# Potential of Maintaining a Healthy Vaginal Environment by TwoLactobacillus Strains Isolated from Cocoa Fermentation

Authors: Ana Clara Correia Melgaço; Wallace Felipe Blohem Pessoa; Herbert Pina Freire; Milena Evangelista de Almeida; Maysa Santos Barbosa; Rachel Passos Rezende; Jorge Timenetsky; Lucas Miranda Marques; Carla Cristina Romano
Journal: BioMed Research International
 (2018)

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## Abstract

Bacteria in the generaMycoplasma andUreaplasma do not have cell walls and therefore interact with host cells through lipid-associated membrane proteins (LAMP). These lipoproteins are important for both surface adhesion and modulation of host immune responses.Mycoplasma andUreaplasma have been implicated in cases of bacterial vaginosis (BV), which can cause infertility, abortion, and premature delivery. In contrast, bacteria of the genusLactobacillus, which are present in the vaginal microbiota of healthy women, are thought to inhibit local colonization by pathogenic microorganisms. The aim of the present study was to evaluate thein vitro interactions between lipoproteins ofMycoplasma andUreaplasma species and vaginal lineage (HMVII) cells and to study the effect ofLactobacillus isolates from cocoa fermentation on these interactions. The testedLactobacillus strains showed some important probiotic characteristics, with autoaggregation percentages of 28.55% and 31.82% forL. fermentum FA4 andL. plantarum PA3 strains, respectively, and percent adhesion values of 31.66 and 41.65%, respectively. The two strains were hydrophobic, with moderate to high hydrophobicity values, 65.33% and 71.12% forL. fermentum FA4 andL. plantarum PA3 in toluene. Both strains secreted acids into the culture medium with pH=4.32 and pH=4.33, respectively, and showed antibiotics susceptibility profiles similar to those of other lactobacilli. The strains were also able to inhibit the death of vaginal epithelial cells after incubation withU. parvum LAMP from 41.03% to 2.43% (L. fermentum FA4) and 0.43% (L. plantarum PA3) and also managed to significantly decrease the rate of cell death caused by the interaction with LAMP ofM. hominis from 34.29% to 14.06% (L. fermentum FA4) and 14.61% (L. plantarum PA3), thus demonstrating their potential for maintaining a healthy vaginal environment.


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## Body

## 1. Introduction

Bacteria of the generaMycoplasma andUreaplasmabelong to the classMollicutes, and these microorganisms are the smallest known free-living organisms. With genomes of only 580–2,200 kb, depending on the species, these bacteria contain only the minimal structures necessary for cell growth and replication and are unable to synthesize some substances that are essential for their growth. Therefore, these substances must be obtained from their hosts [1, 2]. Because they do not have cell walls, these bacteria contact host cells through their plasma membrane, which is composed of a lipid-protein bilayer. Mycoplasmas lipid-associated membrane proteins (LAMP) play an important role in both adhesion to the host cell surface and immune response modulation through the production of proinflammatory cytokines. They also induce apoptosis in different types of cells, such as monocytes and macrophages [3–5]. Mycoplasmas and ureaplasmas are pathogens that are frequently associated with mucosal infections of the respiratory and urogenital tracts [1, 2]. In the female genital tract, mycoplasmas and ureaplasmas have been directly implicated in cases of bacterial vaginosis (BV) [6, 7].BV is a syndrome resulting from an imbalance of the vaginal microbiota, with concomitant proliferation of pathogenic bacteria. These infections mainly affect women of childbearing age, and they have been associated with infertility, preterm birth, endometritis, pelvic inflammatory disease, and susceptibility to infection with human immunodeficiency virus (HIV) [6, 8, 9]. In addition toMycoplasma andUreaplasma species, the other major bacterial species related to BV belong to the generaChlamydia,Neisseria, andGardnerella [10]. Vaginal microbiota is considered to be healthy when specific bacterial community types that have beneficial functions for the host are present, along with the absence of clinical symptoms [11]. The vaginal microbiota of symptom-less women generally includes species of the genusLactobacillus; these bacteria produce lactic acid, hydrogen peroxide (H2O2), bacteriocins, and hydroxyl radicals and thereby inhibit local colonization by pathogenic microorganisms. In addition, the presence of lactobacilli favors a protective environment for the fetus in pregnancy [10].Lactobacilli as well as bacteria of the genusBifidobacterium, Lactococcus lactis, andEscherichia coli as well as the yeastSaccharomyces cerevisiae have been used as probiotics, mainly by the food industry [12]. Probiotics are living microorganisms that, when administered in adequate quantities, confer benefits to host health [13]. Generally, lactobacilli in probiotic formulations were isolated from human microbiota; however, in recent years there has been growing interest in the use of strains isolated from nonhuman sources, including fermented foods, such as cocoa. Thus, in several studies, the probiotic potential of strains isolated from food fermentation has been investigated [14, 15]. Studies previously conducted by our research group showed thatLactobacillus strains derived from the fermentation of cocoa exhibited probiotic potential and antibiotics activity against distinct pathogens. Different strains reduced histological damage and the systemic concentration of inflammatory cytokines and elevated serum levels of immunoglobulin A (IgA) in a model of experimental colitis [16]. Culture supernatants ofL. fermentum TCUESC01 andL. plantarumTCUESC02 inhibited growth and reduced the biofilm formation ability of streptomycin- and dihydrostreptomycin-resistant strains ofStaphylococcus aureus [17]. They also showed antagonistic activity againstG. vaginalis [18]. The aim of this study was to evaluate thein vitro interaction of lipoproteins from genital humanMycoplasma andUreaplasma species with the HMVII vaginal cell line and to study the effect ofLactobacilluson this interaction.


## 2. Materials and Methods

### 2.1. Cell Lines, Microorganisms, and Growth Conditions

HMVII human vaginal epithelial cell line (BCRJ 0316) was obtained from the Rio de Janeiro Cell Bank and was grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FSB), penicillin 100 IU/mL, and streptomycin 100μg/mL.L. fermentum FA4 andL. plantarum PA3 were previously isolated by our research group from a cocoa fermentation [16]. These strains were confirmed to the species level by 16S rRNA sequencing and were deposited in GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers KU244506 and KU244472, respectively. Lactobacilli were grown in de Man, Rogosa, and Sharpe (MRS) medium for 18 h at 37°C in anaerobic jar.M. hominis(ATCC 23114) andM. genitalium(ATCC 33530) were grown in 1 L of SP-4 medium.U. urealyticum serotype 7 (ATCC 27819) andU. parvum serotype 3 (ATCC 27815) were cultured in 200 mL of Ureaplasma Broth (UB) medium. All strains were maintained at 37°C and 5% CO2 until log phase growth. Growth control was observed by the observation of color change in the liquid medium, plus pH indicator (phenol red).


### 2.2. Extraction of Membrane-Associated Lipoproteins (LAMP)

Lipoproteins were extracted according to the method developed by Wang et al. [19] with some modifications. Briefly,M. hominis,M. genitalium,U. parvum, andU. urealyticum were cultured until log phase, until the observation of color change in the liquid medium, pH indicator (phenol red). Then, the cells were recovered by centrifugation at 23,700 ×gfor 30 min at 4°C (Beckman Coulter) and washed with sterile phosphate-buffered saline (PBS) (1X, pH 7,4) to remove the residual culture medium. The cell pellet was suspended in 5 mL of Tris-EDTA (50 mM Tris [pH 8], 0.15 M NaCl, 1 mM EDTA), and Triton TX-114 was added to a final concentration of 2%. The mixture was homogenized by vortexing and incubated at 4°C for 60 min. The lysate was then incubated at 37°C for 10 min and centrifuged at 23,700 ×g at 22°C for 20 min for phase separation. The upper aqueous phase was discarded, and in the final TX-114 step, the volume was adjusted to the original volume by the addition of Tris-EDTA. Then, 2.5 volumes of ethanol were added to precipitate the lipoproteins overnight at −20°C. The precipitated materials were recovered by centrifugation at 23,700 ×gfor 15 min at 4°C. After centrifugation, the pellet was homogenized in PBS by sonication, 6 to 8 cycles per minute at a power of 10W (Coler-Parmer Ultrasonic Processor). Then, the microtubes containing the lipoproteins were stored at −80°C until use. Lipoproteins were quantitated using the 2D Quant Kit (GE Healthcare) according to the manufacturer’s protocol and preincubated for 2 h with polymyxin B at 1000 U/mL prior to the use.


### 2.3. Autoaggregation Assay

To verify the autoaggregation capacity of theLactobacillus strains, the method of Kos et al. [20] was used, with some modifications. Briefly, the strains were cultured in MRS broth for 18 h at 37°C in anaerobic jar. Then, the cells were recovered by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline solution (w/v), and suspended in the same solution to 1 × 108 CFU/mL in a spectrophotometer (Thermo-Scientific). The suspensions were homogenized by vortexing and incubated at 37°C for 5 h. Then, a 1 mL aliquot was gently removed from the top of the suspension every hour, and its absorbance at 600 nm (A600) was read in a spectrophotometer (Thermo-Scientific). Autoaggregation was calculated using the following formula: autoaggregation (%) = [(A0-At)/A0] × 100, where A0 is the absorbance at time 0 h, and At is the absorbance at time t, which was measured every hour, for up to 5 h.


### 2.4. Hydrophobicity Assay

The hydrophobicity of theLactobacillusstrains was verified by testing microbial adherence to hydrocarbons (MATH), using a method adapted from Rodríguez et al. [21].Lactobacillus strains were cultured in MRS broth at 37°C for 18 h in anaerobic jar. Then, the cultures were harvested by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline, and adjusted to an optical density at 600 nm (OD600) of 0.7 in saline. One milliliter of solvent (xylene or toluene) was added to each bacterial suspension, and the mixtures were vortexed for 2 min and then incubated for 2 h at 37°C. The lower aqueous phase was carefully removed, and the A600 was read in a spectrophotometer. Hydrophobicity was calculated using the following formula: hydrophobicity (%) = ((A0-A2)/A0) × 100, where A0 is the absorbance at time 0 (0 h) and A2 is the absorbance after 2 h.


### 2.5.Lactobacillus Adhesion to HMVII Cells

To verify the adhesion capacity of theLactobacillusstrains to HMVII cells, the method of Santos et al. [16] was used, with some modifications. Initially, a monolayer of HMVII cells at a concentration of 1 × 106 cells/well was grown in 24-well plates in an incubator (SANYO) at 37°C and 5% CO2, and the lactobacilli were grown in MRS broth for 18 h at 37°C in anaerobic jar. After culture, the lactobacilli were recovered by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline solution (w/v), and adjusted to 108 CFU/mL in RPMI supplemented with 10% fetal bovine serum (FBS).Lactobacillus suspensions were added to wells containing HMVII cells and were incubated for 2 h at 37°C and 5% CO2. Subsequently, the cells were washed three times with 0.9% saline solution (w/v) and removed from the plates with 0.25% trypsin-EDTA for 5 min. The percentage of adhered lactobacilli was determined by plating serial dilutions on MRS agar. The plates were incubated for 48 h at 37°C, and then the bacteria (CFU/mL) were counted. The percentage of adherent lactobacilli was calculated by the following formula: adhesion (%) = (final CFUend/CFUinitial) × 100.To visualize the adhesion of theLactobacillus strains to HMVII cells, scanning electron microscopy (SEM) was performed. HMVII cells (1 × 106 cells/well) were grown on 24-well plates (containing glass coverslips in each well) in an incubator (SANYO) at 37°C and 5% CO2, with one of the two testedLactobacillusstrains (1 × 108 CFU/mL) and incubated for 2 h at 37°C and 5% CO2. HMVII cells alone were used as controls. After incubation, the coverslips were washed three times with 0.9% saline solution to remove the nonadherent lactobacilli cells.


### 2.6. Evaluation of Acid Production by Lactobacilli

To evaluate acid production by lactobacilli the method of Pessoa el al. was used [18]. Culture supernatants of theLactobacillusstrains were obtained to evaluate acid production. The cultures were grown in MRS broth for 48 h at 37°C and then centrifuged (8000 ×g, 15 min). The supernatant was separated from the pellet, and the pH was measured with a pH meter.


### 2.7. Susceptibility of Lactobacilli to Antibiotics

The antibiotics susceptibility profiles of theLactobacillusstrains were determined by using the modified agar diffusion method of Clinical and Laboratory Standards Institute (CLSI) [22]. Lactobacilli were grown in MRS broth for 18 h at 37°C in anaerobic jar and then centrifuged (8000 ×g, 10 min). The cell pellets were washed twice with 0.9% saline solution (w/v) and adjusted to 0.5 MacFarland. Then, 100 μL of this suspension was spread on MRS agar plates, and antibiotic disks were placed on the plates. The plates were incubated at 37°C for 24 h, and then the diameter of the zone of inhibition surrounding each disk was measured and classified as sensitive (S), moderately sensitive (MS), or resistant (R), according to Charteris et al. [23]. The antibiotics tested were amoxicillin (10 μg), ampicillin (10 μg), cephalothin (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), chloramphenicol (30 μg), erythromycin (10 μg), gentamicin (10 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), penicillin G (10 μg), tetracycline (30 μg), and vancomycin (30 μg).


### 2.8. Interactions between HMVII Cells and LAMP and the Effect ofLactobacillus Strains

To assess the interactions betweenMycoplasma andUreaplasmaLAMP and HMVII cells, first a monolayer of HMVII cells, at a concentration of 1 × 106 cells/well, was grown in 24-well plates in an incubator (SANYO) at 37°C and 5% CO2. After 24 h, the interactions between the lactobacilli strains and the lipoproteins tested were added to the wells of the plates, according to Table 1. The concentration of LAMP used in study (4 μg/mL) was determined from previous tests evaluating the dose response of HMVII cells to the lipoproteins (data not shown). And the suspensions of lactobacilli were adjusted to 1 × 108 CFU/mL in RPMI supplemented with 10% FBS.Table 1
Interactions between vaginal cells andMollicuteslipoproteins in the presence and/or absence of lactobacilli isolated from cocoa fermentation.
Lipoproteins Lactobacilli Lactobacillus fermentum FA4 Lactobacillus plantarum PA3 Ureaplasma parvum (UuLAMP) UpLAMP +L. fermentum FA4 UpLAMP +L. plantarum PA3 Ureaplasma urealyticum (UpLAMP) UuLAMP +L. fermentum FA4 UuLAMP +L. plantarum PA3 Mycoplasma hominis (MhLAMP) MhLAMP +L. fermentum FA4 MhLAMP +L. plantarum PA3 Mycoplasma genitalium (MgLAMP) MgLAMP +L. fermentum FA4 MgLAMP +L. plantarum PA3


### 2.9. Flow Cytometry to Assess the Viability of HMVII Cells

After the HMVII cells were incubated with theLactobacillusstrains and/or lipoproteins for 24 h, they were disrupted by trypsinization, washed twice with PBS and collected by centrifugation (600 xg, 10 min). The cells were labelled using LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions. Fluorescence was analyzed on a FC 500 flow cytometer (Beckman Coulter) with 20,000 events.


### 2.10. Scanning Electron Microscopy (SEM)

Scanning electron microscopy was performed to confirmLactobacillus adhesion to the vaginal cells (after 2 h), as well as demonstrate the physical integrity of the HMIVII cells after interaction with the lipoproteins andLactobacillusstrains (after 24 h). After the incubation period, the culture supernatant was removed from each well, and the cells on the coverslips were fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer for at least 4 h. Next, the samples were washed with 0.1M cacodylate buffer for 5 min, twice, and then dehydrated with increasing concentrations of acetone (50–100%) for 10 min. After dehydration, the samples were taken to the critical point chamber and mounted on the sample holder of the “Stub” SEM with double carbon tape. Then, they were sputter coated with a thin layer of gold, about 20–30 nm thick, with a Sputter Coater SCD 050 (Baltec) for observation in a Quanta 250 Scanning Electron Microscope (FEI Company).


### 2.11. Statistical Analysis

All analyses were performed in triplicate. Quantitative data are expressed as the mean and standard deviation and analyzed with GraphPad Prism software (version 5.01).For the hydrophobicity, autoaggregation, and adhesion tests, the statistical differences were determined by t-test followed by the Mann-Whitney posttest, and a p value less than 0.05 was considered significant. The statistical differences in the flow cytometry results were determined by ANOVA followed by Tukey’s posttest, and a p value less than 0.01 was considered significant.


## 2.1. Cell Lines, Microorganisms, and Growth Conditions

HMVII human vaginal epithelial cell line (BCRJ 0316) was obtained from the Rio de Janeiro Cell Bank and was grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FSB), penicillin 100 IU/mL, and streptomycin 100μg/mL.L. fermentum FA4 andL. plantarum PA3 were previously isolated by our research group from a cocoa fermentation [16]. These strains were confirmed to the species level by 16S rRNA sequencing and were deposited in GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers KU244506 and KU244472, respectively. Lactobacilli were grown in de Man, Rogosa, and Sharpe (MRS) medium for 18 h at 37°C in anaerobic jar.M. hominis(ATCC 23114) andM. genitalium(ATCC 33530) were grown in 1 L of SP-4 medium.U. urealyticum serotype 7 (ATCC 27819) andU. parvum serotype 3 (ATCC 27815) were cultured in 200 mL of Ureaplasma Broth (UB) medium. All strains were maintained at 37°C and 5% CO2 until log phase growth. Growth control was observed by the observation of color change in the liquid medium, plus pH indicator (phenol red).


## 2.2. Extraction of Membrane-Associated Lipoproteins (LAMP)

Lipoproteins were extracted according to the method developed by Wang et al. [19] with some modifications. Briefly,M. hominis,M. genitalium,U. parvum, andU. urealyticum were cultured until log phase, until the observation of color change in the liquid medium, pH indicator (phenol red). Then, the cells were recovered by centrifugation at 23,700 ×gfor 30 min at 4°C (Beckman Coulter) and washed with sterile phosphate-buffered saline (PBS) (1X, pH 7,4) to remove the residual culture medium. The cell pellet was suspended in 5 mL of Tris-EDTA (50 mM Tris [pH 8], 0.15 M NaCl, 1 mM EDTA), and Triton TX-114 was added to a final concentration of 2%. The mixture was homogenized by vortexing and incubated at 4°C for 60 min. The lysate was then incubated at 37°C for 10 min and centrifuged at 23,700 ×g at 22°C for 20 min for phase separation. The upper aqueous phase was discarded, and in the final TX-114 step, the volume was adjusted to the original volume by the addition of Tris-EDTA. Then, 2.5 volumes of ethanol were added to precipitate the lipoproteins overnight at −20°C. The precipitated materials were recovered by centrifugation at 23,700 ×gfor 15 min at 4°C. After centrifugation, the pellet was homogenized in PBS by sonication, 6 to 8 cycles per minute at a power of 10W (Coler-Parmer Ultrasonic Processor). Then, the microtubes containing the lipoproteins were stored at −80°C until use. Lipoproteins were quantitated using the 2D Quant Kit (GE Healthcare) according to the manufacturer’s protocol and preincubated for 2 h with polymyxin B at 1000 U/mL prior to the use.


## 2.3. Autoaggregation Assay

To verify the autoaggregation capacity of theLactobacillus strains, the method of Kos et al. [20] was used, with some modifications. Briefly, the strains were cultured in MRS broth for 18 h at 37°C in anaerobic jar. Then, the cells were recovered by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline solution (w/v), and suspended in the same solution to 1 × 108 CFU/mL in a spectrophotometer (Thermo-Scientific). The suspensions were homogenized by vortexing and incubated at 37°C for 5 h. Then, a 1 mL aliquot was gently removed from the top of the suspension every hour, and its absorbance at 600 nm (A600) was read in a spectrophotometer (Thermo-Scientific). Autoaggregation was calculated using the following formula: autoaggregation (%) = [(A0-At)/A0] × 100, where A0 is the absorbance at time 0 h, and At is the absorbance at time t, which was measured every hour, for up to 5 h.


## 2.4. Hydrophobicity Assay

The hydrophobicity of theLactobacillusstrains was verified by testing microbial adherence to hydrocarbons (MATH), using a method adapted from Rodríguez et al. [21].Lactobacillus strains were cultured in MRS broth at 37°C for 18 h in anaerobic jar. Then, the cultures were harvested by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline, and adjusted to an optical density at 600 nm (OD600) of 0.7 in saline. One milliliter of solvent (xylene or toluene) was added to each bacterial suspension, and the mixtures were vortexed for 2 min and then incubated for 2 h at 37°C. The lower aqueous phase was carefully removed, and the A600 was read in a spectrophotometer. Hydrophobicity was calculated using the following formula: hydrophobicity (%) = ((A0-A2)/A0) × 100, where A0 is the absorbance at time 0 (0 h) and A2 is the absorbance after 2 h.


## 2.5.Lactobacillus Adhesion to HMVII Cells

To verify the adhesion capacity of theLactobacillusstrains to HMVII cells, the method of Santos et al. [16] was used, with some modifications. Initially, a monolayer of HMVII cells at a concentration of 1 × 106 cells/well was grown in 24-well plates in an incubator (SANYO) at 37°C and 5% CO2, and the lactobacilli were grown in MRS broth for 18 h at 37°C in anaerobic jar. After culture, the lactobacilli were recovered by centrifugation (8000 ×g, 10 min), washed twice with 0.9% saline solution (w/v), and adjusted to 108 CFU/mL in RPMI supplemented with 10% fetal bovine serum (FBS).Lactobacillus suspensions were added to wells containing HMVII cells and were incubated for 2 h at 37°C and 5% CO2. Subsequently, the cells were washed three times with 0.9% saline solution (w/v) and removed from the plates with 0.25% trypsin-EDTA for 5 min. The percentage of adhered lactobacilli was determined by plating serial dilutions on MRS agar. The plates were incubated for 48 h at 37°C, and then the bacteria (CFU/mL) were counted. The percentage of adherent lactobacilli was calculated by the following formula: adhesion (%) = (final CFUend/CFUinitial) × 100.To visualize the adhesion of theLactobacillus strains to HMVII cells, scanning electron microscopy (SEM) was performed. HMVII cells (1 × 106 cells/well) were grown on 24-well plates (containing glass coverslips in each well) in an incubator (SANYO) at 37°C and 5% CO2, with one of the two testedLactobacillusstrains (1 × 108 CFU/mL) and incubated for 2 h at 37°C and 5% CO2. HMVII cells alone were used as controls. After incubation, the coverslips were washed three times with 0.9% saline solution to remove the nonadherent lactobacilli cells.


## 2.6. Evaluation of Acid Production by Lactobacilli

To evaluate acid production by lactobacilli the method of Pessoa el al. was used [18]. Culture supernatants of theLactobacillusstrains were obtained to evaluate acid production. The cultures were grown in MRS broth for 48 h at 37°C and then centrifuged (8000 ×g, 15 min). The supernatant was separated from the pellet, and the pH was measured with a pH meter.


## 2.7. Susceptibility of Lactobacilli to Antibiotics

The antibiotics susceptibility profiles of theLactobacillusstrains were determined by using the modified agar diffusion method of Clinical and Laboratory Standards Institute (CLSI) [22]. Lactobacilli were grown in MRS broth for 18 h at 37°C in anaerobic jar and then centrifuged (8000 ×g, 10 min). The cell pellets were washed twice with 0.9% saline solution (w/v) and adjusted to 0.5 MacFarland. Then, 100 μL of this suspension was spread on MRS agar plates, and antibiotic disks were placed on the plates. The plates were incubated at 37°C for 24 h, and then the diameter of the zone of inhibition surrounding each disk was measured and classified as sensitive (S), moderately sensitive (MS), or resistant (R), according to Charteris et al. [23]. The antibiotics tested were amoxicillin (10 μg), ampicillin (10 μg), cephalothin (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), chloramphenicol (30 μg), erythromycin (10 μg), gentamicin (10 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), penicillin G (10 μg), tetracycline (30 μg), and vancomycin (30 μg).


## 2.8. Interactions between HMVII Cells and LAMP and the Effect ofLactobacillus Strains

To assess the interactions betweenMycoplasma andUreaplasmaLAMP and HMVII cells, first a monolayer of HMVII cells, at a concentration of 1 × 106 cells/well, was grown in 24-well plates in an incubator (SANYO) at 37°C and 5% CO2. After 24 h, the interactions between the lactobacilli strains and the lipoproteins tested were added to the wells of the plates, according to Table 1. The concentration of LAMP used in study (4 μg/mL) was determined from previous tests evaluating the dose response of HMVII cells to the lipoproteins (data not shown). And the suspensions of lactobacilli were adjusted to 1 × 108 CFU/mL in RPMI supplemented with 10% FBS.Table 1
Interactions between vaginal cells andMollicuteslipoproteins in the presence and/or absence of lactobacilli isolated from cocoa fermentation.
Lipoproteins Lactobacilli Lactobacillus fermentum FA4 Lactobacillus plantarum PA3 Ureaplasma parvum (UuLAMP) UpLAMP +L. fermentum FA4 UpLAMP +L. plantarum PA3 Ureaplasma urealyticum (UpLAMP) UuLAMP +L. fermentum FA4 UuLAMP +L. plantarum PA3 Mycoplasma hominis (MhLAMP) MhLAMP +L. fermentum FA4 MhLAMP +L. plantarum PA3 Mycoplasma genitalium (MgLAMP) MgLAMP +L. fermentum FA4 MgLAMP +L. plantarum PA3


## 2.9. Flow Cytometry to Assess the Viability of HMVII Cells

After the HMVII cells were incubated with theLactobacillusstrains and/or lipoproteins for 24 h, they were disrupted by trypsinization, washed twice with PBS and collected by centrifugation (600 xg, 10 min). The cells were labelled using LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions. Fluorescence was analyzed on a FC 500 flow cytometer (Beckman Coulter) with 20,000 events.


## 2.10. Scanning Electron Microscopy (SEM)

Scanning electron microscopy was performed to confirmLactobacillus adhesion to the vaginal cells (after 2 h), as well as demonstrate the physical integrity of the HMIVII cells after interaction with the lipoproteins andLactobacillusstrains (after 24 h). After the incubation period, the culture supernatant was removed from each well, and the cells on the coverslips were fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer for at least 4 h. Next, the samples were washed with 0.1M cacodylate buffer for 5 min, twice, and then dehydrated with increasing concentrations of acetone (50–100%) for 10 min. After dehydration, the samples were taken to the critical point chamber and mounted on the sample holder of the “Stub” SEM with double carbon tape. Then, they were sputter coated with a thin layer of gold, about 20–30 nm thick, with a Sputter Coater SCD 050 (Baltec) for observation in a Quanta 250 Scanning Electron Microscope (FEI Company).


## 2.11. Statistical Analysis

All analyses were performed in triplicate. Quantitative data are expressed as the mean and standard deviation and analyzed with GraphPad Prism software (version 5.01).For the hydrophobicity, autoaggregation, and adhesion tests, the statistical differences were determined by t-test followed by the Mann-Whitney posttest, and a p value less than 0.05 was considered significant. The statistical differences in the flow cytometry results were determined by ANOVA followed by Tukey’s posttest, and a p value less than 0.01 was considered significant.


## 3. Results and Discussion

### 3.1. Evaluation of the Probiotic Properties of Lactobacilli Isolated from Cocoa Fermentation

To test whether the testLactobacillusstrains had probiotic properties, their surface properties, including autoaggregation and hydrophobicity, adherence to vaginal epithelial cells, and acid production in culture, were evaluated.Autoaggregation percentages forL. fermentum FA4 andL. plantarum PA3 were 28.55% and 31.82%, respectively (Table 2). Bacterial autoaggregation, defined as the ability of cells to form precipitates, is considered an important probiotic property, as it is directly related to adhesion to host cell surfaces, one of the mechanisms by which probiotics compete with pathogenic bacteria [24]. These values were within the expected range, since there is great variation in autoaggregation among strains of both human vaginal microbiota and nonhuman origin [25, 26]. Gómez et al. [27] found autoaggregation values ranging from 20 to 70%, after only 24 h of incubation, among 8 strains of LAB isolated from different food sources, and the highest percentage, 67%, was aWeissella viridescensstrain isolated from mature cheese. Similar to our study, the two strains ofL. plantarum and one strain ofL. fermentum isolated from cocoa fermentation showed autoaggregation values of approximately 29%, 33%, and 31% after 5 h of incubation [18].Table 2
The surface properties of lactobacilli isolated from cocoa fermentation, their adhesion to vaginal cells, and acidification of the culture medium.
Strain Surface properties Adhesion to HMVII cells ( % ) pH value Autoaggregation ( % ) Hydrophobicity ( % ) Xylene Toluene L. fermentum FA4 28.55 ± 1.08 57.03 ± 1.80 65.33 ± 0.99 31.66 ± 7.82 4.32 L. plantarum PA3 31.82 ± 0.58 66.75 ± 5.11 71.12 ± 3.31 41.65 ± 13.85 4.33The hydrophobicity or microbial adhesion to hydrocarbons (MATH) can be classified as low (MATH <33%), medium (33% <MATH <66%), or high (MATH> 66%) [28]. The hydrophobicity ofL. fermentum FA4 was considered to be average for both xylene (57.03%) and toluene (65.33%), while that ofL. plantarum PA3 was considered high for both xylene and toluene (66.75 and 71.12%, resp.). Some authors present hydrophobicity as microbial adhesion to solvents (MATS), classifying the bacterial surface as hydrophobic (MATS ≥55.00%), amphiphilic (45.00% ≤MATS≤54.99%), or hydrophilic (MATS ≤44.99%) [29, 30]. According to this classification,L. fermentum FA4 andL. plantarumare considered to be hydrophobic. This methodology is a simple way of evaluating the hydrophobicity of potential probiotic cell lines, indicating their ability to adhere to apolar surfaces, such as epithelial cell membranes. Their hydrophobicity justifies the application of these bacteria in probiotic formulations [30]. Similar results were obtained in other studies of isolates from nonhuman sources. For example, Cui et al. [31] determined hydrophobicities of 44% to 78% in lactobacilli isolated from artisanal cheese. In the present study, lactobacilli isolated from a cocoa fermentation showed higher hydrophobicities than several isolates of intestinal origin. In a study conduced by Yadav et al. [32], the highest hydrophobicity amongL. plantarum isolates from human feces was 39.49%.The percentage of cells that adhered to the vaginal cells was 31.66% forL. fermentum FA4 and 41.65% forL. plantarum PA3 after 2 h of incubation. Adhesion was confirmed by SEM, in which it was possible to observe lactobacilli adhered to the surface of the HMVII cells (Figure 1). Adhesion is considered to be one of the major properties of a probiotic strain, because it is thought that the longer the strain remains adhered to the surface of the host cells, the more benefits it can confer. Probiotics are able to induce the expression of adhesins, such as mucin, fibronectin, collagen, laminin, and fibrinogen, which mediate adhesion [32, 33]. Strains ofLactobacillus paracasei subsp.paracasei produce an aggregation promoting factor (AggLb) that contributes to its high aggregation capacity, as well as a strong, specific interaction with host cell collagen, indicating that there is a direct relationship between cell aggregation, hydrophobicity, and collagen binding, as was observed in theLactobacillusstrains used in this study [34].Figure 1
Adhesion of lactobacilli isolated from cocoa fermentation to vaginal epithelial cells after 2 h of incubation. (a) Untreated HMVII cells (×5000); (b) HMVII cells incubated withL. fermentum FA4 (×5000); (c) HMVII cells incubated withL. plantarum PA3 (×5,000, details at ×20,000). Red arrows indicate the lactobacilli attached to the cell surface.
(a) (b) (c)By evaluating the pH of the culture supernatants, we observed that the twoLactobacillus strains were able to reduce the pH of the culture medium from an initial pH=6.6 to similar pH values of 4.32 forL. fermentum FA4 and 4.33 forL. plantarum PA3 (Table 2). This reduction in pH by lactobacilli is mediated by the production of acids, mainly lactic and acetic acids, and is one of the mechanisms by which the growth of pathogenic bacteria is inhibited [35, 36]. In Gram-negative bacteria, lactic acid acts as a permeator of the bacterial outer membrane, releasing lipopolysaccharides (LPS), and increasing their susceptibility to other antibiotics produced by the host [37]. According to the ability to ferment sugars lactobacilli can be classified into homofermentative species (e.g.,L. plantarum), which convert sugars into lactic acid, and heterofermentative species (e.g.,L. fermentum), which produce lactic and acetic acids, ethanol, and CO2 [38]. Thus, contrary to what was observed in the present study, other authors have reported variations in the pH values of the culture medium for different species, indicating differences in acid production and secretion profiles; generally,L. plantarum strains tend to produce more acid thanL. fermentum strains [18]. Among vaginalLactobacillusisolates with inhibitory potential againstG. vaginalis, anL. fermentum strain reduced the pH of the culture supernatant to pH 4.16 after 48 h of incubation [39], whereas 2 strains ofL. plantarum isolated from organic fertilizer, in the same conditions for 24 h, reduced the pH to 3.83 and 3.88 [40]. Two strains ofL. plantarum isolated from a cocoa fermentation reduced the pH to 3.81 and 3.77, whereas anL. fermentumisolate reduced the pH to 4.78 [18].The testedL. fermentum FA4 andL. plantarum PA3 strains did not show any significant differences in the four parameters evaluated, indicating that they have similar probiotic potential.


### 3.2. Susceptibility of Lactobacilli to Antibiotics

In the antibiotics sensitivity profile testing, the twoLactobacillusstrains were sensitive or moderately sensitive to most of the antibiotics tested. They were only resistant to vancomycin, aminoglycosides, and quinolones (Table 3). Although lactobacilli are “generally recognized as safe”, safety tests, such as the determination of antibiotics sensitivity profiles, should always be performed to avoid the transfer of resistance genes, since these profiles vary among species [41]. Similar sensitivity profiles have been described in other studies, corroborating the findings of the present study. Santos et al. [42] found thatL. fermentum andL. plantarum isolates from cocoa fermentation were resistant to vancomycin and quinolone class antibiotics. Similarly, 12 lactobacilli strains isolated from cottage cheese, typical of northeastern China, were resistant to streptomycin, gentamicin, vancomycin, and ciprofloxacin [31]. Strains of human origin also have similar sensitivity profiles. Bouridani et al. [25] showed that all tested vaginalLactobacillusisolates were resistant to ofloxacin, gentamicin, and ciprofloxacin, and almost all strains were sensitive to trimethoprim-sulfamethoxazole, ampicillin, erythromycin, cefotaxime, chloramphenicol, tetracycline, and nitrofurantoin. Bacteria in the genusLactobacillus, like other Gram-positive bacteria, are intrinsically resistant to glycopeptides such as vancomycin. However, the gene responsible for this resistance is chromosomal, and it is not inducible or transferable to other bacteria [43, 44]. Thus, the strains under study could be used in therapeutic applications, as they would not pose a risk to the health of humans or other animals.Table 3
Antibiotics susceptibility profiles ofLactobacillus strains isolated from cocoa fermentation.
Antibiotics Susceptibilitya Group Name Antibiotic concentration (μg) L. fermentumFA4 L. plantarumPA3 Inhibitors of cell wall synthesis  Penicillin Amoxicillin 10 S S Ampicillin 10 S S Penicillin G 10 S MS Cephalosporins Cefalotin 30 S S Glycopeptides Vancomycin 30 R R  Inhibitors of protein synthesis  Aminoglycosides Amikacin 30 R R Gentamicin 10 R R Streptomycin 10 R R Tetracyclines Tetracycline 30 S S Single antibiotics Chloramphenicol 30 S S Macrolides Erythromycin 15 S S Lincosamides Clindamycin 2 S S  Inhibitors of nucleic acid synthesis  Quinolones Ciprofloxacin 5 R R Norfloxacin 10 R R  The broad mechanism of action  Single antibiotics Nitrofurantoin 300 S S


### 3.3. Interaction of HMVII Cells with Lipoproteins and the Effects of Treatment with Lactobacilli

Initially, we evaluated whether lactobacilli isolated from cocoa fermentation reduced the viability of cells of vaginal lineage. After 24 h of incubation withL. fermentum FA4 andL. plantarum PA3, the HMVII cells showed cell death rates of only 0.72% and 0.36%, respectively, and these rates were not significantly different from each other or from that of the control (1.99%; Figure 2).Figure 2
Death of HMVII cells after 24 h of incubation withLactobacillusstrains or membrane-associated lipoproteins (LAMP) fromU. parvum (UpLAMP),U. urealyticum (UuLAMP),M. hominis (MhLAMP), orM. genitalium (MgLAMP). Different letters indicate significant differences (p <0.01).This minimal reduction in HMVII cell viability after incubation with theseLactobacillus strains demonstrates that both can exist in the human vaginal environment without any toxicity. In addition, they reinforce the probiotic potential of these strains in protecting against pathogens that cause BV. Similarly, Abramov et al. [45] showed thatLactobacillus crispatus 2029 did not induce apoptosis in vaginal epithelium cells (VK2/E6E7) and has no cytotoxic effects on them. Strains ofL. fermentum andL. plantaruminhibited apoptosis in HeLa epithelial cells and showed antagonistic activity againstCandida albicans andG. vaginalis [46]. In another study, the viability of cells isolated from epidermoid carcinoma of the cervix (CaSki) and HeLa cells was maintained after incubation with different concentrations ofLactobacillus casei extract [47]. In contrast, cell death of HMVII cells increased significantly after incubation with the membrane-associated lipoproteins (LAMP) ofU. parvum,U. urealyticum,M. hominis, andM. genitalium; LAMP extracted fromU. parvumandM. hominisinduced 41% and 34% cell death, respectively, the highest values among the 4 species (Figure 2).Mollicutes causing genital infections need to adhere and subsequently invade the cells of the genitourinary tract to obtain nutrients from the host cells [1, 2, 47]. During adhesion, human cells first interact with the plasma membrane of these microorganisms, specifically with their lipoproteins. Recently, cytoadhesion and invasion present on the membrane ofM. hominiswere shown to interact with HeLa cells [48]. Hopfe et al. [49] showed that, after 4 h of infection of HeLa cells withM. hominis, genes related to the cell cycle, growth, and cell death are highly regulated, and lipoproteins are generally responsible for inducing inflammation, apoptosis, and cell death [5, 50].The present study is the first to use the HMVII vaginal cell line as a model for interaction with the lipoproteins ofMollicutes, mimicking the infection that occursin vivo. However, the levels of cell death induced by lipoproteins extracted from mycoplasmas and ureaplasmas may vary according to both species and the human cell line used in the experiment. Similarly, to the present study, 2 μg/mLMycoplasma penetrans lipoproteins induced apoptosis in RAW264.7 murine macrophages [51]. LAMP fromMycoplasma salivarium andMycoplasma fermentans caused both apoptosis and necrosis in myeloid (HL-60 and THP-1) and lymphoid (MOLT-4 and Raji cells) leukemia cells at concentrations of 20 and 7 μg/mL, respectively [52]. However, Bai et al. (2007) [4] showed thatMycoplasma hyopneumoniaeLAMP induced apoptosis in the 3D4/21 porcine alveolar macrophage (PAM) cell line at concentrations of 0.4 mg/mL or higher, which is higher than the concentration used in the present study.In this study, we also demonstrated that incubation of HMVII cells with lactobacilli isolated from cocoa fermentation significantly reduced cell death caused by the interaction withUreaplasma species LAMP from 41.03% in UpLAMP to 2.43% (UpLAMP +L. fermentum FA4) and 0.43% (UpLAMP +L. plantarum PA3) and from 25.24% (UuLAMP) to 13.97% (UuLAMP +L. fermentum FA4) (Figure 3). As described for the genusUreaplasma, the treatment of HMVII with lactobacilli also managed to significantly decrease the rate of cell death caused by the interaction with LAMP of the genusMycoplasma. The cell death reduced from 34.29% (MhLAMP) to 14.06% (MhLAMP +L. fermentum FA4) and 14.61% (MhLAMP +L. plantarum PA3) and 25.53 (MgLAMP) to 8.91% (MgLAMP +L. fermentum FA4) and 11% (MgLAMP +L. plantarum PA3) (Figure 4).Figure 3
Death of HMVII cells after 24 h of incubation with LAMP fromU. parvum (UpLAMP) orU. urealyticum (UuLAMP) with and withoutL. plantarum PA3 orL. fermentum FA4. ∗ p<0.01 compared to cells with LAMP alone.Figure 4
Death of HMVII cells after 24 h of incubation with LAMP fromM. hominis (MhLAMP) orM. genitalium(MgLAMP) with and withoutL. plantarum PA3 orL. fermentum FA4. ∗ p<0.01 compared to cells with LAMP alone.The findings of flow cytometry were confirmed by microscopy (Figures5–8). The images showed the vaginal cells after interaction with lipoproteins extracted fromU. parvum,U. urealyticum,M. hominis, andM. genitalium (resp.) and treatment with the two strains of lactobacilli. Cells incubated only with lipoproteins at a concentration of 4 μg / mL (Figures 5(b)–8(b)) presented a large number of cells with altered morphology, when compared to the control (Figures 5(a)–8(a)).Figure 5
Interaction of HMVII cells with 4 μ g/mL ofU. parvumLAMP with and withoutL. fermentum FA4 orL. plantarum PA3.(a) Control (HMVII cells alone). (b) HMVII with UpLAMP. (c) HMVII with UpLAMP andL. fermentum FA4. (d) HMVII with UpLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 6
Interaction of HMVII cells withU. urealyticumLAMP with and withoutL. fermentum FA4 orL. plantarum PA3.(a) Control (HMVII cells alone). (b) HMVII with UuLAMP. (c) HMVII with UuLAMP andL. fermentum FA4. (d) HMVII with UuLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 7
Interaction of HMVII cells with 4 μ g/mL ofM. hominisLAMP with and withoutL. fermentum FA4 orL. plantarum PA3. (a) Control (HMVII cells alone). (b) HMVII with MhLAMP. (c) HMVII with MhLAMP andL. fermentum FA4. (d) HMVII with MhLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 8
Interaction of HMVII cells with 4 μ g/mLM. genitalium lipoproteins with and withoutL. fermentum FA4 orL. plantarum PA3. (a) Control (HMVII cell alone). (b) HMVII with MgLAMP. (c) HMVII with MgLAMP andL. fermentum FA4. (d) HMVII with MgLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy ×2500).
(a) (b) (c) (d)In contrast, in treatments with L. fermentum FA4 (Figures5(c)–8(c)) and L. plantarum PA3 (Figures 5(d)–8(d)), bacteria adhered to HMVII cells were able to reduce cell death. This property was evidenced by normal morphological characteristics of the cells compared to the negative control.Several studies have shown that lactobacilli isolated from the vaginal environment have activity against potentially pathogenic bacteria. Strains ofL. crispatus andL. vaginalis inhibited the growth of several bacterial species causing vaginal and urinary tract infections, includingEnterococcus faecalis,E. coli,S. aureus,Enterococcus faecium,G. vaginalis, andProteus mirabilis[53]. In addition to promoting a protective vaginal environment, strains ofL. crispatusshowed antagonistic activity against several species of the genusCandida [45]. Strains ofLactobacillus rhamnosus andLactobacillus reuteri increased the viability of human epidermal keratinocytes from 8.8% after infection withS. aureus to 42.7% and 53.1%, respectively [54]. However, very few studies have examined the interaction between lactobacilli andMollicutes. Danielle et al. [55] demonstrated that bacteriocins produced byL. fermentum andL. rhamnosus showed antibacterial activity against vaginal isolates ofU. urealyticum andM. hominis. This is the first study to investigate the interaction betweenLactobacillusstrains isolated from cocoa fermentation and theMollicutesclass of bacteria. The antagonist effects of probiotic microorganisms against pathogenic bacteria include competitive adhesion to the mucosa and epithelium; strengthening of the epithelial barrier; secretion of antibiotics substances, such as bacteriocins and organic acids; and modulation of the immune system [36].One possible protective mechanism of the strains in this study againstMycoplasma andUreaplasma lipoproteins is competitive adhesion to host epithelial cells. The excellent adhesion capacity and hydrophobicity of the strains in this study suggest antagonistic effects on the adhesion of LAMP to the plasma membrane of the vaginal epithelium. In addition, this high hydrophobicity, coupled with the self-aggregation ability, of the tested lactobacilli may cause the LAMP to bind to the cell wall of these microorganisms rather than to the epithelial cell membrane [35]. Finally, the cell interaction experiment results indicate that theseL. fermentum andL. plantarum strains isolated from cocoa fermentation have potential for use as vaginal cell protectors against the LAMP of several important pathogens of the genitourinary tract, includingU. parvum,U. urealyticum,M. hominis, andM. genitalium. However, more studies are needed to clarify their protective mechanism(s) of action.


## 3.1. Evaluation of the Probiotic Properties of Lactobacilli Isolated from Cocoa Fermentation

To test whether the testLactobacillusstrains had probiotic properties, their surface properties, including autoaggregation and hydrophobicity, adherence to vaginal epithelial cells, and acid production in culture, were evaluated.Autoaggregation percentages forL. fermentum FA4 andL. plantarum PA3 were 28.55% and 31.82%, respectively (Table 2). Bacterial autoaggregation, defined as the ability of cells to form precipitates, is considered an important probiotic property, as it is directly related to adhesion to host cell surfaces, one of the mechanisms by which probiotics compete with pathogenic bacteria [24]. These values were within the expected range, since there is great variation in autoaggregation among strains of both human vaginal microbiota and nonhuman origin [25, 26]. Gómez et al. [27] found autoaggregation values ranging from 20 to 70%, after only 24 h of incubation, among 8 strains of LAB isolated from different food sources, and the highest percentage, 67%, was aWeissella viridescensstrain isolated from mature cheese. Similar to our study, the two strains ofL. plantarum and one strain ofL. fermentum isolated from cocoa fermentation showed autoaggregation values of approximately 29%, 33%, and 31% after 5 h of incubation [18].Table 2
The surface properties of lactobacilli isolated from cocoa fermentation, their adhesion to vaginal cells, and acidification of the culture medium.
Strain Surface properties Adhesion to HMVII cells ( % ) pH value Autoaggregation ( % ) Hydrophobicity ( % ) Xylene Toluene L. fermentum FA4 28.55 ± 1.08 57.03 ± 1.80 65.33 ± 0.99 31.66 ± 7.82 4.32 L. plantarum PA3 31.82 ± 0.58 66.75 ± 5.11 71.12 ± 3.31 41.65 ± 13.85 4.33The hydrophobicity or microbial adhesion to hydrocarbons (MATH) can be classified as low (MATH <33%), medium (33% <MATH <66%), or high (MATH> 66%) [28]. The hydrophobicity ofL. fermentum FA4 was considered to be average for both xylene (57.03%) and toluene (65.33%), while that ofL. plantarum PA3 was considered high for both xylene and toluene (66.75 and 71.12%, resp.). Some authors present hydrophobicity as microbial adhesion to solvents (MATS), classifying the bacterial surface as hydrophobic (MATS ≥55.00%), amphiphilic (45.00% ≤MATS≤54.99%), or hydrophilic (MATS ≤44.99%) [29, 30]. According to this classification,L. fermentum FA4 andL. plantarumare considered to be hydrophobic. This methodology is a simple way of evaluating the hydrophobicity of potential probiotic cell lines, indicating their ability to adhere to apolar surfaces, such as epithelial cell membranes. Their hydrophobicity justifies the application of these bacteria in probiotic formulations [30]. Similar results were obtained in other studies of isolates from nonhuman sources. For example, Cui et al. [31] determined hydrophobicities of 44% to 78% in lactobacilli isolated from artisanal cheese. In the present study, lactobacilli isolated from a cocoa fermentation showed higher hydrophobicities than several isolates of intestinal origin. In a study conduced by Yadav et al. [32], the highest hydrophobicity amongL. plantarum isolates from human feces was 39.49%.The percentage of cells that adhered to the vaginal cells was 31.66% forL. fermentum FA4 and 41.65% forL. plantarum PA3 after 2 h of incubation. Adhesion was confirmed by SEM, in which it was possible to observe lactobacilli adhered to the surface of the HMVII cells (Figure 1). Adhesion is considered to be one of the major properties of a probiotic strain, because it is thought that the longer the strain remains adhered to the surface of the host cells, the more benefits it can confer. Probiotics are able to induce the expression of adhesins, such as mucin, fibronectin, collagen, laminin, and fibrinogen, which mediate adhesion [32, 33]. Strains ofLactobacillus paracasei subsp.paracasei produce an aggregation promoting factor (AggLb) that contributes to its high aggregation capacity, as well as a strong, specific interaction with host cell collagen, indicating that there is a direct relationship between cell aggregation, hydrophobicity, and collagen binding, as was observed in theLactobacillusstrains used in this study [34].Figure 1
Adhesion of lactobacilli isolated from cocoa fermentation to vaginal epithelial cells after 2 h of incubation. (a) Untreated HMVII cells (×5000); (b) HMVII cells incubated withL. fermentum FA4 (×5000); (c) HMVII cells incubated withL. plantarum PA3 (×5,000, details at ×20,000). Red arrows indicate the lactobacilli attached to the cell surface.
(a) (b) (c)By evaluating the pH of the culture supernatants, we observed that the twoLactobacillus strains were able to reduce the pH of the culture medium from an initial pH=6.6 to similar pH values of 4.32 forL. fermentum FA4 and 4.33 forL. plantarum PA3 (Table 2). This reduction in pH by lactobacilli is mediated by the production of acids, mainly lactic and acetic acids, and is one of the mechanisms by which the growth of pathogenic bacteria is inhibited [35, 36]. In Gram-negative bacteria, lactic acid acts as a permeator of the bacterial outer membrane, releasing lipopolysaccharides (LPS), and increasing their susceptibility to other antibiotics produced by the host [37]. According to the ability to ferment sugars lactobacilli can be classified into homofermentative species (e.g.,L. plantarum), which convert sugars into lactic acid, and heterofermentative species (e.g.,L. fermentum), which produce lactic and acetic acids, ethanol, and CO2 [38]. Thus, contrary to what was observed in the present study, other authors have reported variations in the pH values of the culture medium for different species, indicating differences in acid production and secretion profiles; generally,L. plantarum strains tend to produce more acid thanL. fermentum strains [18]. Among vaginalLactobacillusisolates with inhibitory potential againstG. vaginalis, anL. fermentum strain reduced the pH of the culture supernatant to pH 4.16 after 48 h of incubation [39], whereas 2 strains ofL. plantarum isolated from organic fertilizer, in the same conditions for 24 h, reduced the pH to 3.83 and 3.88 [40]. Two strains ofL. plantarum isolated from a cocoa fermentation reduced the pH to 3.81 and 3.77, whereas anL. fermentumisolate reduced the pH to 4.78 [18].The testedL. fermentum FA4 andL. plantarum PA3 strains did not show any significant differences in the four parameters evaluated, indicating that they have similar probiotic potential.


## 3.2. Susceptibility of Lactobacilli to Antibiotics

In the antibiotics sensitivity profile testing, the twoLactobacillusstrains were sensitive or moderately sensitive to most of the antibiotics tested. They were only resistant to vancomycin, aminoglycosides, and quinolones (Table 3). Although lactobacilli are “generally recognized as safe”, safety tests, such as the determination of antibiotics sensitivity profiles, should always be performed to avoid the transfer of resistance genes, since these profiles vary among species [41]. Similar sensitivity profiles have been described in other studies, corroborating the findings of the present study. Santos et al. [42] found thatL. fermentum andL. plantarum isolates from cocoa fermentation were resistant to vancomycin and quinolone class antibiotics. Similarly, 12 lactobacilli strains isolated from cottage cheese, typical of northeastern China, were resistant to streptomycin, gentamicin, vancomycin, and ciprofloxacin [31]. Strains of human origin also have similar sensitivity profiles. Bouridani et al. [25] showed that all tested vaginalLactobacillusisolates were resistant to ofloxacin, gentamicin, and ciprofloxacin, and almost all strains were sensitive to trimethoprim-sulfamethoxazole, ampicillin, erythromycin, cefotaxime, chloramphenicol, tetracycline, and nitrofurantoin. Bacteria in the genusLactobacillus, like other Gram-positive bacteria, are intrinsically resistant to glycopeptides such as vancomycin. However, the gene responsible for this resistance is chromosomal, and it is not inducible or transferable to other bacteria [43, 44]. Thus, the strains under study could be used in therapeutic applications, as they would not pose a risk to the health of humans or other animals.Table 3
Antibiotics susceptibility profiles ofLactobacillus strains isolated from cocoa fermentation.
Antibiotics Susceptibilitya Group Name Antibiotic concentration (μg) L. fermentumFA4 L. plantarumPA3 Inhibitors of cell wall synthesis  Penicillin Amoxicillin 10 S S Ampicillin 10 S S Penicillin G 10 S MS Cephalosporins Cefalotin 30 S S Glycopeptides Vancomycin 30 R R  Inhibitors of protein synthesis  Aminoglycosides Amikacin 30 R R Gentamicin 10 R R Streptomycin 10 R R Tetracyclines Tetracycline 30 S S Single antibiotics Chloramphenicol 30 S S Macrolides Erythromycin 15 S S Lincosamides Clindamycin 2 S S  Inhibitors of nucleic acid synthesis  Quinolones Ciprofloxacin 5 R R Norfloxacin 10 R R  The broad mechanism of action  Single antibiotics Nitrofurantoin 300 S S


## 3.3. Interaction of HMVII Cells with Lipoproteins and the Effects of Treatment with Lactobacilli

Initially, we evaluated whether lactobacilli isolated from cocoa fermentation reduced the viability of cells of vaginal lineage. After 24 h of incubation withL. fermentum FA4 andL. plantarum PA3, the HMVII cells showed cell death rates of only 0.72% and 0.36%, respectively, and these rates were not significantly different from each other or from that of the control (1.99%; Figure 2).Figure 2
Death of HMVII cells after 24 h of incubation withLactobacillusstrains or membrane-associated lipoproteins (LAMP) fromU. parvum (UpLAMP),U. urealyticum (UuLAMP),M. hominis (MhLAMP), orM. genitalium (MgLAMP). Different letters indicate significant differences (p <0.01).This minimal reduction in HMVII cell viability after incubation with theseLactobacillus strains demonstrates that both can exist in the human vaginal environment without any toxicity. In addition, they reinforce the probiotic potential of these strains in protecting against pathogens that cause BV. Similarly, Abramov et al. [45] showed thatLactobacillus crispatus 2029 did not induce apoptosis in vaginal epithelium cells (VK2/E6E7) and has no cytotoxic effects on them. Strains ofL. fermentum andL. plantaruminhibited apoptosis in HeLa epithelial cells and showed antagonistic activity againstCandida albicans andG. vaginalis [46]. In another study, the viability of cells isolated from epidermoid carcinoma of the cervix (CaSki) and HeLa cells was maintained after incubation with different concentrations ofLactobacillus casei extract [47]. In contrast, cell death of HMVII cells increased significantly after incubation with the membrane-associated lipoproteins (LAMP) ofU. parvum,U. urealyticum,M. hominis, andM. genitalium; LAMP extracted fromU. parvumandM. hominisinduced 41% and 34% cell death, respectively, the highest values among the 4 species (Figure 2).Mollicutes causing genital infections need to adhere and subsequently invade the cells of the genitourinary tract to obtain nutrients from the host cells [1, 2, 47]. During adhesion, human cells first interact with the plasma membrane of these microorganisms, specifically with their lipoproteins. Recently, cytoadhesion and invasion present on the membrane ofM. hominiswere shown to interact with HeLa cells [48]. Hopfe et al. [49] showed that, after 4 h of infection of HeLa cells withM. hominis, genes related to the cell cycle, growth, and cell death are highly regulated, and lipoproteins are generally responsible for inducing inflammation, apoptosis, and cell death [5, 50].The present study is the first to use the HMVII vaginal cell line as a model for interaction with the lipoproteins ofMollicutes, mimicking the infection that occursin vivo. However, the levels of cell death induced by lipoproteins extracted from mycoplasmas and ureaplasmas may vary according to both species and the human cell line used in the experiment. Similarly, to the present study, 2 μg/mLMycoplasma penetrans lipoproteins induced apoptosis in RAW264.7 murine macrophages [51]. LAMP fromMycoplasma salivarium andMycoplasma fermentans caused both apoptosis and necrosis in myeloid (HL-60 and THP-1) and lymphoid (MOLT-4 and Raji cells) leukemia cells at concentrations of 20 and 7 μg/mL, respectively [52]. However, Bai et al. (2007) [4] showed thatMycoplasma hyopneumoniaeLAMP induced apoptosis in the 3D4/21 porcine alveolar macrophage (PAM) cell line at concentrations of 0.4 mg/mL or higher, which is higher than the concentration used in the present study.In this study, we also demonstrated that incubation of HMVII cells with lactobacilli isolated from cocoa fermentation significantly reduced cell death caused by the interaction withUreaplasma species LAMP from 41.03% in UpLAMP to 2.43% (UpLAMP +L. fermentum FA4) and 0.43% (UpLAMP +L. plantarum PA3) and from 25.24% (UuLAMP) to 13.97% (UuLAMP +L. fermentum FA4) (Figure 3). As described for the genusUreaplasma, the treatment of HMVII with lactobacilli also managed to significantly decrease the rate of cell death caused by the interaction with LAMP of the genusMycoplasma. The cell death reduced from 34.29% (MhLAMP) to 14.06% (MhLAMP +L. fermentum FA4) and 14.61% (MhLAMP +L. plantarum PA3) and 25.53 (MgLAMP) to 8.91% (MgLAMP +L. fermentum FA4) and 11% (MgLAMP +L. plantarum PA3) (Figure 4).Figure 3
Death of HMVII cells after 24 h of incubation with LAMP fromU. parvum (UpLAMP) orU. urealyticum (UuLAMP) with and withoutL. plantarum PA3 orL. fermentum FA4. ∗ p<0.01 compared to cells with LAMP alone.Figure 4
Death of HMVII cells after 24 h of incubation with LAMP fromM. hominis (MhLAMP) orM. genitalium(MgLAMP) with and withoutL. plantarum PA3 orL. fermentum FA4. ∗ p<0.01 compared to cells with LAMP alone.The findings of flow cytometry were confirmed by microscopy (Figures5–8). The images showed the vaginal cells after interaction with lipoproteins extracted fromU. parvum,U. urealyticum,M. hominis, andM. genitalium (resp.) and treatment with the two strains of lactobacilli. Cells incubated only with lipoproteins at a concentration of 4 μg / mL (Figures 5(b)–8(b)) presented a large number of cells with altered morphology, when compared to the control (Figures 5(a)–8(a)).Figure 5
Interaction of HMVII cells with 4 μ g/mL ofU. parvumLAMP with and withoutL. fermentum FA4 orL. plantarum PA3.(a) Control (HMVII cells alone). (b) HMVII with UpLAMP. (c) HMVII with UpLAMP andL. fermentum FA4. (d) HMVII with UpLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 6
Interaction of HMVII cells withU. urealyticumLAMP with and withoutL. fermentum FA4 orL. plantarum PA3.(a) Control (HMVII cells alone). (b) HMVII with UuLAMP. (c) HMVII with UuLAMP andL. fermentum FA4. (d) HMVII with UuLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 7
Interaction of HMVII cells with 4 μ g/mL ofM. hominisLAMP with and withoutL. fermentum FA4 orL. plantarum PA3. (a) Control (HMVII cells alone). (b) HMVII with MhLAMP. (c) HMVII with MhLAMP andL. fermentum FA4. (d) HMVII with MhLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy, ×2500).
(a) (b) (c) (d)Figure 8
Interaction of HMVII cells with 4 μ g/mLM. genitalium lipoproteins with and withoutL. fermentum FA4 orL. plantarum PA3. (a) Control (HMVII cell alone). (b) HMVII with MgLAMP. (c) HMVII with MgLAMP andL. fermentum FA4. (d) HMVII with MgLAMP andL. plantarum PA3. Green arrows indicate intact HMVII cells, red arrows indicate HMVII cells with altered morphology, and yellow arrows indicate lactobacilli adhered to whole cells (scanning electron microscopy ×2500).
(a) (b) (c) (d)In contrast, in treatments with L. fermentum FA4 (Figures5(c)–8(c)) and L. plantarum PA3 (Figures 5(d)–8(d)), bacteria adhered to HMVII cells were able to reduce cell death. This property was evidenced by normal morphological characteristics of the cells compared to the negative control.Several studies have shown that lactobacilli isolated from the vaginal environment have activity against potentially pathogenic bacteria. Strains ofL. crispatus andL. vaginalis inhibited the growth of several bacterial species causing vaginal and urinary tract infections, includingEnterococcus faecalis,E. coli,S. aureus,Enterococcus faecium,G. vaginalis, andProteus mirabilis[53]. In addition to promoting a protective vaginal environment, strains ofL. crispatusshowed antagonistic activity against several species of the genusCandida [45]. Strains ofLactobacillus rhamnosus andLactobacillus reuteri increased the viability of human epidermal keratinocytes from 8.8% after infection withS. aureus to 42.7% and 53.1%, respectively [54]. However, very few studies have examined the interaction between lactobacilli andMollicutes. Danielle et al. [55] demonstrated that bacteriocins produced byL. fermentum andL. rhamnosus showed antibacterial activity against vaginal isolates ofU. urealyticum andM. hominis. This is the first study to investigate the interaction betweenLactobacillusstrains isolated from cocoa fermentation and theMollicutesclass of bacteria. The antagonist effects of probiotic microorganisms against pathogenic bacteria include competitive adhesion to the mucosa and epithelium; strengthening of the epithelial barrier; secretion of antibiotics substances, such as bacteriocins and organic acids; and modulation of the immune system [36].One possible protective mechanism of the strains in this study againstMycoplasma andUreaplasma lipoproteins is competitive adhesion to host epithelial cells. The excellent adhesion capacity and hydrophobicity of the strains in this study suggest antagonistic effects on the adhesion of LAMP to the plasma membrane of the vaginal epithelium. In addition, this high hydrophobicity, coupled with the self-aggregation ability, of the tested lactobacilli may cause the LAMP to bind to the cell wall of these microorganisms rather than to the epithelial cell membrane [35]. Finally, the cell interaction experiment results indicate that theseL. fermentum andL. plantarum strains isolated from cocoa fermentation have potential for use as vaginal cell protectors against the LAMP of several important pathogens of the genitourinary tract, includingU. parvum,U. urealyticum,M. hominis, andM. genitalium. However, more studies are needed to clarify their protective mechanism(s) of action.


## 4. Conclusions

In the present study, lactobacilli isolated from cocoa fermentation inhibited cell death of a vaginal cell line (HMVII) induced byMollicutes lipoproteins (Mycoplasma andUreaplasma strains) that cause genital infections. The testedLactobacillusstrains have the fundamental and desirable probiotic properties required to maintain a healthy vaginal environment, including high hydrophobicity and autoaggregation, as well as adherence to epithelial cells and acid production. These characteristics are considered promising for the development of further prophylactic agents.



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