Lacticaseibacillus rhamnosus CA15 (DSM 33960) as a Candidate Probiotic Strain for Human Health

Lactobacilli with probiotic properties have emerged as promising tools for both the prevention and treatment of vaginal dysbiosis. The present study aimed to study the in vitro probiotic potential of the Lacticaseibacillus rhamnosus CA15 (DSM 33960) strain isolated from a healthy vaginal ecosystem. The strain was evaluated for both functional (antagonistic activity against pathogens; H2O2, organic acid, and lactic acid production; antioxidant and anti-inflammatory activities; ability to adhere to intestinal mucus and to both CaCo-2 and VK7/E6E7 cell lines; exopolysaccharide production; surface properties; and ability to survive during gastrointestinal transit) and safety (hemolytic, DNase, and gelatinase activities; mucin degradation ability; production of biogenic amines; and resistance to antimicrobials) characteristics. Data revealed that the tested strain was able to antagonize a broad spectrum of vaginal pathogens. In addition, the adhesion capacity to both vaginal and intestinal cell lines, as well as anti-inflammatory and antioxidant activities, was detected. The ability of the Lacticaseibacillus rhamnosus CA15 (DSM 33960) strain to survive under harsh environmental conditions occurring during the gastrointestinal passage suggests its possible oral delivery. Thus, in vitro data highlighted interesting probiotic properties of the CA15 (DSM 33960) strain, which could represent a valuable candidate for in vivo vaginal infections treatment.


Introduction
Vaginal microbiota, although characterized by few microbial species, is a dynamic ecosystem subjected to changes during a womans lifetime, from birth to pre-and postmenopause [1][2][3]. In addition, intrinsic (e.g., race, immune system imbalance, genetic susceptibility, etc.) and extrinsic factors (such as a diet rich in vitamins and folic acid, use of oral contraceptives, sexual behaviors, hygiene habits, antibiotics, or immunosuppressor therapies) can affect the balance of the vaginal bacterial biota. It is well known that the reduction in lactobacilli and the increase in facultative or obligate anaerobic microorganisms, along with different Candida species, are associated with vaginal microbiota dysbiosis [4,5]. Although most lactobacilli have a GRAS (generally recognized as safe) status and some of them are included in the Qualified Presumption of Safety (QPS) list [6,7], both safety and probiotic features are strain-specific and cannot be generalized to the species. Several studies demonstrated the ability of probiotic lactobacilli to protect vaginal health by ence is often associated with different urogenital tract dysbioses. Each pathogen was cultured using the media and following the conditions suggested by ATCC or DSM. The antagonistic activity of the L. rhamnosus CA15 (DSM 33960) strain against pathogens was evaluated by the agar spot test as previously described by Pino et al. [15]. The antagonistic activity was ranked as absent (−, no inhibition zone); low (+, inhibition zone < 10 mm); intermediate (++, inhibition zone between 11 and 20 mm); and high (+++, inhibition zone > 20 mm). In order to identify the nature of the antimicrobial compounds, 10 µL of the CFS previously treated for pH, heat, catalase, and proteolytic enzymatic, as reported by Scillato and coworkers [31], was used for the antagonistic assay against the aforementioned pathogen stains. All experiments were carried out in triplicate.

Hydrogen Peroxide, Organic Acid, and Lactic Acid Production
The ability to produce hydrogen peroxide (H 2 O 2 ) was evaluated following the protocol reported by Pino et al. [15]. Based on the time required for the appearance of a blue coloration, the tested strain was scored as a nonproducer (absence of blue coloration), low (score 1, time > 20min), medium (score 2, time 10-20min), or high (score 3, time < 10min) H 2 O 2 producer.
Organic acids were quantified by HPLC following the protocol suggested by Chenoll and coworkers [32]. The assay was performed in triplicate, and the results are expressed as mean and standard deviation.

In Vitro Antioxidant Activity
The antioxidant activity, explicated by the L. rhamnosus CA15 (DSM 33960) strain, was tested following the 2,2 ′ -azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS), 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), and Superoxide Dismutase (SOD)-like assays. The scavenging activity toward radical cation 2,2 ′ -azino-di-[3-ethylbenzthiazoline sulfonate] (AB-TS) protocol was performed as reported by Pino et al. [28]. The free radical-scavenging capacity of different concentrations of the tested strain (3 mg/mL; 2.25 mg/mL; 1.5 mg/mL; 0.75 mg/mL) was measured by 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH)-free radical method as previously reported [33]. The SOD-like activity was measured using the pyrogallol autoxidation system as described by Marklund et al. [34]. Briefly, 50 µL of a pyrogallol solution (12 mM, in 1 mM HCl) was rapidly added to a 3 mL mixed solution system (0.05 M Tris-HCl buffer, 1 mM EDTA, and pH 8.2). After samples were thoroughly mixed, the absorbance at 325 nm was measured every 30 s for 5 min at 25 • C. According to the procedure described above, the assays were carried out in the presence of different concentrations of CA15 (DSM 33960) (0.75-3 mg/mL), expressed as mg of protein/mL of filtrate. Proteins were determined by the Nanodrop Protein quantification using a Nan-oDrop UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA). The autoxidation rate of pyrogallol (control) was determined from the slope of the absorbance curve. The change in absorbance in the presence of the antioxidant was compared with that of the control, and SOD-like activity was expressed according to the equation: where A and B are autoxidation rates of pyrogallol in the absence and presence of an antioxidant, respectively. Results are expressed as percentage of inhibition rate ± SEM (Standard Error of the Mean).

Caco-2 Cell Cultures
Caco-2 cells were used as a stable in vitro model for the intestinal epithelium. Caco-2 cells (heterogeneous human epithelial colorectal adenocarcinoma cells) were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA) and grown in DMEM supplemented with 10% fetal bovine serum (FBS), 0.1% streptomycin-penicillin, 1% L-glutamine, and 1% nonessential amino acids. Caco-2 cells were seeded at a concentration of 1.5 × 10 4 cells per well of a 96-well, flat-bottomed 200-µL microplate. Dose- response experiments were performed in cells incubated at 37 • C in a 5% CO 2 -humidified atmosphere and cultured for 24 h in the presence and absence of selected concentrations of CA15 (DSM 33960) (1.5 mg/mL). Cell viability was measured by MTT assay as previously reported [35]. To induce in vitro intestinal inflammation, Caco-2 cells (1.5 × 10 4 ) were seeded in six-well plates and cultured 14-21 days to reach the confluence. An experimental inflammatory condition in Caco-2 cell monolayers was induced by the exposure for 24 h to lipopolysaccharide (LPS) (100 µg/mL). A 24 h pretreatment with a selected concentration of CA15 (DSM 33960) (1.5 mg/mL) was applied before inflammatory stimuli.

Mitochondrial Membrane Potential in Caco-2 Cell Line
Following a 24 h cotreatment of LPS (100 µg), cells were incubated with a 3 µM tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) staining solution (T3168, Invitrogen, Waltham, MA, USA) at 37 • C for 20 min and then washed with PBS in order to visualize the fluorescence with a fluorescence microscope (EVOS Fl AMG). The JC-1 probe aggregates formed a polymer in the mitochondrial matrix of healthy cells, producing a strong red fluorescence (Ex = 585 nm, Em = 590 nm). Otherwise, dysfunctional mitochondria presented JC-1 monomers, resulting in the emission of a green fluorescent signal (Ex = 514 nm, Em = 529 nm). The results are expressed as the ratio of red/green fluorescence.

Anti-Inflammatory Activity
The ability of the tested strain to exert anti-inflammatory activity was evaluated using the human promonocytic U937 cell line as reported by Pino et al. [28]. Gene expression of COX-1, COX-2, IL-8, and IL-10 was evaluated by quantitative real-time PCR (qRT-PCR) using the primer pairs and conditions previously described [28]. The analyses were performed in triplicate, and the results are reported as mean and standard deviation. In addition, conditioned media obtained from Caco-2 cells treated with LPS (100 µg/mL) and the combination with the selected concentration of CA15 (DSM 33960) (1.5 mg/mL) were used for quantitatively determining the IL-6 and IL-2 levels. eBioscience Instant ELISA kits (BMS213INST and BMS221INST, eBioscience, Vienna, Austria) were used following manufacturers instructions. The protein concentration was determined by plotting the OD reading against the standard curve. Results are expressed as pg/mL. Individual measurements were performed in triplicate, and data are reported as mean ± SEM.

Adhesion to Intestinal Mucus
L. rhamnosus CA15 (DSM 33960) was grown overnight at 37 • C on MRS agar (Merck, Darmstadt, Germany). The strain was metabolically labeled by suspending one single colony in MRS broth supplemented with 10 µL/mL of [6-3 H]-Thymidine. After overnight incubation at 37 • C, cells were harvested by centrifugation (2500× g for 10 min) and washed twice with phosphate-buffered saline (PBS; pH 7.2). The OD 600 of the bacterial suspension was adjusted with PBS to 0.250 ± 0.02, which corresponded approximately to 10  total of 75 ng of mucus was immobilized on polystyrene microtiter plate wells (Maxisorp, Nunc, Roskilde, Denmark) by overnight incubation at 4 • C. The mucus-coated microtiter plate wells were washed three times with 200 µL of PBS, and then 100 µL of cells was added to the wells and incubated at 37 • C for 1 h. Unattached bacteria were removed by washing the wells three times with 200 µL of PBS, whereas adhered cells were released and lyzed with 1% (w/v) sodium dodecyl sulfate (SDS) in 1 M NaOH (100 µL per well) by incubation at 37 • C overnight. The contents of the wells were transferred to microfuge tubes containing scintillation liquid (OptiPhase HiSafe 3, Wallac, Turku, Finland), and radioactivity was measured by liquid scintillation counter. Adhesion was expressed as the percentage of radioactivity recovered after adhesion relative to the radioactivity of the bacterial suspension added to the immobilized mucus. Adhesion was determined in three independent experiments, and each assay was performed in six replicates to calculate the intra-assay variation. The strains were chosen based on their ability to adhere to mucus with percentages higher than 3% (data not shown). Except for L. rhamnosus DSM 33960, the strains were grown overnight using BHI (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 2% of Yeast Extract (Sigma-Aldrich, St. Louis, MO, USA). Adhesion inhibition and displacement were performed using mucus immobilized into polystyrene microtiter plate wells as above.
Adhesion inhibition was tested by adding unlabeled L. rhamnosus CA15 (DSM 33960) cells (100 µL, 10 8 CFU/mL) to the mucus wells. After incubation at 37 • C for 1 h, unbound cells were removed by washing the wells three times with 200 µL of PBS after which radiolabeled pathogens (100 µL, 10 8 CFU/mL) were added to the wells. After incubation at 37 • C for 1 h, unbound radiolabeled pathogens were removed by washing the wells three times with PBS, whereas the bound pathogens were released and lyzed with 1% (w/v) SDS in NaOH (0.1 mol/L) (100 µL per well) by incubation at 37 • C overnight. Radioactivity was assessed by liquid scintillation. Results are expressed as the percentage of radioactivity recovered after adhesion, relative to the radioactivity of the bacterial suspension added to the wells. The percentage of adhesion inhibition was calculated as the difference between the adhesion of the pathogen in the absence and presence of L. rhamnosus CA15 (DSM 33960) cells.
The ability of L. rhamnosus CA15 (DSM 33960) to displace adhered pathogens was evaluated by adding radiolabeled pathogens (100 µL, 10 8 CFU/mL) to the mucus wells. After incubation at 37 • C for 1 h, unbound pathogens were removed by washing three times with PBS, and then unlabeled L. rhamnosus CA15 (DSM 33960) (100 µL, 10 8 CFU/mL) was added to the wells. After incubation at 37 • C for 1 h, the wells were washed three times with PBS. After release and lysis of bound pathogens, the radioactivity was measured as described above. Displacement was calculated as the difference between the adhesion of pathogens before and after the addition of the L. rhamnosus CA15 (DSM 33960) cells. All assays were performed in three independent experiments, and each assay was performed in six replicates to calculate the intra-assay variation.

Biofilm Formation Assay
The ability of the L. rhamnosus CA15 (DSM 33960) strain to produce biofilms was evaluated following the method proposed by dos Santos et al. [37] with slight modifications. An overnight microbial culture (10 9 CFU/mL) was used as inoculum (5% v/v) in 200 µL of MRS broth medium with and without the addition of TWEEN 80 (1% v/v) and incubated at 37 • C, for 24 h and 72 h. For the assay, 96-well polystyrene microplates were used. After incubation, the wells were washed three times with PBS (pH 7.0), and 200 µL of 0.1% (w/v) crystal violet, prepared in an isopropanol-methanol-PBS (1:1:18 v/v) solution, was added and allowed to stain for 30 min. Excess dye was washed with sterile water and left to dry for 2-3 h. For the reading of the adhered biofilm inside the wells, 200 µL of 30% glacial acetic acid was added. The optical density (OD) was measured at 595 nm using an iMark™ Microplate Absorbance Reader (Biorad). A sterile medium was used as negative control (OD c ). The strain was considered as nonbiofilm producers (OD ≤ OD c ); weak biofilm producers (OD c < OD ≤ 2 × OD c ); moderate biofilm producers (2 × OD c < OD ≤ 4 × OD c ); strong biofilm producers (4 × OD c < OD ≤ 8 × OD c ), and very strong biofilm producers (8 × OD c < OD).

Exopolysaccharide Production, Hydrophobicity, Autoaggregation, and Coaggregation Abilities
The phenol/sulfuric acid method was used to test the ability to produce exopolysaccharide (EPS). The amount (mg/L) of produced EPS was evaluated using glucose (50-500 mg/L) as standard [19,39].

Tolerance to Lysozyme, Low pH, and Bile Salts
Lysozyme tolerance was tested following the method previously described [19]. Tolerance to pH 2.0 and 3.0, as well as bile salt tolerance, was assessed as previously described [19]. For each assay, three independent experiments were performed using 9 log cfu/mL bacterial suspension of the L. rhamnosus CA15 (DSM 33960) strain previously cultured twice in MRS broth. Results are expressed as survival rate percentage (SR%) based on the initial and the final numbers of viable cells enumerated on MRS agar after incubation at 37 • C for 48 h. The analyses were performed in triplicate.

Ability to Survive during Simulated Gastrointestinal Transit
The L. rhamnosus CA15 (DSM 33960) strain was tested for the ability to survive during the in vitro gastrointestinal transit. The survivability was evaluated on simulated gastric juice (SGJ, pH 2.5) and simulated intestinal fluid (SIF, pH 7.5) using 9 log cfu/mL bacterial suspension of the tested strain previously cultured twice in MRS broth, according to Pino et al. [19]. The assay was performed in triplicate, and results are expressed as survival rate percentage (SR%), based on the initial and the final numbers of viable cells. The analyses were performed in triplicate.

Statistical Analysis
All data are expressed as a mean and standard deviation or as frequencies (percentages) of triplicate independent experiments. Data related to RT-PCR of COX-1, COX-2, IL-8, and IL-10 gene-level expression were subjected to one-way ANOVA followed by Tukeys multiple comparison test. Differences were considered statistically significant at p ≤ 0.05. All statistical analyses were performed using SPSS Version 25.0 (Armonk, NY, USA, IBM Corp.).

Genetic Analysis of the L. rhamnosus CA15 (DSM 33960) Strain
The alignment of the nucleotide sequence of the 16S rRNA gene of the L. rhamnosus CA15 (DSM 33960) strain showed 100% identity (i.e., 613 nucleotides out of 613 were identical) with the 16S rRNA gene of the L. rhamnosus-type strain ATCC 7469 (sequence accession no. JQ580982.1). The CA15 (DSM 33960) strain was therefore identified as belonging to the species Lacticaseibacillus rhamnosus (old name Lactobacillus rhamnosus). The genetic fingerprint via PFGE is shown in Figure 1.
All data are expressed as a mean and standard deviation or as frequencies (percentages) of triplicate independent experiments. Data related to RT-PCR of COX-1, COX-2, IL-8, and IL-10 gene-level expression were subjected to one-way ANOVA followed by Tukey's multiple comparison test. Differences were considered statistically significant at p ≤ 0.05. All statistical analyses were performed using SPSS Version 25.0 (Armonk, NY: IBM Corp.).

Genetic Analysis of the L. rhamnosus CA15 (DSM 33960) Strain
The alignment of the nucleotide sequence of the 16S rRNA gene of the L. rhamnosus CA15 (DSM 33960) strain showed 100% identity (i.e., 613 nucleotides out of 613 were identical) with the 16S rRNA gene of the L. rhamnosus-type strain ATCC 7469 (sequence accession no. JQ580982.1). The CA15 (DSM 33960) strain was therefore identified as belonging to the species Lacticaseibacillus rhamnosus (old name Lactobacillus rhamnosus). The genetic fingerprint via PFGE is shown in Figure 1.

Safety Evaluation
The L. rhamnosus CA15 (DSM 33960) strain was considered safe because it showed neither hemolysis halos after growth on blood agar plates nor zones of gelatin hydrolysis on gelatin agar plates overlaid with saturated ammonium sulfate solution. In addition, the CA15 (DSM 33960) strain did not show DNAse activity and mucin degradation ability.
The antibiotic susceptibility profile (MIC values) of the tested CA15 (DSM 33960) strain is shown in Table 1. Susceptibility to all the antibiotics suggested by the EFSA was detected, whereas high MIC values for Metronidazole (> 600 µg/mL), Clotrimazole (> 256

Safety Evaluation
The L. rhamnosus CA15 (DSM 33960) strain was considered safe because it showed neither hemolysis halos after growth on blood agar plates nor zones of gelatin hydrolysis on gelatin agar plates overlaid with saturated ammonium sulfate solution. In addition, the CA15 (DSM 33960) strain did not show DNAse activity and mucin degradation ability.
The antibiotic susceptibility profile (MIC values) of the tested CA15 (DSM 33960) strain is shown in Table 1. Susceptibility to all the antibiotics suggested by the EFSA was detected, whereas high MIC values for Metronidazole (>600 µg/mL), Clotrimazole (>256 µg/mL), and Boric acid (>10.000 µg/mL) were observed. In addition, as reported in Table 1, no relevant amounts of the assayed biogenic amines were detected by quantification performed according to ISO19343:2017.

Antagonistic Activity against Pathogens
The results of the antimicrobial activity against pathogens displayed by the L. rhamnosus CA15 (DSM 33960) strain are reported in Table 2. Overall, a broad spectrum of antagonistic activity was detected except for Enterobacter cloaceae DSM 30054, Enterococcus faecalis DSM 2570, Escherichia coli ATCC 25922, Escherichia coli DSM 105393, and Proteus mirabilis DSM 30116. The highest antimicrobial activity was displayed against Candida albicans ATCC10231, Candida krusei ATCC 14243, Gardnerella vaginalis ATCC 14018, and Streptococcus agalactiae DSM 2134. Based on the CFS treatments, the antagonistic activity was, in all cases, attributed to organic acid production.

Hydrogen Peroxide, Organic Acid, and Lactic Acid Production
The L. rhamnosus CA15 (DSM 33960) strain, with a score of three, was classified as a high hydrogen peroxide producer because the appearance of an intense blue coloration was observed less than 10 min after plate air exposure (Table 3). Regarding organic acid production, a high concentration of lactic acid (799.29 mmol/L) was quantified. In addition, propionic (98.54 mmol/L), succinic (98.48 mmol/L), and butyric (39.61 mmol/L) and acetic (18.31 mmol/L) acids were detected (Table 3).

In Vitro Antioxidant and Anti-Inflammatory Activities and Adhesion Ability to Caco-2 and Human Vaginal Epithelial Cells
The antioxidant activity was evaluated in cell-free models by ABTS, DPPH Radical Scavenging, and SOD-like assays. Figure 2 shows the results of the ABTS test suggesting the ability of the L. rhamnosus CA15 (DSM 33960) strain to inhibit the peroxidation of the linoleic acid. Interestingly, no statistically significant difference (p > 0.05) was found in antioxidant activity between the tested strain and the well-known antioxidant compound α-tocopherol. DPPH Radical Scavenging and SOD-like assays were performed using different concentrations (3, 2.25, 1.5, and 0.75 mg/mL) of the filtered broth, obtained after the growth of the L. rhamnosus CA15 (DSM 33960) strain, and the results are displayed in Figure 3A,B). Overall, similar DPPH radical scavenging activity, with a percentage of about 80%, was detected using 3, 2.25, and 1.5 mg/mL of the L. rhamnosus CA15 (DSM 33960) strain culture filtrate ( Figure 3A). On the contrary, the tested strain was able to scavenge superoxide anion in a dose-dependent manner, reaching about 80% at a concentration of 3 mg/mL ( Figure 3B).  The ability of LPS to induce oxidative stress in the Caco-2 cell line was evaluated by measuring the levels of JC-1, a cationic dye that accumulates in the mitochondria, as dimer JC-1 or monomer JC-1. As shown in Figure 3C, a low signal of JC-1 dimer (red fluorescence) and a high presence of JC-1 monomer (green fluorescence) were detected in LPS-treated Caco-2 cells, indicating the induction of oxidative damage and mitochondrial dysfunction. In addition, as shown in Figure 3D, the LPS treatment was able to induce oxidative stress in the Cac-2 cell line with slight cytotoxicity. No significant differences were observed in the CA15 (DSM 33960) cells treated and in the CA15 (DMS 33960)-LPStreated cells, compared to the control group.  The ability of LPS to induce oxidative stress in the Caco-2 cell line was evaluated by measuring the levels of JC-1, a cationic dye that accumulates in the mitochondria, as dimer JC-1 or monomer JC-1. As shown in Figure 3C, a low signal of JC-1 dimer (red fluorescence) and a high presence of JC-1 monomer (green fluorescence) were detected in LPS-treated Caco-2 cells, indicating the induction of oxidative damage and mitochondrial dysfunction. In addition, as shown in Figure 3D  A significant reduction in the IL-6 levels was detected in LPS-treated Caco-2 cell lines after treatment with the L. rhamnosus CA15 (DSM 33960) strain culture filtrate, suggesting the ability of the tested strains to reduce both oxidative stress and inflammation (Figure 3E). On the contrary, the treatment of LPS-treated Caco-2 cell lines with the L. rhamnosus CA15 (DSM 33960) strain culture filtrate was able to induce an increase in the IL-2 levels ( Figure 3F).
As reported in Table 4, the L. rhamnosus CA15 (DSM 33960) strain showed anti-inflammatory activity in differentiated human macrophages treated with LPS. In detail, compared to the untreated cells, the treatment with the L. rhamnosus CA15 (DSM 33960) strain culture filtrate determined the downregulation of both COX-2 and IL-8 genes, whereas the IL-10 gene was upregulated. In addition, the tested strain exhibited binding ability to both Caco-2 and VK2/E6E7 cell lines, with rates of 36.15% ± 0.32 and 34.71% ± 0.09, respectively (Table 4). Table 4. Antioxidant, anti-inflammatory, and adhesion to Caco-2 and VK2/E6E7 cell lines exhibited by the L. rhamnosus CA15 (DSM 33960) strain.

Anti-Inflammatory Activity on U937 Cells
Adhesion % Antioxidant activity (Abs at 500 nm) was measured under a linoleic acid oxidation system for 8 days. α-Tocopherol (1 mg/mL) was used as a positive control. Data are reported as means of three independent experiments. Table 5 shows the results of the adhesion ability of the L. rhamnosus CA15 (DSM 33960) strain to intestinal mucus as well as the results of the ability to displace and inhibit the adhesion of selected potentially pathogen strains. In detail, the L. rhamnosus CA15 (DSM 33960) strain showed the ability to adhere to intestinal mucus with a percentage equal to 11.99% (Table 5). In addition, the L. rhamnosus CA15 (DSM 33960) strain showed the ability to inhibit the adhesion as well as to displace all the tested pathogens with percentages higher than 50% (Table 5). The results are expressed in percentage as means and standard deviation of the three independent experiments.

Biofilm Formation
The results of the biofilm formation are reported in Table 6. In detail, the tested strain exhibited strong biofilm production in MRS after both 24 and 72 h of incubation, whereas when using MRS with Tween 80, a very strong biofilm production was recorded after 72 h of incubation (Table 6).

Exopolysaccharide Production, Hydrophobicity, Autoaggregation, and Coaggregation Abilities
The results of the exopolysaccharide (EPS) production and surface properties shown by the L. rhamnosus CA15 (DSM 33960) strain are reported in Table 8. The tested strain was able to produce 292 mg/L of EPS. Both cell surface hydrophobicity (86.13%) and autoaggregation properties (71.80%) were detected. In addition, the CA15 (DSM 33960) strain was able to coaggregate with all the tested pathogens. The highest coaggregation percentage was observed in the presence of C. albicans ATCC 10231 (86.12%), whereas the lowest value was observed in the case of coaggregation with E. coli 555 (64.18%) ( Table 8).

Survival under In Vitro Gastrointestinal Conditions
The tolerance to lysozyme, acidic conditions, and bile salts as well as the ability to survive during simulated gastrointestinal (GI) transit is reported in Table 9. Overall, the L. rhamnosus CA15 (DSM 33960) strain showed the ability to survive under the tested stressful conditions with a survival rate (SR%) higher than 80%. In detail, the L. rhamnosus CA15 (DSM 33960) strain was able to survive under acidic conditions (pH 2.0 and 3.0) after both 2 and 4 h of incubation. Regarding the tolerance to lysozyme, the CA15 (DSM 33960) strain was classified as lysozyme-resistant [41] because it showed a survival rate of 95% and 93% after 30 and 120 min of incubation, respectively. The tolerance to both 0.5% and 1% of bovine bile salts, with an SR% higher than 80%, was recorded after both 2 h and 4 h of incubation (Table 9). A survival rate higher than 90% was observed after treatment with simulated gastric juice (SGJ) and simulated intestinal fluid (SIF). In fact, starting from an initial number of viable cells of 9.13 log cfu/mL, the L. rhamnosus CA15 (DSM 33960) strain exhibited several viable cells of 9.10 log cfu/mL after SGJ treatment (SR% of 99.7%) and 8.74 log cfu/mL after SIF treatment (SR% of 95.7%) ( Table 9).

Discussion
It is well known that, during reproductive age, a balanced vaginal microbiota is dominated by lactobacilli, while their reduction, and the increase in pathogens, is often associated with a dysbiotic state [42]. Although antimicrobial drugs have been traditionally used to treat dysbiosis, several side effects, such as short-term relapse and the promotion of antibiotic resistance, are frequently reported. For these reasons, alternative therapeutic strategies, able to modulate the microbiota and correct the imbalance, from an ecological approach, have been investigated. In this context, the probiotic supplementation, alone or complementary to antibiotic therapy, has gained increased attention to restore vaginal health avoiding the recurrence of bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) [43,44]. Although several probiotic strains have been characterized, only few are host-specific. It has been suggested that probiotic vaginal isolates, when administered, have increased colonization and adaptation abilities to the vaginal niche. The workflow of the present study was designed to evaluate the potential probiotic properties of the vaginal isolate L. rhamnosus CA15 (DSM 33960) strain as a potential candidate for womens health.
Noteworthy, probiotics must satisfy safety requirements. In this context, the inability to produce hemolysins and gelatinases, as well as to exert mucin degradation and DNase activities, suggest that the L. rhamnosus CA15 (DSM 33960) strain can be safely used. In addition, regarding the horizontal transfer of genes encoding antibiotic resistance [45,46], no transferable antibiotic resistance should be carried by promising probiotic strains. In the present study, the L. rhamnosus CA15 (DSM 33960) strain did not show resistance to the antibiotics suggested by the EFSA, whereas resistance to metronidazole, clotrimazole, and boric acid was detected. The high resistance to antibiotics routinely used to treat BV and The ability of probiotics to counteract pathogen invasion and colonization is a prerequisite to modulate the vaginal microbiota in a homeostatic way. Antimicrobial substances, such as hydrogen peroxide (H 2 O 2 ) and organic acids, produced by probiotic bacteria, play a key role in the maintenance of a healthy vaginal ecosystem. Although hydrogen peroxide is able to exert a microbicide effect, there is no consensus about its activity against pathogens due to both a low physiological concentration in the vagina and the neutralizing effects of semen and vaginal fluid [47]. It is well established that lactic acid, by acidifying the vagina, acts as a virucide and microbicide along with an immunomodulatory agent [48]. In the present study, we observed that the tested strain was able to produce both H 2 O 2 and lactic acid. In particular, the amount of lactic acid produced could be related to the broad antagonistic effect displayed by the CA15 (DSM 33960) strain against the tested pathogens [48]. The absence of antimicrobial activity after CFS neutralization suggests that the acid environment had a substantial action against the growth of the tested pathogens. However, other components, not yet identified, may occur in CFS. Further study based on the metabolomic approach will be carried out to characterize in depth the components of CFS and to evaluate their synergistic action to inhibit pathogens [49,50].
Along with the production of metabolites with antimicrobial properties, probiotic strains should be able to colonize and survive in harsh environments. The ability to adhere to the intestinal mucosa is considered a prerequisite for probiotics, allowing the colonization, albeit transient, of the human intestinal tract [9,14]. In vitro experiments showed the ability of the CA15 (DSM 33960) strain to adhere to intestinal mucus as well as to both Caco-2 and VK2/E6E7 cell lines with adhesion percentages higher than those previously obtained by L. rhamnosus GG [15]. In addition, the tested strain showed hydrophobicity and the ability to autoaggregate and coaggregate with pathogens. A higher hydrophobicity and autoaggregation ability was displayed by the CA15 strain compared to L. rhamnosus GG [40]. It is well known that the ability to adhere is related to the predisposition to self-aggregate, which, in turn, can exert a protective role by inhibiting the growth and adhesion of pathogens [14]. Moreover, the exclusion/competition behavior of probiotics is also related to coaggregation with pathogens [37]. According to that, the L. rhamnosus CA15 (DSM 33960) strain showed a significant capability to coaggregate with all the tested pathogens as well as to displace and inhibit their adhesion.
Recently, several health benefits (e.g., immunomodulation, antioxidant, antiviral, and antiyeast properties) have been associated with exopolysaccharide (EPS) produced by lactobacilli [51]. According to that, the ability of the CA15 (DSM 33960) strain to produce EPS could be useful to maintain vaginal health. Other interesting properties of the L. rhamnosus CA15 (DSM 33960) strain are the antioxidant and anti-inflammatory profiles. The ability to counteract the lipid peroxidation, and in turn oxidative stress, is considered central for the prevention of tissue damage and the development of human pathologies. In particular, the superoxide radical scavenging activity is an important enzymatic antioxidant defense mechanism catalyzed by the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into oxygen and hydrogen peroxide to protect the body from oxygen toxicity. In this study, we measured the scavenging activity of the superoxide radical generated by the self-oxidation of pyrogallol. In addition, the anti-inflammatory profile suggests a potential role of the CA15 (DSM 33960) strain to decrease the levels of proinflammatory cytokines and maintain the inflammatory homeostasis by increasing the levels of the anti-inflammatory molecules. In particular, the tested strain was able to induce a decrease in the IL-6 and, in turn, determine an increase in the IL-2 in LPS-treated Caco-2 cells. It is well known that IL-6 is an important proinflammatory cytokine involved in the development of inflammatory bowel diseases (IBD) [52,53], whereas considerable evidence suggest the important role played by IL-2 in maintaining a healthy immune response in the gut. In fact, a high rate of colitis, with striking clinical and morphological similarities to ulcerative colitis, was reported in a IL-2-deficient mouse