± 3.9	9.3 ± 4.1
Fa	0.959	1.365	1.15
P value	0.443	0.267	0.351

^aF statistics of one-way ANOVA test

Discussion

In this study, L. crispatus and L. gasseri were documented to be the most common vaginal species among women in northern Iran. Martinez et al. determined the most dominant species of Lactobacillus in the vagina to be L. gasseri, L. crispatus and L. jensenii among Mexican women [24]. Moreover, Vitali et al. [25] found that L. acidophilus, L. gasseri, L. vaginalis, and L. iners were the most common species in the vaginal microbiota among healthy Italian women. Overall, various factors, such as individual genetics, nutrition, personal hygiene and sexual activity, can be effective in the colonization of certain species of lactobacilli in the vagina among healthy women [26, 27].

Probiotics are defined as “Live microorganisms that, when administered in sufficient amounts, have beneficial effects on human health [28]. Genitourinary lactobacilli are known as probiotic agents that protect the vagina against invading pathogens that are responsible for urinary tract infections or sexually transmitted diseases [19]. The survival potential of lactobacilli in low pH and bile salts is very important, because probiotics confront a range of gastrointestinal stresses that intensify in the stomach; therefore, resistance to stomach acid and bile salts ensures the successful administration of probiotics [29]. In the study by Kiray et al. [29], it was observed that none of the strains were able to tolerate pH = 2; however, the survival of the strains at pH = 2.5 was equal to 67.6%, and this ratio increased to 89.3% at pH = 3. Our results showed that L. gasseri species had the highest percentage of survival at pH = 2 for 4 h. It has been found that some strains can maintain a high viability in stomach pH and thus maintain sufficient numbers to compete and bind with pathogenic bacteria when they reach the digestive tract [30]. Bile salt tolerance is another essential criterion for selecting probiotic strains. Bile salts play an important and fundamental role in the specific defense of the intestine by destroying microorganisms. Therefore, investigating the viability of the strain in different concentrations of bile salts is necessary to prove the probiotic potential [17, 31]. The resistance of Lactobacilli to different concentrations of salt indicates their higher stability [32]. In our study, Lactobacillus species tolerated 0.3% bile salt for 24 h.

One of the most important features of probiotics is the production of compounds, such as short-chain fatty acids and bacteriocins, which lead to antimicrobial properties against pathogens. In the case of lactobacilli, the presence of compounds such as bacteriocins, hydrogen peroxide and organic acids and competition for nutrients with pathogenic microorganisms lead to antimicrobial properties [21, 29]. Vaginalis Lactobacillus strains control vaginal microorganisms, including Candida albicans, by colonizing the vaginal epithelium and inhibiting the growth of other microorganisms. Therefore, as candidates for vaginal probiotics, Lactobacillus strains are usually tested in vitro for their ability to adhere to the vaginal epithelium and their antimicrobial activity against Candida albicans [29]. The species L. crispatus and L. jensenii are high H₂O₂ producers compared to L. gasseri [33]. These results are in accordance with the findings of the present study. The production of H₂O₂ is one of the most important defense mechanisms against the colonization of pathogenic or opportunistic microorganisms by vaginal Lactobacillus [34]. Absence of H₂O₂ producing Lactobacillus increases the risk of bacterial vaginosis (BV), such as recurrent urinary tract infection by E. coli [22, 32, 34]. Kiray et al. investigated the antimicrobial activity of vaginal probiotics and the antagonistic effect of the strains on pathogenic microorganisms causing urinary tract and genital tract infections. The results of their meta-analysis showed that vaginal lactobacilli are effective in treating bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) [29]. Hutt et al. showed that L. crispatus species had significantly higher anti-microbial activity compared to L. jensenii, while L. gasseri species showed lower anti-Candida activity compared to L. crispatus. and L. jensenii species [22]. Our results were in line with these findings, showing the antagonist effect of vaginal lactobacilli strains against Candida albicans. Only a small number of Lactobacillus species were found to gather in clusters and prevent the growth of pathogenic agents. The aggregation of lactobacilli species with Candida strains is very important in protecting against vaginal yeast infections, because lactobacilli can create a small environment around pathogens through co-aggregation, and by synthesizing large amounts of inhibitory factors, they can prevent the pathogen microorganisms from distribution [33, 35]. Moreover, one of the most important tests in potential probiotics is the adhesion of the probiotic microorganism to mucin and human epithelial cells. The attachment of lactobacilli to vaginal epithelial cells is defined as the first step in the formation of a barrier to prevent the colonization of opportunistic pathogenic microorganisms [34, 36]. Various models, including Caco-2 and HT29 cells, have been used to evaluate the ability of bacteria to attach to cells in vitro [13, 22]. In the present study, Hela cells were used to evaluate the adhesion capacity of vaginal lactobacilli. As reported in previous studies, different species of lactobacilli have different abilities to attach to the cells [37]. Similarly, a lectin-like protein in the cell wall of L. plantarum may play a role in adhesion [38]. In pervious literature, Lactobacillus surface factors, such as lipoteichoic acid of L. johnsonii and S-layer protein of L. crispatus, were reported to play roles in adhesion to epithelial cells. They may be involved in sticking to epithelial cells. Therefore, different mechanisms could exist for different Lactobacillus strains to adhere to epithelial cells [13, 39]. In the present study, the highest percentage of adhesion to Hela cells was seen in L. gasseri species; in contrast, Mousavi et al. [23] reported that L. rhamnosus species adhered to the epithelial cells to a greater degree compared to other Lactobacillus species and L. gasseri, which showed weak adherence.

Conclusion

Based on the results, each Lactobacillus strain isolated from the vagina of healthy Iranian women had different probiotic properties. In particular, four Lactobacillus strains (C7, C12, G5 and G10) exhibited stronger potential probiotic characteristics such as tolerance to a low pH, H₂O₂ production, adherence to Hela cells and better antagonistic activity against Candida albicans and E. coli strains; there fore, they are effective in maintaining vaginal health and preventing infections of the genitourinary system. Each vaginal strain is specific and needs to be clinically well investigated through in vitro and in vivo studies.

Appendix

See Table 5.

Table 5.

The relationship between delivery types and Lactobacillus species frequencies for study sample

	Delivery types			Total
	Only natural	Only caesarean	Both
	n (%)	n (%)	n (%)
L. acidophilus	2 (33.3)	4 (66.7)	0 (0)	6 (100)
L. crispatus	5 (38.5)	8 (61.5)	0 (0)	13 (100)
L. gasseri	5 (50.0)	5 (50.0)	0 (0)	10 (100)
L. jensenii	3 (60.0)	2 (40.0)	0 (0)	5 (100)
L. johnsonii	1 (25.0)	1 (25.0)	2 (50)	4 (100)
Total	16 (42.1)	20 (52.6)	2 (5.3)	38 (100)

${\chi }_{df=8}^2$ = 19.12; P value = 0.014

Author Contributions

All authors contributed to the study conception and design and experiments. Material preparation, performed experiments, data collection and analysis were performed by HZ, RIA, MR, HK. The first draft of the manuscript was written by HZ. FZ Supervised, directed and managed the study. All authors read and approved the final manuscript.

Funding

Not applicable.

Availability of Data and Materials

Not applicable.

Code Availability

Not applicable.

Declarations

Conflict of interest

The authors declare that there are no conflict of interest.

Ethical Approval

The study protocol was approved by the Ethics Committee of Amol Azad University (ID: IR.IAU.AMOL.REC.1400.051). At the beginning, all study participants were given adequate information about the study. Then informed consent was obtained.

Consent to Participate

The procedure was explained to all the recruited participate who signed written informed consent forms.

Consent for Publication

Not applicable.