Lactobacillus Rhamnosus AD3 As a Promising Alternative for Probiotic Products

biomolecules

Article Lactobacillus rhamnosus AD3 as a Promising Alternative for Probiotic Products

Aldo Stivala , Giuseppe Carota, Virginia Fuochi *,† and Pio Maria Furneri †

Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, 95123 Catania, Italy; [email protected] (A.S.); [email protected] (G.C.); [email protected] (P.M.F.) * Correspondence: [email protected] † These authors work equally to this study.

Abstract: Lactobacillus strains dominate the vaginal habitat and they are associated with a lower risk of genital infections. In addition, they contribute to the conservation of the vaginal microbiota by producing postbiotic agents. Previous studies have shown that their predominance involves antimicrobial activity against urogenital pathologies. In this context, probiotics may improve treatment outcomes. The aim of this study was to evaluate the probiotic properties of lactobacilli strains of vaginal origin using a multidisciplinary approach. For this purpose, safety criteria, ability to resist at low pH and bile salts, antimicrobial activity, ability to produce bioﬁlm, capacity to produce hydrogen peroxide and more importantly, auto-aggregation, co-aggregation (with Candida spp.) and adhesion to human cells were evaluated. The strains belonged to the species of L. crispatus, L. gasseri, L. rhamnosus and L. delbruckii. Among these, a strain of L. rhamnosus named AD3 showed the best probiotic properties. As probiotics are already in use in many clinical practice and there are no major safety concerns, L. rhamnosus AD3 showed promise in becoming a prevention and complementary treatment option for urogenital diseases. Indeed, these results suggest that strain L. rhamnosus AD3 is non-pathogenic and likely to be safe for human consumption. This study revealed the great amensalistic properties of a new L. rhamnosus strain which can aim to be used as probiotic in pharmaceutical applications.

Citation: Stivala, A.; Carota, G.; Keywords: L. rhamnosus; antimicrobial activity; probiotic properties; urogenital infections; S. agalactiae; Fuochi, V.; Furneri, P.M. Candida spp. Lactobacillus rhamnosus AD3 as a Promising Alternative for Probiotic Products. Biomolecules 2021, 11, 94. https://doi.org/10.3390/biom11010094 1. Introduction

Received: 23 December 2020 Urogenital infections are common concerns in young women [1,2]. Among all sexu- Accepted: 11 January 2021 ally transmitted diseases, bacterial vaginosis and vulvovaginal candidiasis are the most Published: 13 January 2021 frequent [3,4]. These infections make-up the most common reasons for a woman to need to visit to gynaecologist, with the outcome of discomfort and also enormous costs for Publisher’s Note: MDPI stays neu- health care treatment [5]. Moreover, these kinds of diseases most often include treatment tral with regard to jurisdictional clai- with antibiotics and/or antimycotics, which have led to a raised concern regarding drug ms in published maps and institutio- resistance among pathogenic species [6,7]. nal afﬁliations. The human vaginal ecosystem is dominated by Lactobacillus species and an altered vaginal microbiota can cause a greater chance of symptomatic conditions. Furthermore, there is a clear relationship between vaginal microbiota and urogenital infections [8], in fact most of the microorganisms in the vagina also derive from the gastrointestinal tract, and Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. this does not depend only on sexual activity but also on personal hygiene and nutrition [9]. This article is an open access article The protective role of lactobacilli is increasingly accepted and several clinical studies are distributed under the terms and con- underway to evaluate the use of lactobacilli as probiotics. Further studies have shown that ditions of the Creative Commons At- the composition of the vaginal microbiota can undergo changes throughout life. The vaginal tribution (CC BY) license (https:// environment is dynamic and it changes in relation to events such as sexual activity, the creativecommons.org/licenses/by/ menstrual cycle or menopause [10]. During the fertile age, there is a vaginal epithelium rich 4.0/). in glycogen and mucins that act as nutrients and that allow colonization and domination

Biomolecules 2021, 11, 94. https://doi.org/10.3390/biom11010094 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 94 2 of 14

of the lactobacilli. Furthermore, the fertile age is characterized by monthly ovulation and therefore high estrogenic levels which, also thanks to the action of the proton pumps, bring the vaginal lumen to be particularly acidic, which leads to better conditions for the growth of the lactobacilli [11]. In postmenopausal women, especially in those who show signs of vaginal dryness and atrophy, a reduction in the vaginal Lactobacilli component can be noted [12]. Sexual activity can also influence the vaginal microbial composition, causing a reduction in the lactobacilli component with relative loss of their protective action, and an increase in the predisposition to dysbiosis [13]. In this context, the use of probiotics consisting of Lactobacillus spp. is increasing in the gynaecological field to restore the physiologic vaginal microbiota and treat bacterial vaginosis and vulvovaginal candidiasis even if there is conflicting evidence between them [14–16]. Although the local administration should be the best and fastest solution to the problem of vaginal dysbiosis, main scientific concept associated with the use of probiotics is the oral administration of live microorganisms in order to restore the environment by competition [17]. Of the many probiotic microorganisms available commercially, L. rhamnosus GG and L. rhamnosus GR-1 are the most studied and they have been used successfully to treat and prevent diarrhoea and urogenital infections in women, respectively [18,19]. Nonetheless, the strains of Lactobacilli used in such treatments must respect certain rules so that they can be considered potential probiotics: they should be able to adhere to the cells, to prevent or decrease adherence of pathogenic microorganisms, to secrete acids, hydrogen peroxide, and bacteriocins so as to prevent growth of pathogens. Moreover, they should be safe, and should not be invasive, carcinogenic, and pathogenic and finally, they should be able to form clusters so as to produce normal-balanced microbiota [20]. In view of the fact that the market can offer different products with various probiotic components, the main goal of the study had been the characterization of Lactobacillus strains in order to clarify their ecological role in the female genital tract and to focus on selected Lactobacillus strains with probiotic properties to finding a valid prevention and complementary treatment option for urogenital diseases, and to improve the normal vaginal microbiota in healthy women. Moreover, the project included in vitro studies on L. rhamnosus GG for a comparison on the probiotic properties of the strains investigated.

2. Materials and Methods 2.1. Bacterial Strains and Growth Conditions In this study, twelve strains of Lactobacillus spp., previously isolated from vaginal swabs, were used. L. rhamnosus GG was used as internal positive control for probiotic properties. Moreover, ATCC strains of pathogenic bacteria were used to assess antibacterial activity: E. coli ATCC 25922, E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, S. agalactiae ATCC 13813 and K. pneumoniae ATCC 700603. Finally, clinical strains of Candida spp., previously isolated from women with vulvovaginal candidiasis, were used to assess antifungal activity: C. albicans, C. glabrata and C. tropicalis. All strains are available to Laboratory of Applied Microbiology, Department of Biomedical and Biotechnological Sciences, Università degli Studi di Catania. Lactobacillus spp. were grown in de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Thermo Fisher Scientific Inc., Rodano (MI), Italy, CM0359) at 37 ◦C for 48 h under micro- aerobic conditions before proceeding with subsequent investigations. Candida strains were grown on Sabouraud Dextrose Agar with Chloramphenicol (Oxoid, Thermo Fisher Scientific Inc., Rodano (MI), Italy, PO0358) at 35 ◦C for 48 h under aerobic conditions before proceeding with subsequent investigations and finally, bacteria strains were grown in Muller-Hinton (MH) broth (Oxoid, Thermo Fisher Scientific Inc., Rodano (MI), Italy, CM0405) at 37 ◦C overnight before proceeding with subsequent investigations. Biomolecules 2021, 11, 94 3 of 14

2.2. LAB Identiﬁcation 2.2.1. DNA Extraction RTP Bacterial DNA Mini Kit (Stratec Biomedical AG, Birkenfeld, Germany) was used to extract bacterial DNA following manufacturer instruction. Then, DNA samples obtained were visualized by electrophoresis (70 V) in agarose gel (1.5%).

2.2.2. Molecular Identification 16S rDNA PCR/RFLP and, multiplex-PCRs to amplify different loci of the 16S-23S gene region and ITSregion flanking the 23S rDNA gene were used to make molecular identification as previously described [21].

2.3. Safety Criteria of Potential Probiotics 2.3.1. Antibiotic Resistance Ampicillin, erythromycin, tetracycline, gentamicin, clindamycin, chloramphenicol, streptomycin and kanamycin, were used to assess antibiotic resistance according to European Food Safety Authority [22]. Moreover, ciprofloxacin was used as internal negative control.

2.3.2. Gelatinase Activity and Haemolytic Activity Gelatinase activity and haemolytic activity were evaluated as previously described [23]. Brieﬂy, overnight cultures of strains were streaked onto Nutrient Gelatin agar plates (Oxoid, Thermo Fisher Scientiﬁc Inc., Rodano (MI), Italy, CM0635) and Columbia +5% sheep blood agar plates (bioMérieux Corporate, Bagno a Ripoli (FI), Italy). Plates were incubated at 37 ◦C under micro-aerobic conditions for 48 h. Bacillus subtilis ATTC 23857 and Streptococcus pyogenes ATCC 19615 were used as internal positive control for gelatinase activity and haemolytic activity, respectively.

2.4. Evaluation of Probiotic Properties 2.4.1. Ability to Resist at Low pH and Bile Salts The evaluation of the resistance to low pH and bile salts was carried out by bacterial counts and relative survival rate after stressed growth [24]. Brieﬂy, Lactobacillus strains and E. coli ATCC 25922 (as positive internal control) were grown in LSM broth and MH broth, respectively, at 37 ◦C overnight. Then, two different conditions were evaluated: growth in bile salts agar, and growth in bile salts agar after one hour in acidic condition (pH 3.0). Subsequently, serial ten-fold dilutions in NaCl (0.85% w/v) were made and plated on agar for enumeration. Each experiment was made six times in duplicate and the growth in LSM broth (and MH broth for E. coli) without stress was used as control.

2.4.2. Antimicrobial Activity Cells-free supernatants produced by Lactobacillus strains (grown at 37 ◦C, 18 h in micro-aerobic condition) were neutralized with NaOH 1 mol·L−1 and inoculated into wells in pre-inoculated MH agar plates and Sabouraud dextrose agar with chloramphenicol, with bacteria and fungi, respectively [25–27]. For quality control ciproﬂoxacin and itraconazole were used as positive internal controls for antibacterial and antifungal activity, respectively. MH plates were incubated at 37 ◦C overnight in aerobic condition to evaluate antibacterial activity, and SAB plates were incubated at 33 ◦C for 24 h in aerobic condition to evaluate antifungal activity. Inhibition zones were measured by a gauge [23,28]. Results were expressed in mm ± SD.

2.4.3. Ability to Produce Bioﬁlm Lactobacillus strains were tested for the ability to form bioﬁlm. An inoculum (1.5 × 105 CFU mL−1) of each strain was incubated in MRS broth at 37 ◦C under micro- aerobic condition. Then, the absorbance at 600 nm (BioRad Microplate Reader Model 680, ©2021 Bio-Rad Laboratories, Inc., Segrate (MI), Italy) was recorded and the broth was discharged. The plates were washed, with PBS in order to remove suspended cells, and in- Biomolecules 2021, 11, 94 4 of 14

cubated at 60 ◦C for 1 h. Subsequently, 200 µL of crystal violet were added in each well and after 10/15 min crystal violet residues were dissolved with a solution of methanol/acetic acid 2:1.5. A second read was recorded at 570 nm. The following formula

OD570/OD600 × 0.4

was used to calculate bioﬁlm and OD 0.061 was considered as cut-off value.

2.4.4. Capacity to Produce Hydrogen Peroxide Horseradish peroxidase (Sigma, Merck KGaA, Darmstadt, Germany, P6782) and 3,30,5,50-tetramethylbenzidine (Sigma, Merck KGaA, Darmstadt, Germany, T2885) were added to MRS agar to evaluate and quantify hydrogen peroxide production by Lactobacillus strains. Brieﬂy, a bacterial inoculum (1.5 × 108 CFU mL−1) was streaked on this MRS modiﬁed agar and, the plates were incubated under micro-aerobic condition for 48 h at 3 ◦C, and further at room temperature for other 24 h in the dark. Negative producers gave colourless colonies, while positive producers gave colour colonies from brown to dark blue.

2.4.5. Ability to Adhere to Human Hep-2 and Caco-2 Cell Lines Hep-2 cells (Human epithelial type 2 cells, human laryngeal carcinoma, ATCC®CCL- 23™, University Boulevard Manassas, VA, USA) and Caco-2 cells (Human epithelial cells, human colorectal adenocarcinoma, ATCC®HTB-37™, University Boulevard Manassas, VA, USA) were cultured in DMEM (Dulbecco’s MEM—Biochrom GmbH, Berlin, Germany). Medium were supplemented with 6% FBS, 2 mM L-glutamine, 100 U·mL−1 of penicillin and 100 µg·mL−1 of streptomycin. Plates were incubated at 37 ◦C in a humidified, 95% air/5% CO2 atmosphere. The medium was changed every 2–3 days. One day before the experiment, cells were trypsinized, counted and inoculated in 129AX-1 tubes with a slide (SterilinTM, Thermo Fisher Scientific Inc., Rodano (MI), Italy); approximately 1.5 × 105 cells/tube. After 48 h the cells were washed in PBS, and a bacterial suspension (1.0 × 108 CFU mL−1) was added and the mixture incubated for 30 min at room temperature. Then, three washes with PBS were done and cells were fixed with ethanol 1 mL/tube for 15 min at 3 ◦C, stained with 20% Giemsa (v/v in phosphate-buffered water) for 20 min (in the dark) and examined microscopically under oil immersion. For each slide 200 cells were observed and the results are expressed as the average of bacteria per cells. The adhesion test was repeated at least six times and data were summarized using mean (±SD/cell) [29].

2.4.6. Auto-Aggregation Study Auto-aggregation study was performed with slightly modiﬁed protocol previously described by Gil et al. [30]. Brieﬂy, the strains were grown overnight at 37 ◦C in MRS agar, then suspensions of 0.6 at 600 nm (BioRad Microplate Reader Model 680, © 2021 Bio-Rad Laboratories, Inc., Segrate (MI), Italy) in PBS were done. Variation of OD was monitored every hour for 4 h. Then, the percentage of auto-aggregation was calculated as follows:

% Autoaggregation = (ODin − ODﬁn/ODﬁn) × 100

Gram staining was used to visualize the aggregates under oil immersion microscopy (1000×).

2.4.7. Co-Aggregation Study Co-aggregation study was evaluated as follows: 129AX-1 tubes with slides (SterilinTM, Thermo Fisher Scientiﬁc Inc., Rodano (MI), Italy) were added with 500 µL of an overnight culture of Lactobacillus strains grown at 37 ◦C in MRS broth (Oxoid, Thermo Fisher Scien- tiﬁc Inc., Rodano (MI), Italy, CM0359) under micro-aerobic conditions and 500 µL of an overnight culture of Candida spp. grown at 33 ◦C in DMEM (Dulbecco’s MEM—Biochrom GmbH, Berlin, Germany). Tubes were incubated at 37 ◦C for 4 h. Then, Gram staining was Biomolecules 2021, 11, 94 5 of 14

used to visualize the co-aggregates under oil immersion microscopy (1000×)[30]. Scoring was done according to Reid et al. [31]. Moreover, the co-aggregation were also evaluated on Hep-2 and Caco-2 cells. Tubes with monolayer cells were incubated at 37 ◦C for 4 h after inoculation with Candida and Lactobacillus strains, then Giemsa staining was used to visualize co-aggregates and adhesion on cells.

2.5. Statistical Analysis All experiments were performed at least three times and data were summarized using mean (±SD). Where applicable, data were analysed by one-way ANOVA with correction for multiple comparisons by Bonferroni. All results with p-value < 0.05 were considered signiﬁcant. All results and graphs were generated using GraphPad® Prism ver. 6 software.

3. Results 3.1. LAB Identification The identification of the species of Lactobacillus isolates was conducted with the amplification of 16S rDNA. Four species were identified among which L. crispatus was the most representative (6 strains), followed by L. gasseri (4 strains), L. rhamnosus (1 strain) and L. delbrueckii (1 strain).

3.2. Safety Criteria of Potential Probiotics 3.2.1. Antibiotic Resistance Antibiotic resistance was evaluated to verify if the twelve Lactobacillus strains could be accepted to use as potential probiotic. Strains were inhibited by the eight antibiotics tested according to the microbiological cut-off values deﬁned by EFSA (Table1). Therefore, all strains were susceptible to all antibiotics. Concurrently, the strains resulted resistant for the antibiotic CIP used as quality control.

Table 1. MIC values expressed as µg mL−1 for the evaluation of antibiotic resistance of the Lactobacillus strains.

Strains AM ERY TET GM CM CAF S KAN CIP BG 2 L. crispatus ≤0.03 0.12 0.25 1.0 0.12 0.12 2.0 2.0 >16 EB 1 L. crispatus ≤0.03 0.12 0.25 2.0 0.12 0.12 2.0 2.0 >16 EB 3 L. crispatus ≤0.03 0.25 0.50 2.0 0.12 0.12 2.0 1.0 >16 EB 4 L. crispatus ≤0.03 0.12 0.25 2.0 0.12 0.12 2.0 2.0 >16 CC 1 L. crispatus ≤0.03 0.12 0.25 4.0 0.12 0.25 4.0 2.0 >16 CC 4 L. crispatus ≤0.03 0.12 0.25 2.0 0.12 0.25 2.0 2.0 >16 BA 3 L. gasseri 0.12 0.06 1.0 2.0 0.12 0.12 2.0 2.0 >16 MS 1 L. gasseri 0.12 0.06 1.0 4.0 0.12 0.12 2.0 2.0 >16 SG 1 L. gasseri 0.12 0.06 1.0 4.0 0.12 0.12 2.0 2.0 >16 SG 3 L. gasseri 0.12 0.06 0.25 1.0 0.12 0.25 2.0 1.0 >16 RB 1 L. delbrueckii 0.5 0.06 0.25 2.0 0.25 0.25 4.0 2.0 >16 AD 3 L. rhamnosus 1.0 0.06 1.0 2.0 0.12 0.12 4.0 2.0 8 GG L. rhamnosus 1.0 0.06 1.0 4.0 0.12 0.25 4.0 2.0 8 AM ampicillin; ERY erythromycin; TET tetracycline; GM gentamicin; CM clindamycin; CAF chloramphenicol; S streptomycin; KAN kanamycin; CIP ciproﬂoxacin.

3.2.2. Gelatinase Activity and Haemolytic Activity Gelatinase activity was not found for the Lactobacillus strains tested. Furthermore, lysis on blood agar plates were not shown for any Lactobacillus strain under examination.

3.3. Evaluation of Probiotic Properties 3.3.1. Ability to Resist at Low and Bile Salts As already stated, according to FAO [32], bacterial strains must resist to gastrointestinal conditions and remain viable to be considered probiotics. Figures1 and2 show the reduction to exposition to low pH and different concentrations of bile salts, respectively. Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 14

3.3. Evaluation of Probiotic Properties 3.3.1. Ability to Resist at Low and Bile Salts As already stated, according to FAO [32], bacterial strains must resist to gastroin‐ testinal conditions and remain viable to be considered probiotics. Figures 1 and 2 show the reduction to exposition to low pH and different concentrations of bile salts, respec‐ tively. First of all, all strains survived one hour at pH 3.0 but with substantial differences: the strain that showed the greatest resistance was L. rhamnosus AD3 (−15.39%), followed by L. gasseri SG3 (−18.16%) and L. crispatus CC4 (−21.95%). In contrast, the most suscepti‐ ble strains were L. rhamnosus GG (−88.33%) and L. delbrueckii RB1 (−99.28%). All isolates showed good tolerance to bile salts at difference concentration pre‐exposition at low pH. In particular, L. rhamnosus AD3 proved to be the most resistant, Biomolecules 2021, 11, 94 with a survival of 69.2% with bile salts at 0.5% and even 130.8% with bile salts at 0.12%.6 of 14 Oddly, the only strain that showed significant reduction in bile salt treatment was L. rhamnosus GG (−93.7% with BS 0.5%; −90% with BS 0.12%). After exposure to acid pH for one hour, the consequent exposure to bile salts showed a slight decrease for all strains includingFirst of all, L. all rhamnosus strains survived GG. Additionally one hour in at this pH case, 3.0 but L. withrhamnosus substantial AD3 proved differences: to be the moststrain resistant that showed strain the (−0%). greatest This resistance means that was afterL. GI rhamnosus tract passage,AD3 ( −all15.39%), strains were followed able byto surviveL. gasseri butSG3 not (− at18.16%) the same and initialL. crispatus concentrationCC4 (− 21.95%).of inoculum. In contrast, the most susceptible strains were L. rhamnosus GG (−88.33%) and L. delbrueckii RB1 (−99.28%).

Biomolecules 2021, 11, x FOR PEER REVIEW 7 of 14 Figure 1. Comparison between controlcontrol growthgrowth and and growth growth after after one one hour hour of of exposure exposure to to pH pH 3 by3 by associating associating the the strains strains by byspecies: species: (A )(AL.) crispatusL. crispatusstrains,( strains,(B) L.B) delbrueckiiL. delbrueckiistrain, strain, (C) (L.C) gasseri L. gasseristrains strains and and (D )(DL.) rhamnosus L. rhamnosusstrains. strains.

Figure 2. Percentage of survival at different concentrations of bile salts before and after exposure to acid pH. Data are Figure 2. Percentage of survival at different concentrations of bile salts before and after exposure to showed as meanacid± pH.SD; Datap values are showed were calculated as mean ± bySD; applying p values were one-way calculated ANOVA by applying with Bonferroni one‐way ANOVA correction for multiple comparisons. Signiﬁcancewith Bonferroni vs. control correction * p

3.3.2. Antimicrobial Activity The antagonistic activity of putative probiotic strains was assessed against the fol‐ lowing target microorganisms: Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, a clinical isolate of Streptococcus agalactiae, three clinical isolates of Candida (C. albicans 10, C. glabrata 14, C. tropicalis 21). The strains showed good activity both against Gram‐positive and Gram‐negative pathogens, and also excellent activity against Candida strains (Table 2). L. gasseri SG3 showed the highest antagonistic activity against both bacteria and fungi, fol‐ lowed by L. rhamnosus AD3 which however had no effect against E. coli and C. tropicalis. Finally, no inhibition zones were shown by L. rhamnosus GG.

Table 2. Antimicrobial activity of cell‐free supernatants produced by Lactobacillus strains against bacterial and fungal urogenital pathogens. Results were expressed in mm (mean ± SD).

Escherichia Enterococcus Pseudomonas Klebsiella Streptococcus Candida Candida Candida Strains coli faecalis aeruginosa pneumoniae agalactiae albicans glabrata tropicalis BG 2 L. crispatus ≤6 18.3 ± 0.3 ≤6 ≤6 9.3 ± 0.3 10.2 ± 1.5 11.2 ± 1.7 17.7 ± 0.4 EB 1 L. crispatus 9.6 ± 1.9 23.5 ± 1.3 ≤6 ≤6 13.2 ± 1.6 16.3 ± 0.4 18.5 ± 1.5 22.1 ± 1.7 EB 3 L. crispatus 12.2 ± 0.2 28.5 ± 0.3 ≤6 ≤6 9.8 ± 1.6 22.3 ± 1.4 23.4 ± 1.8 19.6 ± 0.8 EB 4 L. crispatus 9.5 ± 1.3 16.7 ± 1.1 ≤6 ≤6 10.1 ± 2.2 20.5 ± 2.3 24.5 ± 0.7 21.2 ± 1.6 CC 1 L. crispatus 9.7±0.3 17.4 ± 0.2 12.4 ± 0.2 9.5 ± 0.5 11.2 ± 0.5 18.4 ± 0.4 21.4 ± 0.3 19.1 ± 0.6 CC 4 L. crispatus 10.0 ± 2.3 24.3 ± 0.3 23.6 ± 0.1 18.5 ± 0.6 11.5 ± 0.4 19.6 ± 0.9 21.3 ± 2.4 20.5 ± 0.5 BA 3 L. gasseri 12.3 ± 0.4 20.3 ± 0.4 23.5 ± 0.2 14.4 ± 0.2 12.3 ± 1.1 17.9 ± 1.4 15.5 ± 2.4 20.1 ± 1.3 MS 1 L. gasseri ≤6 19.3 ± 1.3 21.4 ± 1.7 20.4 ± 0.8 12.5 ± 1.9 20.3 ± 1.7 21.6 ± 2.3 21.5 ± 3.6 SG 1 L. gasseri ≤6 20.4 ± 0.3 15.5 ± 0.6 ≤6 11.3 ± 0.4 20.2 ± 0.4 21.6 ± 0.5 21.3 ± 0.3 SG 3 L. gasseri 10.1 ± 0.2 22.3 ± 0.4 26.4 ± 0.3 25.3 ± 0.2 17.6 ± 0.3 21.4 ± 0.6 22.4 ± 0.3 25.4 ± 0.1 RB 1 L. delbrueckii ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 AD 3 L. ≤6 21.4 ± 0.5 20.4 ± 1.4 16.4 ± 0.3 11.5 ± 2.3 17.4 ± 1.2 21.3 ± 0.5 ≤6 rhamnosus GG L. rhamnosus ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6

Biomolecules 2021, 11, 94 7 of 14

All isolates showed good tolerance to bile salts at difference concentration pre-exposition at low pH. In particular, L. rhamnosus AD3 proved to be the most resistant, with a survival of 69.2% with bile salts at 0.5% and even 130.8% with bile salts at 0.12%. Oddly, the only strain that showed significant reduction in bile salt treatment was L. rhamnosus GG (−93.7% with BS 0.5%; −90% with BS 0.12%). After exposure to acid pH for one hour, the consequent exposure to bile salts showed a slight decrease for all strains including L. rhamnosus GG. Additionally in this case, L. rhamnosus AD3 proved to be the most resistant strain (−0%). This means that after GI tract passage, all strains were able to survive but not at the same initial concentration of inoculum.

3.3.2. Antimicrobial Activity The antagonistic activity of putative probiotic strains was assessed against the following target microorganisms: Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, a clinical isolate of Streptococcus agalactiae, three clinical isolates of Candida (C. albicans 10, C. glabrata 14, C. tropicalis 21). The strains showed good activity both against Gram-positive and Gram- negative pathogens, and also excellent activity against Candida strains (Table2). L. gasseri SG3 showed the highest antagonistic activity against both bacteria and fungi, followed by L. rhamnosus AD3 which however had no effect against E. coli and C. tropicalis. Finally, no inhibition zones were shown by L. rhamnosus GG.

Table 2. Antimicrobial activity of cell-free supernatants produced by Lactobacillus strains against bacterial and fungal urogenital pathogens. Results were expressed in mm (mean ± SD).

Escherichia Enterococcus Pseudomonas Klebsiella Streptococcus Candida Candida Candida Strains coli faecalis aeruginosa pneumoniae agalactiae albicans glabrata tropicalis BG 2 ≤6 18.3 ± 0.3 ≤6 ≤6 9.3 ± 0.3 10.2 ± 1.5 11.2 ± 1.7 17.7 ± 0.4 L. crispatus EB 1 9.6 ± 1.9 23.5 ± 1.3 ≤6 ≤6 13.2 ± 1.6 16.3 ± 0.4 18.5 ± 1.5 22.1 ± 1.7 L. crispatus EB 3 12.2 ± 0.2 28.5 ± 0.3 ≤6 ≤6 9.8 ± 1.6 22.3 ± 1.4 23.4 ± 1.8 19.6 ± 0.8 L. crispatus EB 4 9.5 ± 1.3 16.7 ± 1.1 ≤6 ≤6 10.1 ± 2.2 20.5 ± 2.3 24.5 ± 0.7 21.2 ± 1.6 L. crispatus CC 1 9.7 ± 0.3 17.4 ± 0.2 12.4 ± 0.2 9.5 ± 0.5 11.2 ± 0.5 18.4 ± 0.4 21.4 ± 0.3 19.1 ± 0.6 L. crispatus CC 4 10.0 ± 2.3 24.3 ± 0.3 23.6 ± 0.1 18.5 ± 0.6 11.5 ± 0.4 19.6 ± 0.9 21.3 ± 2.4 20.5 ± 0.5 L. crispatus BA 3 12.3 ± 0.4 20.3 ± 0.4 23.5 ± 0.2 14.4 ± 0.2 12.3 ± 1.1 17.9 ± 1.4 15.5 ± 2.4 20.1 ± 1.3 L. gasseri MS 1 ≤6 19.3 ± 1.3 21.4 ± 1.7 20.4 ± 0.8 12.5 ± 1.9 20.3 ± 1.7 21.6 ± 2.3 21.5 ± 3.6 L. gasseri SG 1 ≤6 20.4 ± 0.3 15.5 ± 0.6 ≤6 11.3 ± 0.4 20.2 ± 0.4 21.6 ± 0.5 21.3 ± 0.3 L. gasseri SG 3 10.1 ± 0.2 22.3 ± 0.4 26.4 ± 0.3 25.3 ± 0.2 17.6 ± 0.3 21.4 ± 0.6 22.4 ± 0.3 25.4 ± 0.1 L. gasseri RB 1 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 L. delbrueckii AD 3 ≤6 21.4 ± 0.5 20.4 ± 1.4 16.4 ± 0.3 11.5 ± 2.3 17.4 ± 1.2 21.3 ± 0.5 ≤6 L. rhamnosus GG ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 ≤6 L. rhamnosus BiomoleculesBiomolecules 2021 2021, 11, 11, x, FORx FOR PEER PEER REVIEW REVIEW 8 of8 of 14 14 Biomolecules 2021, 11, 94 8 of 14

3.3.3.3.3.3. Ability Ability to to Produce Produce Biofilm Biofilm 3.3.3. Ability to Produce Bioﬁlm TheThe strains strains were were divided divided by by means means of of BI: BI: non non‐producers‐producers (OD (OD < <0.061), 0.061), weak weak pro pro‐ ‐ The strains were divided by means of BI: non-producers (OD < 0.061), weak producers ducersducers (0.061 (0.061 < >0.300) 0.300) [23]. [23]. All All strains strains showed showed moderate moderate biofilm biofilm production production after after over over‐ ‐ (OD > 0.300) [23]. All strains showed moderate bioﬁlm production after overnight growth nightnight growth growth at at 37 37 °C, °C, with with the the exception exception of of L. L. crispatus crispatus BG2 BG2 which which instead instead proved proved to to be be at 37 ◦C, with the exception of L. crispatus BG2 which instead proved to be the weakest thethe weakest weakest producer producer (Figure (Figure 3). 3). producer (Figure3).

Figure 3. Bioﬁlm formation by Lactobacillus strains recorded after overnight growth at 37 ◦C under FigureFigure 3. 3. Biofilm Biofilm formation formation by by Lactobacillus Lactobacillus strains strains recorded recorded after after overnight overnight growth growth at at 37 37 °C °C under under ± microaerobicmicroaerobicmicroaerobic condition. condition. Data Data Data are are are showed showed showed as as mean as mean mean ± ±SD; SD; pSD; pvalues valuesp values were were calculated were calculated calculated by by applying applying by applying oneone-wayone‐way‐way ANOVA ANOVAANOVA with withwith Bonferroni BonferroniBonferroni correction correctioncorrection for for multiple multiple comparisons. comparisons. Significance Significance Signiﬁcance vs. vs. AD AD AD 3 3and 3 and and GGGGGG * *p* pp< <<0.05, 0.05, ** ** p p< <0.01. 0.01. 3.3.4. Capacity to Produce Hydrogen Peroxide 3.3.4.3.3.4. Capacity Capacity to to Produce Produce Hydrogen Hydrogen Peroxide Peroxide Production of hydrogen peroxide was tested in 12 vaginal lactobacilli strains and ProductionProduction of of hydrogen hydrogen peroxide peroxide was was tested tested in in 12 12 vaginal vaginal lactobacilli lactobacilli strains strains and and L. L. L. rhamnosus GG by the semi-qualitative TMB peroxidase assay (Figure4). Only three rhamnosusrhamnosus GG GG by by the the semi semi‐qualitative‐qualitative TMB TMB peroxidase peroxidase assay assay (Figure (Figure 4). 4). Only Only three three strains out of 13 produced great amount of hydrogen peroxide: two L. crispatus (BG2, EB3) strainsstrains out out of of 13 13 produced produced great great amount amount of of hydrogen hydrogen peroxide: peroxide: two two L. L. crispatus crispatus (BG2, (BG2, and one L. gasseri (BA3). L. rhamnosus GG showed a moderate production. EB3)EB3) and and one one L. L. gasseri gasseri (BA3). (BA3). L. L. rhamnosus rhamnosus GG GG showed showed a amoderate moderate production. production.

FigureFigureFigure 4. 4.4. Hydrogen Hydrogen peroxide peroxideperoxide production productionproduction of ofof Lactobacillus LactobacillusLactobacillus isolates. isolates.isolates. White White-colorless:White‐colorless:‐colorless: negative; negative; Brown: Brown: slightlyslightlyslightly positive; positive;positive; Light LightLight blue: blue:blue: moderate; moderate;moderate; Dark DarkDark blue: blue:blue: strong. strong.strong. Data Data are are showed showedshowed as asas mean meanmean ± ±±SD. SD.SD. 3.3.5. Ability to Adhere to Human Hep-2 and Caco-2 Cell Lines 3.3.5.3.3.5. Ability Ability to to Adhere Adhere to to Human Human Hep Hep‐2‐ 2and and Caco Caco‐2‐ 2Cell Cell Lines Lines The ability of a bacterial strain to adhere to human epithelial cells is one of the most TheThe ability ability of of a abacterial bacterial strain strain to to adhere adhere to to human human epithelial epithelial cells cells is is one one of of the the most most important properties for such a strain to be considered probiotic. Indeed, this feature will importantimportant properties properties for for such such a astrain strain to to be be considered considered probiotic. probiotic. Indeed, Indeed, this this feature feature will will allow it to colonize the mucous membrane and perform important modulating functions allowallow it itto to colonize colonize the the mucous mucous membrane membrane and and perform perform important important modulating modulating functions functions on the immune system. In this study, Lactobacillus strains were tested for adhesion to Hep-2 onon the the immune immune system. system. In In this this study, study, Lactobacillus Lactobacillus strains strains were were tested tested for for adhesion adhesion to to and Caco-2 cells (Table3). Among all isolates tested, L. rhamnosus AD3 and L. crispatus EB1 Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 14

Biomolecules 2021, 11, 94 Hep‐2 and Caco‐2 cells (Table 3). Among all isolates tested, L. rhamnosus AD3 and9 of 14 L. crispatus EB1 showed heavy in vitro adhesion towards both epithelial cell lines compared to L. rhamnosus GG (≈47/cell). showed heavy in vitro adhesion towards both epithelial cell lines compared to L. rhamnosus Table 3. Adhesion of Lactobacillus strains to Hep‐2 cells and Caco‐2 [29] and co‐aggregation rates obtained with three GG (≈47/cell). different strains of Candida [31].

Table 3. AdhesionAdhesion of Lactobacillus on Hepstrains‐2 Adhesion to Hep-2 on cells Caco and‐2 Caco-2 [29] and co-aggregation rates obtained with three Strains Candida albicans * Candida krusei * Candida tropicalis * different strains of Candida(n Bacteria/Cell)[31]. (n Bacteria/Cell) BG 2 L. crispatus 37 ± 12 43 ± 11 1 1 2 Adhesion on Hep-2 Adhesion on Caco-2 Strains Candida albicans * Candida krusei * Candida tropicalis * EB 1 L. crispatus (n Bacteria/Cell)50 ± 15 (n Bacteria/Cell)50 ± 14 1 1 3 EB 3 L. crispatus 33 ± 13 38 ± 4 1 2 2 BG 2 L. crispatus 37 ± 12 43 ± 11 1 1 2 EBEB 4 1 L. crispatuscrispatus 5039± ±15 8 50 ±4114 ± 8 12 12 32 CCEB 31 L.L. crispatus crispatus 3331± ± 1310 3835± ±4 11 11 22 23 CCEB 4 L.L. crispatus crispatus 3949± ± 86 4149± 8± 9 22 22 23 ± ± CCBA 1 3L. L. crispatus gasseri 3124 ±10 5 3531 11± 10 12 22 33 CC 4 L. crispatus 49 ± 6 49 ± 9 2 2 3 MSBA 31 L.L. gasseri gasseri 24 ± 135 3137± 10± 11 21 21 34 SGMS 1 L. gasserigasseri 2425± ±13 9 3729± 11± 15 12 11 42 SGSG 13 L.L. gasseri gasseri 3025 ± 119 2943± 15± 12 21 11 22 SG 3 L. gasseri 30 ± 11 43 ± 12 1 1 2 RB1RB1 L. delbrueckiidelbrueckii 3030± ± 33 4545± 16± 16 22 11 33 AD3AD3 L. rhamnosusrhamnosus 8282± ± 1212 108108± 20± 20 11 22 44 GGGG L. rhamnosusrhamnosus 4545± ± 1515 5050± 16± 16 11 44 22 ** co-aggregation co‐aggregation rates: rates: No aggregationNo aggregation (0); small (0); aggregates small aggregates (1); aggregates (1); aggregates with larger numberswith larger of bacteria numbers (2); of macroscopically bacteria (2); visiblemacro‐ clumpsscopically with largervisible groups clumps of bacteriawith larger (3) and groups macroscopically of bacteria visible (3) and clumps macroscopically (4). visible clumps (4).

3.3.6.3.3.6. Auto-Aggregation Auto‐Aggregation Study Study TheThe ability ability of auto-aggregationof auto‐aggregation of vaginal of vaginal lactobacilli lactobacilli was evaluated was atevaluated ﬁve time-points at five (0–4time h).‐points This (0–4 ability h). was This conﬁrmed ability was at confirmed all time-points at all but time the‐points strains but showed the strains the highestshowed degreethe highest of auto-aggregation degree of auto after‐aggregation 4 h of incubation. after 4 h Some of incubation. strains have Some a greater strains ability have to a self-aggregategreater ability than to self others:‐aggregate in this studythan others:L. rhamnosus in thisAD3 study and L.L. rhamnosus crispatus CC4AD3 showed and L. thecris‐ highestpatus CC4 auto-aggregating showed the highest aptitude, auto even‐aggregating greater than aptitude,L. rhamnosus even greaterGG. Figurethan L.5 rhamnosusshowed auto-aggregationGG. Figure 5 showed observed auto for‐aggregationL. rhamnosus observedAD3, L. for crispatus L. rhamnosusCC4 and AD3,L. rhamnosus L. crispatusGG CC4 in twoand time-points L. rhamnosus (T0 GG and in T4).two time‐points (T0 and T4).

Figure 5. Auto-aggregation of vaginal lactobacilli at two time-points: T0 and T4. L. rhamnosus AD3, L. crispatus CC4 and L. rhamnosus GG showed greater ability to self-aggregate than others strains. Strains were observed under the microscope (1000×) after Gram staining. Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 14 Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 14

Figure 5. Auto‐aggregation of vaginal lactobacilli at two time‐points: T0 and T4. L. rhamnosus AD3, Biomolecules 2021, 11, 94 L.Figure crispatus 5. Auto CC4‐aggregation and L. rhamnosus of vaginal GG showed lactobacilli greater at two ability time to‐points: self‐aggregate T0 and T4. than L. othersrhamnosus strains.10 AD3, of 14 StrainsL. crispatus were CC4 observed and L. underrhamnosus the microscope GG showed (1000×) greater after ability Gram to self staining.‐aggregate than others strains. Strains were observed under the microscope (1000×) after Gram staining. 3.3.7. Co‐Aggregation Study 3.3.7. Co Co-Aggregation‐Aggregation Study The auto‐aggregation ability of Lactobacillus strains was reflected in the ability to createThe co ‐ auto-aggregationaggregatesauto‐aggregation with Candida ability ability of ofcells.Lactobacillus Lactobacillus Vaginalstrains strains strains was were was reﬂected evaluatedreflected in the inwith ability the Candida ability to create alto‐ bicanscreateco-aggregates (Figureco‐aggregates with6A), Candida with Candida krusei cells. cells.(Figure Vaginal Vaginal 6B) strains and strains were Candida were evaluated tropicalisevaluated with (Figure withCandida Candida 6C). albicans The al‐ cobicans(Figure‐aggregation (Figure6A), Candida 6A),scores kruseiCandida were(Figure obtainedkrusei6B) (Figure and afterCandida 46B) h of and tropicalisincubation Candida(Figure attropicalis 376 C).°C The(Table (Figure co-aggregation 3). 6C).Figure The 7 ◦ showedcoscores‐aggregation were macroscopically obtained scores after were visible 4 hobtained of incubationclumps after when at4 h 37 L. of rhamnosusC incubation (Table3). AD3 Figure at and37 7°C showedGG (Table have macroscopi- 3).been Figure tested 7 withshowedcally all visible threemacroscopically clumps Candida when strains. visibleL. rhamnosus It isclumps possibleAD3 when that and L. GGco rhamnosus‐aggregates have been AD3 testedare and able withGG to have allcreate three been microenCandida tested‐ vironmentswithstrains. all Itthree is around possible Candida the that strains. pathogen co-aggregates It is possiblecapable are that ableof blocking co to‐aggregates create its microenvironments diffusion are able towards to create around epithelialmicroen the‐ cells.vironmentspathogen Indeed, capable around the study of the blocking ofpathogen the itsco‐ diffusionaggregation capable towardsof betweenblocking epithelial Lactobacillusits diffusion cells. Indeed,strains towards and the epithelial studyCandida of strainscells.the co-aggregation Indeed, on Hep the‐2 studyand between Caco of theLactobacillus‐2 monolayersco‐aggregationstrains has between andshown,Candida Lactobacillusin somestrains cases, on strains Hep-2 how and andthe Candida Caco-2bacte‐ riastrainsmonolayers‐fungus on coHep has‐aggregates‐ shown,2 and Caco in inhibited some‐2 monolayers cases, the how adhesion the has bacteria-fungus shown,of Candida in someon co-aggregates epithelial cases, how cells inhibited the(Figure bacte the 8‐ Candida AD3).riaadhesion‐fungus of co‐aggregateson epithelial inhibited cells the (Figure adhesion8 AD3). of Candida on epithelial cells (Figure 8 AD3).

Figure 6. Candida albicans (A), C. krusei (B) and C. tropicalis (C) observed under a microscope (1000(1000×)×) after after Giemsa Giemsa staining. Figure 6. Candida albicans (A), C. krusei (B) and C. tropicalis (C) observed under a microscope (1000×) after Giemsa staining.

Figure 7. Macroscopically visible clumps after Giemsa staining of L. rhamnosus AD3 and GG with all three Candida strains. CoFigure‐aggregates 7. Macroscopically of L. rhamnosus visible AD3 clumps with C. after albicans Giemsa (AD3 staining ‐6), C. kruseiof L. rhamnosus (AD3‐8) and AD3 C. and tropicalis GG with (AD3 all all‐ 16).three three Co CandidaCandida‐aggregates strains.strains. of Co‐aggregates of L. rhamnosus AD3 with C. albicans (AD3‐6), C. krusei (AD3‐8) and C. tropicalis (AD3‐16). Co‐aggregates of L.Co-aggregates rhamnosus GG of withL. rhamnosus C. albicansAD3 (GG with‐6), C. krusei albicans (GG(AD3-6),‐8) andC. C. krusei tropicalis(AD3-8) (GG‐ and16). C. tropicalis (AD3-16). Co-aggregates of L. rhamnosus GG with C. albicans (GG‐6), C. krusei (GG‐8) and C. tropicalis (GG‐16). L. rhamnosus GG with C. albicans (GG-6), C. krusei (GG-8) and C. tropicalis (GG-16). Biomolecules 2021, 11, 94 11 of 14 Biomolecules 2021, 11, x FOR PEER REVIEW 11 of 14

Figure 8. L. rhamnosus AD3 on Hep‐2 cell monolayer inhibited adhesion of Candida on epithelial Figure 8. L. rhamnosus AD3 on Hep-2 cell monolayer inhibited adhesion of Candida on epithelial cells cells (AD3). Instead, the adhesion of L. rhamnosus GG on the Hep‐2 monolayer did not inhibit the (AD3). Instead, the adhesion of L. rhamnosus GG on the Hep-2 monolayer did not inhibit the adhesion adhesion of the Candida (dark purple cells) (GG). Strains were observed under the microscope × of(1000×) the Candida after Giemsa (dark purple staining. cells) (GG). Strains were observed under the microscope (1000 ) after Giemsa staining. 4. Discussion 4. Discussion Maintaining an adequate concentration of Lactobacilli is a necessary requirement to Maintaining an adequate concentration of Lactobacilli is a necessary requirement to ensure the balance and health of the urogenital tract, especially in women [33]. ensure the balance and health of the urogenital tract, especially in women [33]. Apparently, as already mentioned, alterations in the composition of the microbiota Apparently, as already mentioned, alterations in the composition of the microbiota in women determine a greater susceptibility to infections. Therefore, re‐establishing the in women determine a greater susceptibility to infections. Therefore, re-establishing the Lactobacilli prevalence in the vaginal microbiota is necessary for the maintenance of the Lactobacilli prevalence in the vaginal microbiota is necessary for the maintenance of the woman’s state of health [34]. woman’s state of health [34]. The aim of this work was to find strains of Lactobacilli with probiotic characteristics The aim of this work was to find strains of Lactobacilli with probiotic characteristics adequate to respond to the physiological demands of women. adequate to respond to the physiological demands of women. Firstly,Firstly, the the strains strains were were tested tested for for their their susceptibility susceptibility to to antibiotics antibiotics as as well well as as for for haemolytichaemolytic and and gelatinase gelatinase activity. activity. These These tests tests were were necessary necessary to evaluate to evaluate the safetythe safety of the of strains,the strains, in fact in no fact strain no havingstrain having antibiotic antibiotic resistance resistance genes or genes haemolytic/gelatinase or haemolytic/gelatinase activity canactivity be marketed can be marketed [22,32]. All [22,32]. the strains All the tested strains met tested these met safety these qualities. safety qualities. TheThe strains strains studied studied belonged belonged to the to following the following species: species:L. crispatus L., L.crispatus gasseri,, L.L. rhamnosus gasseri, L. andrhamnosusL. delbrueckii and L.. These delbrueckii species. These are among species the are most among encountered the most in encountered the vaginas in of the healthy vagi‐ womennas of healthy [35,36]. women Usually, [35,36].L. crispatus Usually,and L. L.crispatus gasseri andare strongL. gasseri producers are strong of producers lactic acid of andlactic inhibitory acid and molecules inhibitory against molecules pathogenic against species pathogenic that causespecies urethritis, that cause vaginitis urethritis, or candidiasisvaginitis or [ 37candidiasis,38]. In our [37,38]. study, In a our strain study, of L. a strain gasseri of, named L. gasseri SG3,, named showed SG3, the showed highest the antagonistichighest antagonistic activity against activity both against bacteria both and bacteria fungi, followed and fungi, by L.followed rhamnosus by AD3.L. rhamnosus Oddly enough,AD3. Oddly the positive enough, control the positive strain L. control rhamnosus strainGG L. did rhamnosus not show GG any did inhibitory not show activity. any in‐ Inhibitory addition, activity.L. gasseri In addition,and L. rhamnosus L. gasseri areand the L. rhamnosus species most are resistantthe species to gastrointestinalmost resistant to conditions,gastrointestinal as well conditions, as strong producersas well as strong of biofilm producers [23,24]. of biofilm [23,24]. InIn this this study, study, the the strain strain that that showed showed the the greatest greatest resistance resistance at at low low pH pH and and bile bile salts salts (pre-(pre‐ andand post-exposition) post‐exposition) was wasL. L. rhamnosus rhamnosusAD3, AD3, followed followed by byL. L. gasseri gasseriSG3. SG3. In In contrast, contrast, thethe positive positive control controlL. L. rhamnosus rhamnosusGG GG showed showed low low resistance resistance to to these these factors. factors. AsAs far far as as biofilm biofilm production production is is concerned, concerned, all all strains strains have have been been shown shown to be to moderatebe moder‐ orate strong or strong (AD3 (AD3 and GG) and producersGG) producers except except for CC1, for BG2CC1, and BG2 RB1. and The RB1. latter The didlatter not did even not showeven resistance show resistance to gastrointestinal to gastrointestinal conditions conditions proving to beproving the weakest to be strainthe weakest among thosestrain testedamong and those therefore tested the and strain therefore with the less strain probiotic with properties. less probiotic properties. Moreover,Moreover, the the most most of of the the strains strains under under examination examination showed showed moderate moderate accumulation accumula‐ oftion H2 Oof2 H, with2O2, with the exceptionthe exception of SG3 of SG3 (very (very low) low) and and BG2, BG2, EB3 EB3 and and BA3 BA3 which which showed showed toto be be great great producers. producers. However, However, the the role role of of hydrogen hydrogen peroxide peroxide as as a a protective protective agent agent is is controversial.controversial. Indeed, Indeed, despite despite the the statement statement that thatLactobacillus Lactobacillusspp. spp. can can exert exert its its probiotic probiotic propertiesproperties also also through through the the H H2O2O2 2production production is is accepted, accepted, there there are are several several evidences evidences as as Biomolecules 2021, 11, 94 12 of 14

support the implausibility of “in vivo” action as an antimicrobial factor in the vaginal environment [39]. Hence, this property cannot be taken as a predisposing factor for a strain’s probiotic activity. The ability of auto-aggregation of Lactobacillus strains can help to increase the colonization of vaginal environment by adhesion and sub-sequentially by bioﬁlm formation and postbiotic production to inhibit the overgrowth pathogenic microorganisms [30,40]. More- over, the ability to create co-aggregates with pathogenic microorganisms can modulate the destiny of pathologies among which stand out recurrent vulvovaginal candidiasis [41,42]. L. rhamnosus AD3 and L. crispatus CC4 showed the highest capacity to adhesion and auto- aggregation (Figure5). Moreover, L. rhamnosus AD3 has shown an intended ability to form co-aggregates with all three Candida species, especially C. tropicalis (Figure7). Although L. rhamnosus GG’s co-aggregation capacity is visibly greater (Figure7), L. rhamnosus AD3 would seem capable of binding Candida cells more consistently. This property is evident from Candida absence on monolayer cells after co-culture exposure (Figure8 AD3). On the contrary, bound Candida cells can be seen on cells despite the formation of co-aggregates with L. rhamonsus GG (Figure8 GG). In summary, our results indicated that among all vaginal lactobacilli strains tested, L. rhamnosus AD3 revealed the required properties as potential probiotic strain. Indeed, it has responded to all safety features, and it has shown the highest levels of resistance to the gastrointestinal tract, production of active postbiotics and bioﬁlm against fungi and bacteria, ability to adhere to epithelial mucosa, capacity to form strong aggregates and co-aggregates with Candida. Instead, L. delbrueckii RB1 proved to be the weakest strain. Strangely, as already said, the strain used as a positive control, L. rhamonosus GG, already on the market, showed low resistance to the gastrointestinal tract and no production of postbiotics active against pathogenic strains. Nonetheless, it showed high adhesion, auto- aggregation and co-aggregation ability, although candida cells were still present on the epithelial mucosa. These results suggest that L. rhamnosus AD3 may be a promising strain to aid vaginal microbiota recovery as complementary treatment option for urogenital diseases, and to enhance the normal vaginal microbiota in healthy young women. Future studies are required to assess technological method to make pharmaceutical preparations able to respond to market demands.

Author Contributions: Conceptualization, V.F. and P.M.F.; data curation, A.S. and V.F.; investigation, A.S., G.C. and V.F.; methodology, A.S. and V.F.; software, V.F.; writing—original draft, V.F.; writing— review and editing, A.S., V.F. and P.M.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: This work was partially supported by Piano per la Ricerca 2016–2018 Linea di Intervento 2 “Dotazione Ordinaria” UPB 20722142122 University of Catania (PI: Giovanni Li Volti). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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