Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach

Do nomadic lactobacilli fit as potential vaginal probiotics? 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The answer lies in a successful selective multi-step and scoring approach Claudia Cappello, Marta Acin-Albiac, Daniela Pinto, Andrea Polo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2201461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2023 Read the published version in Microbial Cell Factories → Version 1 posted 7 You are reading this latest preprint version Abstract Background The goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens Streptococcus agalactiae and Candida albicans , which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed in vitro for their ability to reduce the inflammation caused by C. albicans infection on the reconstituted human vaginal epithelium (HVE). Results Lactiplantibacillu s plantarum produced both isomers of lactic acid, while Lacticaseibacillus paracasei produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within Lact. paracasei strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. S. agalactiae 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of C. albicans UNIBZ54 was less frequent. Overall, L. plantarum strains had the highest pathogen inhibition and functional scoring. L. plantarum C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by C. albicans infection in the HVE model. Conclusions Our multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains ( L. plantarum C5 and POM1), being applicable to preserve and improve human vaginal health. Vaginal microbiota vaginal ecosystem nomadic lactobacilli probiotics pathogens inhibition screening Figures Figure 1 Figure 2 Figure 3 Background The human vaginal microbiome is a key determinant of vaginal health. The vagina ecosystem is often dominated by highly adapted lactobacilli. Vaginal microbiota can be clustered into five community state types (CSTs). Four of these CSTs are dominated by Lactobacillus crispatus (CST-I), Lactobacillus iners (CST-III), Lactobacillus gasseri (CST-II) or Lactobacillus jensenii (CST-V), while CST-IV is characterized by strict and facultative anaerobes not belonging to Lactobacillus genus (Smith and Ravel 2017). The presence of Lactobacillus spp. is correlated with a healthy vaginal microbiome, whereas the CTS-IV is associated with a status of dysbiosis and a consequently higher risk of infections and obstetric complications. Functional traits of lactobacilli involve the production of lactic acid and hydrogen peroxide, synthesis of bacteriocins as a response to the imbalance of the vaginal microbiome, host protection against vaginal pathogens, promotion of immunomodulation mechanisms by triggering the innate immunity system, and stimulation of anti-inflammatory mechanisms (Graf et al. 2019 ; Kovachev 2018 ; Borges, Silva, and Teixeira 2014; Han and Ren 2021). Infections of the host such as bacterial vaginosis (BV), urinary tract infections, yeast vaginitis, and sexually transmitted diseases like human immunodeficiency virus are all prevented in large part by the microorganisms that colonize the vaginal environment (Cribby, Taylor, and Reid 2008). Aerobic vaginitis defined by disruption in Lactobacillus dominance is accompanied by more extreme inflammatory changes than BV and the presence of mainly aerobic enteric commensals or pathogens, including Streptococcus agalactiae (Group B Streptococcus [GBS]). S. agalactiae is a member of the commensal microbiota of the human intestinal and genitourinary tracts and rarely causes infections in healthy adults. Infrequently it may cause morbidity in the elderly, pregnant women, and patients with underlying predisposing factors. Pregnancy-related maternal GBS colonization during birth is linked to newborn pneumonia, meningitis, and sepsis (Kaambo et al. 2018 ). Candida albicans is a widespread fungus that lives in human mucosa and a few other environmental reservoirs. The human mouth, vagina, and stomach colonization normally begins in infancy, mostly after vaginal delivery or breastfeeding. The vagina is the primary mucocutaneous surface affected by C. albicans , resulting in vulvovaginal candidiasis (VVC) (Lopes and Lionakis 2022). Vaginal microbiota transits among different CSTs during a woman's lifespan, being Lactobacillus -dominated, are more common among reproductive age and, particularly, in a pregnant woman. In addition, other lactobacilli may also be found, especially nomadic species like Lactiplantibacillus plantarum and Lacticaseibacillus casei group (Borges, Silva, and Teixeira 2014; Cribby, Taylor, and Reid 2008). A nomadic lifestyle is formally defined as a dynamic, generalist way of life of a species that involves both environmental and host niches, with no signs of specialization (Duar et al. 2017; Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, van Hijum, et al. 2016). Consequently, nomadic lactobacilli are found in contrasting environments, from vertebrates and invertebrate hosts to fermented vegetable and dairy products (Atassi et al. 2006 ; Ciocia et al. 2013 ; Kang et al. 2020 ; Khemariya et al. 2016 ; Muñoz-Quezada et al. 2013 ). These bacteria did not undergo a reductive evolution strategy and thus their large genomes encompass increased metabolic flexibility, which enables them to thrive in diverse environments (Duar et al. 2017; Inglin, Meile, and Stevens 2018; Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, van Hijum, et al. 2016). L. plantarum exemplifies the paradigm of the nomadic lifestyle and possesses one of the largest genomes among lactobacilli (3.3 Mbp). Strains isolated from different environments could reshape their phenotype expression towards a common direction under the same environmental condition (Acin-Albiac et al. 2021 ). In the same way, Lacticaseibacillus rhamnosus and Lact. casei genotypes and phenotypes do not correlate properly with their source of isolation (Broadbent et al. 2012 ; Cai et al. 2009 ; Ceapa et al. 2016 ; 2015 ). Due to their ability to thrive under contrasting conditions when compared to host-adapted lactobacilli, nomadic lactobacilli hold a huge potential as probiotics capable of positively modulating several aspects of human health (Fidanza, Panigrahi, and Kollmann 2021 ). Hence, the aim of this study is the selection of nomadic lactobacilli to be used as vaginal probiotics. A multistep approach was followed, where the first step encompassed a wise panel of assays for functional traits and pathogens inhibition using a custom scoring procedure to select the most promising strains. Then, eight candidate strains were further investigated for biofilm production. Subsequently, the two final promising candidate strains were included in a gel formulation prototype alone or as a binary combination. The ability of these prototypes to reduce the expression of the genes involved in the inflammatory cascade using the reconstituted human vaginal epithelium (HVE) infected with C. albicans was evaluated. Results Lactic acid isomers and hydrogen peroxide quantification The capacity of lactobacilli to produce lactic acid highly varied at species and strain levels (Fig. 1 A). The highest value of total lactic acid was found for L. plantarum POM1 (1.28 ± 0.21 g·L − 1 ), followed by L. plantarum C5 (1.17 ± 0.16 g·L − 1 ) and D3.15 (1.11 ± 0.06 g·L − 1 ). Within the L. pentosus species, E1.4 strain showed the highest total lactic acid yield (0.82 ± 0. 06 g·L − 1 ). Lact. rhamnosus B4.2 and B6.19 produced almost equal quantities (0.82 ± 0.03 and 0.78 ± 0.02 g·L − 1 , respectively). Overall, the production of total lactic acid from Lact. paracasei strains was quite low, with the highest amount produced from Lact. paracasei 93j (0.83 ± 0.09 g·L − 1 ). A significant difference (P < 0.005) was observed between L. pentosus and Lact. paracasei , and between Lact. paracasei and Lact. rhamnosus . Moreover L. plantarum and Lact. paracasei showed a significant difference with P < 0.00005. Further investigation of lactic acid production revealed different patterns of lactic acid isomer production (Fig. 1 B). Among L. plantarum strains, POM1 produced the highest amount of D-lactate (0.86 ± 0.12 g L − 1 ), followed by C5 (0.83 ± 0.17 g·L − 1 ), and P1 (0.74 ± 0.10 g L − 1 ). The production of D-lactate by L. pentosus species ranged from 0.48 ± 0.07 g L − 1 (strain E1.4) to 0.40 ± 0.20 g L − 1 (strain D2.15). The two Lact. rhamnosus strains showed an opposite behavior where Lact. rhamnosus B4.2 produced 0.42 ± 0.10 g L − 1 of D-lactate, whereas Lact. rhamnosus B6.19 produced 0.05 ± 0.01 g L − 1 . Almost no strains of Lact. paracasei produced D-lactate, with eight strains failing to produce both isomers. Lact. paracasei strains produced the highest levels of L-lactate, especially Lact. paracasei 84f (0.76 ± 0.09 g L − 1 ). The highest L-lactate yields came mostly from the Lact. paracasei group, and in the specific Lact. paracasei 84f showed the highest production (0.76 ± 0.09 g L − 1 ). After it came the L-lactate yields from L. plantarum strains, with the highest yield found for L. plantarum AFI (0.51 ± 0.21 g L − 1 ) and the lowest for L. plantarum CB5 (0.21 ± 0.02 g L − 1 ). Lact. rhamnosus B6.19 and B4.2 produced 0.73 ± 0.01 and 0.40 ± 0.07 g L − 1 of L-lactate, respectively. Within the L. pentosus species, the L-lactate highest yield was found for the E1.4 strain (0.34 ± 0.01 g L − 1 ), and the lowest for L. pentosus D2.15 (0.29 ± 0.02 g L − 1 ). H 2 O 2 production also showed high variability at species and strains level (Fig. 1 C). Overall, Lact. paracasei produced the highest amounts of H 2 O 2 ranging from 0.72 ± 1.02 (25h) to 22.44 ± 2.07 (ALII8) µM whereas Lact. paracasei 22e was the only one within this species unable to produce H 2 O 2 . No H 2 O 2 was produced from the strains belonging to L. pentosus species. Most of the L. plantarum examined did not produce H 2 O 2 , with L. plantarum D2.6 (6.07 ± 6.28 µM) having the highest production within the species. Lact. rhamnosus B4.2 and B6.19 strains produced 2.45 ± 3.20 and 1.59 ± 2.24 µM, respectively. Based on statistical analysis at the species level, a significant difference (P < 0.05) was observed between L. plantarum and Lact. paracasei , and between Lact. paracasei and Lact. rhamnosus . Auto-aggregation capacity and hydrophobicity characteristics The results from auto-aggregation and hydrophobicity assays were not species-dependent (Fig. 1 D ) . Approximately 60% of lactobacilli strains isolated from various sources and belonging to different species, showed an auto-aggregation capacity above the average value (63.71 ± 4.18%). The highest aggregation capacity was reached by L. plantarum D9.46 (81.77 ± 1.01%). A significant difference (P < 0.05) was observed between Lact. paracasei and Lact. rhamnosus species. The capability of the bacteria to potentially adhere to the epithelial cells was further investigated through the characterization of cell wall hydrophobicity (Fig. 1 D). L. plantarum S6w5 had the highest hydrophobicity capacity (88.40 ± 0.29%). All the other strains were almost equally distributed around the average value, showing a value lower (41.1% of all the strains) or higher (43.2% of all the strains) than the average (31.5 ± 8.7%) value. Only a few strains (15.8%), belonging to different species and isolated from different sources, did not show any hydrophobicity characteristics. A significant difference (P < 0.05) was observed between L. plantarum and Lact. paracasei . Pathogen growth inhibition screening Growth kinetics of S. agalactiae 88II3 and C. albicans UNIBZ54 were assessed in the presence of cell-free supernatants (CFSs) from all Lactobacillus strains and compared against the control conditions. The inhibitory activity was tested using CFSs at the original pH of ca. 4.0 (o-CFSs) or neutralized CFSs at pH 7 (n-CFSs), to exclude pH-dependent effects. S. agalactiae 88II3 grown at pH 7.0 as a control condition reached a maximum absorbance ( A ) of 0.62 ± 0.03, with the lag phase ( λ ) 1.34 ± 0.72 h, and growth rate ( µ ) 0.24 ± 0.02 h -1 . At pH ca. 4.0, lower A (0.47 ± 0.05), longer λ (8.67 ± 3.98 h), and smaller µ (0.04 ± 0.01 h -1 ) were reached. Three common patterns of inhibition of S. agalactiae 88II3 were observed with n-CFSs, while no common pattern of inhibition was observed with o-CFSs. n-CFSs from seventy lactobacilli strains caused a statistically significant (P < 0.05) reduction of the A value when compared to the control condition at pH 7.0. n-CFSs from Lact. paracasei HHI10 had the strongest repressive effect on S. agalactiae 88II3 in terms of A (0.50 ± 0.02). n-CFSs from 25 strains most of which belonged to L. plantarum species, did not inhibit S. agalactiae 88II3 in terms of A. Most of the n-CFSs were collected from Lact. paracasei significantly (P < 0.05) extended the lag phase ( λ ) of S. agalactiae 88II3, with the highest λ value detected when S. agalactiae 88II3 was grown with n-CFS from Lact. paracasei 104g (2.58 ± 0.02 h). When S. agalactiae 88II3 was cultured with n-CFSs from sixteen Lact. paracasei strains no lag phase was observed. Negative values of λ can be interpreted biologically as the pathogen starting to grow right after inoculation. Significant (P < 0.05) reduction of µ at pH 7.0 was found mostly with n-CFSs collected from Lact. paracasei strains, with the lowest (P < 0.05) value for Lact. paracasei F23 (0.20 ± 0.00). By monitoring the A parameter, complete inhibition of S. agalactiae 88II3 growth was found during culturing with o-CFSs from L. plantarum (36 strains), Lact. paracasei (20), L. pentosus (3), and Lact. rhamnosus (2). C. albicans UNIBZ54 cultured at pH 7.0 as a control condition reached a maximum A of 1.31 ± 0.02, with a λ of 6.86 ± 0.30 h, and µ of 0.21 ± 0.01 h -1 . At pH 4.0 as the control condition, C. albicans UNIBZ54 reached lower A (1.18 ± 0.08), longer λ (8.22 ± 1.26 h), and smaller µ (0.16 ± 0.02 h -1 ). Eight n-CFSs that were obtained from L. plantarum (1 strain) and Lact. paracasei (7 strains), led to a higher A parameter when compared to the control condition at pH 7.0, while 87 strains had a repressive effect (P < 0.05) on the A parameter. The n-CFS that was obtained from Lact. paracasei wI10 had the strongest inhibition effect in terms of A at pH 7.0 (1.13 ± 0.14). n-CFSs from 19 strains, mostly L. plantarum , extended the lag phase ( λ parameter), with the longest value reached with L. plantarum 1LS16 (7.68 ± 0.06 h). No significant (P > 0.05) reduction of the µ parameter of C. albicans UNIBZ54 was observed at pH 7.0. Lower significant inhibition of the growth curve of C. albicans UNIBZ54 was observed with o-CFSs under acidic condition (pH 4). Inhibition was obtained from only 12 o-CFSs collected from Lact. paracasei (5), L. plantarum (6), and L. pentosus (1). The lowest value of A was reached with o-CFSs from Lact. paracasei 41j (0.39 ± 0.17). Only five o-CFSs that were collected from 5 strains of L. pentosus (3) and Lact. paracasei (2) affected the λ value of C. albicans UNIBZ54. The highest value of λ was detected during incubation with o-CFS from L. plantarum D2.6 (9.33 ± 0.18 h). No significant inhibition on the µ parameter for C. albicans was attributable to the o-CFSs under acidic condition (pH ca. 4.0). To evaluate the differences among all the strains and the overall characteristics investigated, we carried out a principal component analysis (PCA) (Fig. 1 E). L. plantarum differ from Lact. paracasei in their functional and pathogen inhibition traits of interest in vaginal health. Furthermore, particularly interesting is the position of L. plantarum POM1 and C5 in the corner of the PCA. These two strains differentiate also from other standalone L. plantarum strains. Candidate selection approach and scoring procedure A scoring procedure was set for both the functional and the pathogen inhibition assays for the selection of the best-performing strains through the definition of a cumulative score (see Materials and Methods). Overall, the best performing assays for the selection were the D-lactic acid production and hydrophobicity characteristics, where a total of 45 CFSs showed a relative score of 1 or 2, meaning that the value is either between Q2 and Q3, or higher than Q3. For the H 2 O 2 production and auto-aggregation evaluation, a total of 44 CFSs showed a relative score equal to 1 or 2, and lastly total lactic acid production, where only 9 strains showed positive results (Table 1 ). Table 1 Single or double hit of inhibition . C. albicans S. agalactiae Single hit Double hit Single hit Double hit Species o-CFS (original acidic pH) n-CFSs (neutral pH) o-CFS (original acidic pH) n-CFSs (neutral pH) o-CFS (original acidic pH) n-CFSs (neutral pH) o-CFS (original acidic pH) n-CFSs (neutral pH) Lact. paracasei 5 42 0 1 20 4 0 45 L. pentosus 2 3 0 0 3 0 0 3 L. plantarum 6 23 0 16 36 18 0 4 Lact. rhamnosus 0 0 0 2 2 0 0 0 Grand Total 13 68 0 19 61 22 0 52 The total number of strains categorized per species whose cell-free supernatants at the original acidic pH (o-CFSs) and CFSs at neutral pH (n-CFSs) showed a single and double hit of inhibition on the growth of each pathogen. o-CFS and n-CFS had a significant effect on A or λ of C. albicans UNIBZ54, but never on its µ , thus resulting in a single hit of inhibition (Table 1 ). On the other side, some n-CFS had a significant effect on more than one growth parameter of S. agalactiae 88II3 at pH 7.0, giving a double hit of inhibition (Table 1 ). Moreover, some o-CFS had a remarkable effect on A of S. agalactiae 88II3 at acidic pH (pH ca. 4.0) (Table 1 ). Based on their pathogen inhibition and functional assays score (Table 2 ), eight best-performing Lactobacillus strains were selected: L. plantarum OE1, C5, D3.15, 1LS16, POM1, E3.8 and D2.6, and Lact. paracasei 41j. Table 2 Scoring overview for the first eight best-performing lactobacilli Specie Sub-source of isolation Code Pathogen inhibition scoring Functional scoring Cumulative scoring L. plantarum Carrot C5 5 8 13 L. plantarum Olives OE1 9 4 13 L. plantarum Sourdough E3.8 10 3 13 L. plantarum Sourdough D2.6 6 6 12 L. plantarum Sourdough D3.15 6 5 11 L. plantarum Tomato POM1 5 6 11 Lact. paracasei Milk 41j 6 5 11 L. plantarum Pineapple 1LS16 5 5 10 Biofilm imaging by confocal laser scanning microscopy Three-dimensional images of the biofilm colonies grown on membrane filters were collected through CLSM (confocal laser scanning microscopy) observations of the mature biofilms resulting from the 8 selected strains after 48 h of incubation. All biofilms appeared as widespread agglomerates of sessile cells (green fluorescence) embedded in exopolysaccharides (EPS) (red fluorescence) (Fig. 2 ). Biofilm architecture was strain-dependent, with a lower quantity of red fluorescence visible in the images captured from L. plantarum E3.8 and L. plantarum POM1. Biofilm growth and extracellular matrix characterization Biomass, cell density, and extracellular matrix (ECM) (protein, extracellular DNA (eDNA), and total saccharides) content from the mature biofilms of the eight selected strains formed after 48 h of incubation are shown in Table 3 . The highest biofilm formation in terms of biomass was achieved by L. plantarum POM1 (36.20 ± 12.02 mg), whereas L. plantarum C5 led to the lowest one (11.30 ± 1.70 mg). In terms of cell density and ECM protein content, there was no statistically significant difference (P > 0.05) among the strains. The eDNA concentration ranged between a minimum for Lact. paracasei 41j (0.59 ± 0.00 µg mg − 1 ) to a maximum for L. plantarum C5 (1.93 ± 0.00 µg mg − 1 ) (P < 0.05). The highest production of EPS was found in ECMs extracted from the biofilm of L. plantarum C5 (80.11 ± 0.02 µg mg − 1 ), being significantly (P < 0.05) higher than EPS extracted from L. plantarum D2.6 (35.00 ± 0.00 µg mg − 1 ), L. plantarum POM1 (26.11 ± 0.00 µg mg − 1 ), L. plantarum E3.8 (25.09 ± 0.01 µg mg − 1 ), L. plantarum 1LS16 (22.44 ± 0.00 µg mg − 1 ), and Lact. paracasei 41j (17.91 ± 0.02 µg mg − 1 ) (P < 0.05). Taking into consideration all these results, two strains, L. plantarum C5 and POM1 , were further investigated for more specific analysis to understand their behavior in the vaginal environment. Table 3 Biofilm characterization Strain Biomass (mg) Cell density (Log CFU mL − 1 ) eDNA (µg mg − 1 ) Proteins (µg mg − 1 ) EPS (µg mg − 1 ) L. plantarum C5 11.30 ± 1.70 b 9.22 ± 0.27 a 1.93 ± 0.38 a 6.87 ± 1.27 a 80.11 ± 16.98 a L. plantarum POM1 36.20 ± 12.02 a 10.13 ± 0.22 a 0.62 ± 0.10 ab 1.72 ± 0.36 a 26.11 ± 1.12 b L. plantarum 1LS16 34.70 ± 4.81 ab 9.28 ± 0.15 a 0.66 ± 0.24 ab 2.33 ± 0.06 a 22.44 ± 0.58 b L. plantarum D3.15 24.15 ± 5.59 ab 9.40 ± 0.24 a 1.03 ± 0.21 ab 3.50 ± 0.16 a 38.07 ± 5.00 ab L. plantarum OE1 16.35 ± 9.40 ab 9.54 ± 0.48 a 1.16 ± 0.22 ab 4.77 ± 0.03 a 44.11 ± 10.84 ab L. plantarum D2.6 30.85 ± 0.49 ab 9.56 ± 0.02 a 0.86 ± 0.34 ab 2.66 ± 0.02 a 35.00 ± 1.64 b L. plantarum E3.8 34.50 ± 1.70 ab 9.55 ± 0.35 a 0.75 ± 0.02 ab 2.45 ± 0.06 a 25.08 ± 4.79 b Lact. paracasei 41j 26.80 ± 4.81 ab 9.44 ± 0.04 a 0.59 ± 0.18 b 3.28 ± 0.39 a 17.91 ± 20.87 b Biomass (mg), cell density (Log CFU mL − 1 ), and ECMs (eDNA, proteins, and exopolysaccharide concentration, expressed as µg/mg of the total biomass) characterization of the newly formed biofilm, and reported as mean ± standard deviation. (a–b Means within the columns with different letters are significantly different (P < 0.05)). Evaluation of CFSs preservation of HVE metabolic activity after infection with C. albicans Compared to the negative control, C. albicans UNIBZ54 without the addition of CFS (positive control) sharply decreased the metabolic activity of the HVE (25.91 ± 0.78 %) according to the MTT assay ( Table 4 ). Table 4 Percentage of metabolic activity of Human Vaginal Epithelium model . Treatment Viability (%) Negative control 100.00 ± 0.16 a Positive control 25.91 ± 0.78 c L. plantarum C5 56.78 ± 11.09 b L. plantarum POM1 64.94 ± 5.24 b Combination ( L. plantarum C5 + POM1) 71.55 ± 9.85 b Negative control is the untreated epithelium, positive control is the epithelium infected with Candida albicans . L. plantarum C5, L. plantarum POM1, and C5 + POM1 are the epithelium models infected with the pathogen and then treated with the gel containing the respective strain or the combination of both. Data represent the mean ± standard deviation. (a–c Means with different letters are significantly different (P < 0.05)). Generally, both L. plantarum C5 and POM1 strains were able to prevent HVE metabolic activity loss either when cultured in single or in combination. In detail, the metabolic activity of the infected HVE treated with a gel containing L. plantarum C5 and L. plantarum POM1 reached 56.78 ± 11.09% and 64.94 ± 5.24%, respectively, with a statistically (P < 0.05) significant difference from the viability of cells of positive control. The combination of the two strains showed a metabolic activity of 71.55 ± 9.85%, demonstrating a synergistic effect of nomadic lactobacilli, still significantly different (P < 0.05) than the positive control. Gene expression profiling HVEs showed a significant decrease (P < 0.05) in interleukin 8 (IL-8) expression, compared to the positive control when HVE models were treated with gels containing the single lactobacilli strains. In addition, a synergistic effect was found when HVEs were treated with the gel containing both lactobacilli strains, also showing a significant difference from the control. A positive effect of the treatment with the gels containing the lactobacilli was visible also from the analysis of the E-cadherin expression. The E-cadherin expression, which sharply decreased in the HVEs infected with C. albicans UNIBZ54, increased when the epithelium model was treated with a gel containing lactobacilli, both in single and in combination (P < 0.05). The results of the gene expression analyses of HVE infected with C. albicans UNIBZ54 and treated with a gel containing selected lactobacilli were shown in Fig. 3 . The negative control represents the gene expression in the untreated epithelium model, and it is standardized as equal to 1. Under-expression or over-expression of the genes are represented by lower or higher values, respectively. The blank sample is the epithelium model treated with blank gel, positive control is the epithelium infected with C. albicans , while the last three are epithelium models infected with the pathogen and treated with the gel containing the respective strains ( L. plantarum C5 or L. plantarum POM1), or combination of the two (C5 + POM1). Discussion A healthy human vaginal microbiome is dominated by homofermentative Lactobacillus spp., which reflects vaginal community states (De Seta et al. 2019; Vaneechoutte 2017 ). The choice of optimum probiotic strains for therapeutic use is crucial since their use can lessen the possibility of the rapid growth of non-native lactobacilli in the urogenital tract in the vaginal environment (He et al. 2020 ). The lactobacilli group is the major source of probiotic strains because of their professed benefits on human health (Vitali et al. 2012 ), and because many of them have been granted the status of qualified presumption of safety (QPS) by the European food safety authority (EFSA) (EFSA Panel on Biological Hazards - BIOHAZ, 2013). Until now, many commercial vaginal probiotics have been focusing on using exogenous lactobacilli neglecting the fact of their lifestyle and inherent ability to thrive in contrasting environments (van de Wijgert and Verwijs 2020). However, nomadic lactobacilli ( Lactiplantibacillus spp. and Lacticaseibacillus spp.) may also thrive and colonize successfully vaginal niches (Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, Hijum, et al. 2016). Moreover, their upstream processing scalability for product development is more feasible when compared to native vaginal lactobacilli, such as Lb. crispatus (Fidanza, Panigrahi, and Kollmann 2021 ). The vaginal epithelial barrier is shielded from pathogen colonization and invasion by lactobacilli, which produce antibacterial chemicals including lactic acid and hydrogen peroxide, stick to vaginal epithelial cells to form a protective film, and block pathogen adhesion (He et al. 2020 ). In this study, we screened 95 nomadic lactobacilli isolated from different sources using a multistep approach, for desirable metabolic traits to maintain and restore vaginal health. L. plantarum POM1 isolated from tomatoes was the highest producer of lactic acid and D-lactate. Lact. paracasei spp. produced significantly lower amounts of lactic acid. This might be because Lact. paracasei spp. only synthesizes L- lactic acid isomer since it lacks the gene coding for D-lactate dehydrogenase (Liu et al., 2021). The D-lactate production within the vaginal microbiome suppresses the vaginal extracellular matrix metalloproteinase inducer, adding another level of protection against upper genital tract infections (Witkin et al. 2013 ; Tozetto-Mendoza et al. 2020 ). Analogously, most of the native vaginal lactobacilli ( Lb. gasseri, Lb. crispatus , and Lb. jensenii ) produce both isomers of lactic acid. Therefore, we favored total and D-lactic acid producers in our scoring procedure. The highest production of H 2 O 2 , a key factor in maintaining the balance of a healthy vaginal environment, was observed for Lact. paracasei spp., all isolated from dairy, fruits, and vegetables. Auto-aggregation is suggested to be necessary for the adhesion of probiotic microorganisms to the intestinal epithelium. Both auto-aggregation and hydrophobicity characteristics can be considered a pre-test for selecting probiotic strains which potentially adhere to epithelial cells (Krausova et al., 2019). L. plantarum D9.46 showed the highest auto-aggregation capacity, while L. plantarum DM was the only strain showing none. No common characteristics among the strains were observed for both auto-aggregation and hydrophobicity. S. agalactiae (GBS) causes an important life-threatening infection in infants, which is transmitted during pregnancy (Rosen et al. 2017 ; Kaambo et al. 2018 ). On the other hand, C. albicans is an opportunistic pathogen, which can overgrow in the vaginal ecosystem causing VVC (Mendling 2016 ). Given the importance and prevalence of these vaginal pathogens, we assessed the capacity of nomadic lactobacilli CFSs to inhibit them. To exclude pH-dependent inhibition we also included neutralized CFSs. The pathogens' growth inhibition differed substantially across the lactobacilli and pathogens examined (Osset, Bartolome, and Garcı 2014). Our findings suggest that the studied Lactobacillus strains showed inter-strain differences in anti- Candida activity. Lactobacillus strains that produce the highest quantities of lactic acid have the strongest antagonistic effect on GBS (Marziali et al. 2019 ). These findings imply that S. agalactiae is sensitive to organic acids and that individual lactobacilli's capacity to acidify is critical for anti-GBS action. Microecological preparations, when administered orally or vaginally, have been shown in numerous clinical studies to significantly lower incidence and recurrence rates, prolong the time between recurrences, increase recovery rates, relieve symptoms, and enhance the vaginal microecological patterns of BV and VVC (He et al., 2020 ). More in general, some lactobacilli adhere to the vaginal epithelial cells, while others can prevent uropathogens attachment to these cells and suppress uropathogens’ growth. Indeed, certain Lactobacillus strains can restrict the growth of vaginal pathogens and excrete chemicals that prevent them from multiplying, which are two crucial phases in the pathogenesis of urinary infections (Osset, Bartolome, and Garcı 2014). Biofilm formation by lactobacilli is responsible for their stable maintenance of a stable ecosystem, as it grants long-term permanence to the host’s vaginal mucosa (Ventolini, Mitchell, and Salazar 2015 ). The accessory genome of Lb. crispatus contains genomic islands that encode enzymes involved in EPS biosynthesis (Ojala et al. 2014 ). In fact, this species is associated with the healthiest vaginal community state (De Seta et al. 2019). Hence, we determined the relative composition of protein, eDNA, and total saccharides biofilm components. As a result, all the eight previously selected strains were able to form biofilms with distinct macrostructure and composition, which highly diverged between L. plantarum strains and Lact. paracasei 41j. The main component of ECM is EPS, for all the eight strains, with significant differences (P < 0.05) observed among the strains. We further selected L. plantarum POM1 and C5 due to their differentiation from other L. plantarum strains (Fig. 1 E), and also due to their high biomass production and quantity of EPS (Table 3 ), respectively, for further investigation. Topical gel formulations containing the two selected strains, in single and in combination, were formulated as a prototype method to be applied to the vaginal surface, using the HVE in vitro model previously infected with C. albicans UNIBZ54. C. albicans endocytosis has been linked to the proteolytic breakdown of E-cadherin which is the major protein in vaginal epithelial cell junctions (Naglik et al. 2011 ). Compared to negative control, infection with C. albicans UNIBZ54 decreased the HVEs cell viability up to 25%, while significant (P < 0.05) higher metabolic activity was found when treating HVEs with the topical gel. HVE treated with the gel showed an improved cell viability compared to the untreated one, due to an antagonistic effect of lactobacilli against C. albicans UNIBZ54, as already demonstrated by Allonsius et al. ( 2017 ). The positive role of the two selected strains was confirmed by the expression analysis of genes encoding for anti-inflammatory cytokines in HVEs. Common vaginal pathogens, including C. albicans have been previously associated with elevated expression of IL-8, a proinflammatory chemokine capable of attracting neutrophils to sites of infection (Agace et al. 1993 ; Spear et al. 2008 ). IL-8 gene was overexpressed in cells infected with C. albicans UNIBZ54, while the cells treated with the gels containing lactobacilli showed a statistically significant decrease in IL-8 gene expression. A synergistic effect was observed when the cells were treated with the gel containing the combination of the two L. plantarum strains. On the other hand, the treatment with the gels containing the two L. plantarum strains, both in single and in combination, prevented the reduction of E-cadherin gene expression normally caused by the pathogen. Conclusions To summarize, we highlighted the potential of nomadic lactobacilli isolated from different foods, animal, and human sources as suggested probiotic strains for the vaginal environment, thanks to their functional properties and their inhibitory activity against vaginal pathogens ( C. albicans UNIBZ54 and S. agalactiae 88II3). This investigation was carried out to find the isolates with most of the qualities required for Lactobacillus to operate as biotherapeutic microorganisms. By using a cumulative scoring-based approach, the pool of potential probiotic candidates was narrowed down. In the end, the ability to form biofilms led to the selection of two L. plantarum strains. We provided in vitro evidence on the use of a topic gel containing the two selected potential probiotic strains that can reduce the inflammation caused by C. albicans UNIBZ54 infection in the HVE model, either alone or in combination, and thus appear to be promising probiotic strains for vaginal health. Nonetheless, comprehensive clinical investigations will be required to confirm their actual therapeutic advantages. Methods Bacterial cultures and growth conditions For this study, a total of 95 lactobacilli were investigated; 48 strains were isolated from dairy products, 31 from fruits and vegetables, 15 from sourdough, and 1 from other sources (Table 5 ). These strains, together with the pathogenic strain S. agalactiae 88II3 and C. albicans UNIBZ54, belong to the Micro4Food collection from the University of Bolzano-Bozen. All the cultures were maintained as frozen stocks at -20°C in their specific broth medium with 20% glycerol for subsequent analysis. Before their use, the lactobacilli were propagated twice in MRS broth (Sigma Aldrich) at 37°C (body temperature) for 24 h. C. albicans UNIBZ54 was refreshed in Sabouraud broth (Scharlau, Spain) with 5% Tween ® 80 (Sigma Aldrich) and incubated overnight at 37°C. S. agalactiae 88II3 was refreshed in Brain-Heart Infusion (BHI) broth and incubated at 37°C until it reached the stationary phase (ca. 24 h). Table 5 Lactobacilli strains (n = 95) used in this study . Number of strains Species Source and sub-source of isolation 50 Lacticaseibacillus paracasei 31a, 25h, 100i, 104g, 22e, 25e, 25g, 28g, 41j, 45j, 50a, 52i, 76d, 83e, 84f, 93j, 99a, AAI9, AII8, BBII10, bI5, dII1, eI3, GII3, HHI10, iiII4, iiII9, JJI8, kI12, LII1, MMII7, wI10, WWI9, zzI10, zzI3, zzI4, zzI6 Dairy (Milk) AFI10, AFI7, AFII5, ALII8 Fruits and Vegetables (Apple-by-products) S4d8 Fruits and Vegetables (Sauerkraut) F1, F10, F12, F13, F2, F23, F25, F5 Dairy (Cheese) 3 Lactiplantibacillus pentosus D2.15, E1.4, E3.10 Sourdough 40 Lactiplantibacillus plantarum P1, CB5 Dairy (Cheese) 11j Dairy (Milk) C5 Fruits and Vegetables (Carrot) CIL6 Fruits and Vegetables (Cherry) Fin6, Fin10 Fruits and Vegetables (Fennel) IT1, IT5 Fruits and Vegetables (Grape) K1, K13, K2, K9, KI-5 Fruits and Vegetables (Kiwi) OE1 Fruits and Vegetables (Olives) P3 Fruits and Vegetables (Papaya) 1LS16 Fruits and Vegetables (Pineapple) PR14, PR3, PR6 Fruits and Vegetables (Prune) S6w5, AFI5 Fruits and Vegetables (Sauerkraut) POM1, POM20, POM27, POM35, POM42, POM43, POM40 Fruits and Vegetables (Tomato) DM Other E3.13, E3.19, D9.30, D9.40, D9.46, D3.15, C5.10, D9.18, E3.8, D2.6 Sourdough 2 Lacticaseibacillus rhamnosus B6.19, B4.2 Sourdough Cell-free supernatants collection Lactobacillus strains were grown in MRS broth for 24 h at 37°C, and then the CFSs were recovered by centrifugation (7,500 rpm, 10 min). Further, CFSs were fractionated in two aliquots. One aliquot was neutralized at pH 7.0 (n-CFSs) with NaOH and sterilized by using a 0.22 µm filter. The other aliquot was preserved at the original acidic pH of ca. 4.0 (o-CFSs) and was further divided into two aliquots: one was sterilized by using a 0.22 µm filter, and one was kept not sterile. All the aliquots of CFSs were stored at -20°C until needed. Sterile CFSs were used for pathogen inhibition screening, while non-sterile CFSs were used for functional assays (H 2 O 2 and lactic acid quantification). Lactic acid isomers and hydrogen peroxide quantification The quantification of lactic acid and H 2 O 2 in the CFSs was done using two commercial kits, respectively the Megazyme D-Lactate and L-Lactate Assay Kit (Megazyme International Ireland Ltd., Wicklow) and the Peroxide assay kit (Sigma Aldrich), following the instructions provided by the manufacturers. Auto-aggregation capacity and hydrophobicity characterization The candidate strains were grown in MRS at 37°C for 18–22 hours. The pellets were harvested by centrifugation (10,000 g, 10 mins, 5°C), washed with PBS (phosphate buffer solution) (pH 7.0), and re-suspended in the same buffer. Cell suspensions were adjusted to an optical density (OD) of 620 nm of ca. 0.25 and used for both analyses ( A 0 and H 0 ). Cell auto-aggregation was performed according to Gil-Rodríguez, Carrascosa, and Requena (2015) as modified by Di Cagno et al. ( 2020 ). Each cell suspension was left to settle at room temperature, then the OD 620 was measured after 2, 4, and 24 h (A t ). The percentage of auto-aggregation ( A ) was calculated according to the following formulae: Estimation of hydrophobicity was performed according to Burns et al. ( 2008 ) as modified by Di Cagno et al. ( 2020 ). Xylene was used to determine the hydrophobicity of the cell surface. A total volume of 0.4 mL of xylene (Sigma Aldrich) was added to the cell suspension (2 mL), and vortexed for 120s. After phase stabilization and separation (1 h, 37°C), the OD of the aqueous phase was measured at 620 nm (Ht). The percentage of hydrophobicity ( H ) was calculated according to the following formulae: High-throughput pathogen inhibition screening Growth inhibitions of C. albicans UNIBZ54 and S. agalactiae 88II3 by CFSs, previously collected and stored, were screened. Briefly, overnight cultures of the pathogens were washed twice with saline solution (0.9% NaCl) and resuspended in saline solution to a final OD 600 of 0.1 for C. albicans UNIBZ54, or OD 620 of 0.25 for S. agalactiae 88II3. The 96-well plates were set using one volume of CFSs and three volumes of each pathogen. The analysis was done in triplicate using CFSs previously neutralized (pH 7.0) and the acidic CFSs (pH ca. 4.0). Control wells were prepared using MRS medium at pH 4.0, and MRS at pH 7.0, and run in triplicates. Control MRS was acidified using a racemic solution of lactic acid to pH 4.0 and adjusted back to pH 7.0 using NaOH 1M to exclude the osmolyte effect. Growth kinetics of the pathogens were recorded for 46 h at 37°C measuring the absorbance (at a wavelength of 600 nm for C. albicans UNIBZ54, and 620 nm for S. agalactiae 88II3) every 15 min with the Infinite ® M Nano + Spectrophotometer (TECAN, Austria). Candidate selection approach and scoring procedure The best-performing lactobacilli were selected through a scoring procedure based on the results of the previously mentioned tests and considering the measure of central tendency along with quartiles. Strains were considered “positive” if the value of each strain was higher than the third quartile (e.g., a value ≥ 25% of the highest values in the dataset for one assay). $$Score= \frac{\sum positive}{total number of essays} \times 100$$ Strains were given a score according to their relative performance on each functional assay: score 1 if Q2 Q3 and score 2 if X > Q3. Double hits (DH = 1 point) of inhibition by the n-CFSs against S. agalactiae 88II3 (SA) or C. albicans UNIBZ54 (CA) corresponded to a significant effect (P < 0.05) towards a given pathogen detectable for two or more growth parameters. Strains whose n-CFSs at pH 7.0 showed a double hit (DH) inhibition and o-CFSs that showed a single inhibition effect (H) on A at pH 4.0 were scored according to the following calculation: $$Inhibition Score =\left(4\times {DH}_{CA}+ {DH}_{SA}\right)+({4 \times H}_{CA}+ {H}_{SA})$$ Double hit (DH) or hit (H) on A for C. albicans UNIBZ54 were multiplied by 4 since the frequency of a double hit of inhibition on C. albicans UNIBZ54 was ca. 4 times lower than the one on S. agalactiae 88II3. Biofilm preparation Overnight planktonic cultures of the lactobacilli were centrifuged (7,500 rpm, 10 min, 4°C) and the pellets from each strain were collected and washed twice in saline solution. The suspensions were diluted to an OD 620 of ca. 0.25 with saline solution. One 5-µL drop of diluted culture was used to inoculate individual sterile membrane filters (pore size, 0.22 µm, Whatman) resting on MRS agar Petri dishes. The membranes were sterilized by UV exposure (15 min per side) before inoculation. The plates were inverted after the inoculum and incubated at 37°C, with the membrane-supported biofilms transferred to a fresh MRS agar Petri dish every 8 to 10 h (Anderl, Franklin, and Stewart 2000 ). Five membranes were prepared for each strain. Biofilm imaging by confocal laser scanning microscopy One membrane-supported biofilm for each strain culture was visually inspected and photographed by CLSM (Leica SP8LIA, Leica Microsystems). The membrane-supported biofilms were carefully mounted on glass slides. Bacterial cells and the polysaccharide fraction of ECM were stained with 15 µM SYTO® 9 (Invitrogen) and 200 µg mL − 1 Texas Red®-labeled Concanavalin A (ConA, Invitrogen, stock solution, 5 mg mL − 1 in 0.1 M sodium bicarbonate) solution in PBS (pH 7.5). Samples were incubated under dark conditions for 1 h at room temperature. Then, biofilm images were collected with a CLSM with excitation at 488 nm and emission > 552 nm lasers. Fluorescence emission was observed between 500–565 nm (for SYTO® 9) and 565–645 nm (for ConA). Images were captured with a 40x lens using immersion oil and analyzed with the software LAS X (Leica). Biofilm growth and ECM characterization At the end of the incubation, two membrane-supported biofilms for each strain were inserted in separate falcon tubes containing saline solution (9 mL) to quantify the biomass (mg). Cells were then detached and suspended through the vortex (1 min at maximum speed) and serially diluted. Dilutions were plated on MRS agar Petri dishes and the cell density (Log colony forming unit [CFU] mL − 1 ) was enumerated after 48 h of incubation at 37°C. The ECM components of the biofilm colonies were collected and characterized following the method described by Chiba et al. ( 2015 ). Briefly, the biofilm colonies were scraped from the membrane filters and suspended in NaCl solution (1.5 M). The suspensions were centrifuged (5,000 g, 10 min, 25°C) and the supernatants were collected as ECM fractions for the quantification of proteins, total saccharides, and eDNA. The concentration of protein was measured following the Bradford assay (Bradford, 1976), using bovine serum albumin (BSA) as a standard. The protein concentration was measured at 590 nm with the UV-1800 Spectrophotometer (SHIMADZU). The total saccharide concentration in the ECMs was measured by the phenol sulfuric acid method (Chiba et al. 2015 ), using glucose as a standard. Briefly, the isolated ECM fractions (20 µL) were mixed with 5% phenol (20 µL) in a 96-well plate, before adding sulfuric acid (100 µL). The plate was then incubated for 10 min at room temperature, and then the absorbance was read at 492 nm with an Infinite® M Nano + Spectrophotometer (TECAN, Austria). The concentration of the eDNA in the ECM fractions was measured with NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Gel formulation From the results of the second screening, L. plantarum C5 and POM1 were selected to be inserted in gel formulations that were developed in collaboration with the R&D Innovation center of Giuliani S.p.A. (Milan, Italy). The cell densities of overnight cultures of the selected lactobacilli were calculated by measuring the OD 620 using the 6715 UV/VIS Spectrophotometer (Jenway, UK). A final pellet of ca. 11 Log CFU mL -1 was collected, then resuspended in 10 mL of vegetal glycerol (Acef s.p.a., Italy), to a final cell density of ca. 10 Log CFU mL -1 . The glycerol suspension containing lactobacilli was then inserted in an aqueous-based gel formulation. The final formulation contained deionized water (77%), vegetal glycerol suspension (20%) (Solagum tara, Seppic, France), containing lactobacilli (2%), and EUXYL K712 (1%), a liquid cosmetic preservative, containing sodium benzoate and potassium sorbate. The final cell density of the lactobacilli in the probiotic gel was about 9 Log CFU mL -1 . The viability of the lactobacilli in the gel was assessed by preparing serial dilutions and by plating these on MRS agar medium before any treatment. A total of four gels were prepared, one for each strain previously selected, L. plantarum C5 and POM1, one with the combination of the two strains, and one with no cell suspensions. Human Vaginal Epithelium infection and treatment The SkinEthic ™ HVE model was obtained from EpiSkin (Lyon, France). The HVE model is based on the vulvar epidermoid carcinoma cell line A431 cells which form a 3-D tissue like the human in vivo vaginal mucosa when cultivated in vitro on a polycarbonate filter in a chemically defined medium (de Brugerolle 2007 ). After the arrival of the HVEs, the inserts were placed in a 24-well plate, containing maintenance medium (SkinEthic, Episkin) (1 mL), and re-equilibrated for 24 h at 37°C, in a humidified, 5% carbon dioxide (CO 2 ) atmosphere. The HVEs were inoculated with C. albicans UNIBZ54 previously adjusted to an OD 600 ca. 6 Log CFU mL -1 using a maintenance medium (SkinEthic, Episkin). The HVEs were previously incubated with C. albicans UNIBZ54 (30 µL) in a humidified, 5% CO 2 atmosphere, at 37°C for 24 h, to allow the pathogen adhesion and infection. At the end of the incubation period, the HVEs were treated with the gel formula previously prepared (within 24 h from the moment of preparation): two gel formulations containing the single strains of L. plantarum C5 and POM1, one gel containing the combination of these two strains, and a gel not containing any strain. Also, some HVEs were left untreated, while HVEs infected with C. albicans UNIBZ54 were used as a positive control. After the analysis, the inserts were all incubated for the same time and in the same conditions previously described. The MTT assay for cell viability determination The cell viability of HVEs after infection with C. albicans UNIBZ54 and treatment with the gels were determined by the MTT assay (Grela, Kozłowska, and Grabowiecka 2018). Briefly, the yellow water-soluble salt is reduced by mitochondrial dehydrogenases to purple water-insoluble formazan, according to the viability of cells. A final formazan extraction step is required, using an organic solvent (e.g., isopropanol). A stock solution of MTT (5 mg mL -1 in PBS) was diluted (1:10) in the cell culture medium to prepare the MTT solution. The MTT assay was assessed using untreated HVEs (negative control), HVEs treated with blank gel (blank), HVEs infected with C. albicans UNIBZ54 (positive control), and HVEs infected with C. albicans UNIBZ54 and treated separately with L. plantarum C5 and POM1, and with the gel containing a combination of these two strains. After 24 h of incubation, the inserts were rinsed with PBS and placed in a new 12-well plate containing MTT solution (300 µL). After 2 h of incubation in a humidified, 5% CO 2 atmosphere, at 37°C, isopropanol (800 µL) was added to each well. The plate was incubated again for 1.5 h, in the same conditions above mentioned. Two aliquots (200 µL) were then taken from each well and placed in 96-well plates. The OD 570 was measured with a BioTek Micro-volume Plate Reader (BioTek Instruments Inc., Bad Friedrichshall, Germany) and elaborated with the ELX808 software (BioTek Instruments Inc., Bad Friedrichshall, Germany) (reference filter: 630 nm). Results were expressed as a percentage of viability compared to the negative control (mean ± standard error of triplicate cultures), using the following formula: viability (%) = [OD (570 nm – 630 nm) test product / OD (570 nm – 630 nm) negative control] x 100 Gene expression profiling The total RNA was extracted from the HVEs, previously infected with C. albicans UNIBZ54 and treated with the gels, using the RNeasy mini kit (Qiagen, Valencia, CA, USA), according to De Vuyst ( 2014 ). Briefly, the circumference of the polycarbonate filter was dissected from the bottom of the insert using a sharp surgical blade and then transferred into a 12-well culture plate containing RLT buffer (600 µL). After 1 or 2 min, the stratum corneum, detached from the epidermis, was removed using a pair of tweezers and discarded. For disrupting keratinocytes, the epidermis was gently scratched with a micropipette tip in the lysis buffer, provided by the kit. The lysate was homogenized by pipetting and then transferred into a spin column placed in a 2.0 mL collection tube. This procedure allows the recovery of enough RNA from the HVEs for real-time (RT) polymerase chain reaction (PCR) analysis of gene expression. Following the instructions provided by the manufacturer, 2 µg of RNA templates were used to synthesize complementary DNA (cDNA) in a 20 µL reaction volume, using the PrimeScriptTM RT Reagent Kit (TakaraBioInc., Japan). The cDNA was amplified and detected by the Stratagene Mx3000P RT-PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). PCR conditions were the following: 37°C for 15 min, 85°C for 5 sec, and 25°C for 2 min. Afterward, the TaqMan® Gene Expression Assays were carried out for RT-PCR using the following genes: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1, IL-8 Hs00174103_m1, the cadherin 1 (CDH1) Hs01023894_m1. The GAPDH was used as a housekeeping gene. PCR amplifications were carried out in 20 µL of total volume. The mixture of reaction contained 10 µL of 2X Premix Ex Taq (Takara, Japan), 1 µL of 20× TaqMan Gene Expression assay, 0.4 µL of RoX Reference Dye II (Takara, Japan), 4.6 µL of water, and 4 µL of cDNA. Data and statistical analysis All the analyses were performed considering three biological replicates analyzed in triplicate. The growth parameters of each pathogen were determined using the grofit R package (Kahm et al. 2010 ) for each replicate of CFSs and controls. Growth parameters were determined using the free splines approach and bootstrapping with 100 resamplings. The inhibition effect was assessed through a non-parametric one-way Kruskal-Wallis followed by the Dunn Control post hoc test provided by the PMCMR R package (Pohlert, 2016). The resulting p-values were adjusted for multiple hypothesis testing using Benjamini and Hochberg false discovery rate correction (FDR). Data from the determination of biofilm formation were submitted for analysis of variance by the General Linear Model (GLM) of R statistical package (R, version 1.6.2 rcompanion.org/handbook/). Multi-comparison of treatment means was achieved by a Tukey-adjusted comparison procedure with a p-value < 0.05 (Mangiafico 2016 ). For the gene expression profiling, the average value of the target gene was normalized using the GAPDH gene, and the relative quantification of the levels of gene expression was determined by comparing the Δ cycle threshold (ΔC t ) value (Vigetti et al. 2008 ). The statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc). Data are expressed as the mean, mean ± standard error of the mean (SEM), or mean fold change ± SEM. p-values < 0.05 were considered as statistically significant. Abbreviations BHI Brain Heart Infusion BSA Bovine serum albumin BV Bacterial vaginosis CDH1 cadherin 1 cDNA Complementary DNA CFS Cell-free supernatant CFU Colony Forming Unit CLSM Confocal laser scanning microscopy CO 2 Carbon Dioxide ConA Concanavalin A CST Community state type ECM Extracellular matrix eDNA Extracellular DNA EFSA European Food Safety Authority EPS exopolysaccharides FDA Food and drug administration FDR False discovery rate GBS Group B Streptococcus GAPDH glyceraldehyde-3-phosphate dehydrogenase GRAS Generally recognized as safe H 2 O 2 Hydrogen peroxide HVE Human vaginal epithelium IL-8 Interleukin 8 MRS De-Man-Rogosa-Sharpe MTT 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide OD Optical density PCR Polymerase Chain Reaction PBS Phosphate buffer solution QPS Qualified Presumption of Safety RT Real-Time SEM Standard error of mean VVC vulvovaginal candidiasis Declarations Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the Open Access Publishing Fund of the Free University of Bozen-Bolzano. Author’s contributions RDC, MG, and FR conceived the idea. MAA designed the experiments. RDC and MG were the supervisors and coordinators of the research units. MAA, CC, DP, and AP carried out the experiments. MAA and CC analyzed the data and wrote the original manuscript draft. RDC and PF reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript. References Acin-Albiac, Marta, Pasquale Filannino, Rossana Coda, Carlo G. Rizzello, Marco Gobbetti, and Raffaella Di Cagno. 2021. ‘How Water-Soluble Saccharides Drives the Metabolism of Lactic Acid Bacteria during Fermentation of Brewers´ Spent Grain’. Microbial Biotechnology . 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Vitali, Beatrice, Giovanna Minervini, Carlo Giuseppe Rizzello, Enzo Spisni, Simone Maccaferri, Patrizia Brigidi, Marco Gobbetti, and Raffaella Di Cagno. 2012. ‘Novel Probiotic Candidates for Humans Isolated from Raw Fruits and Vegetables’. Food Microbiology 31 (1): 116–25. https://doi.org/10.1016/j.fm.2011.12.027. Wijgert, Jhhm van de, and Mc Verwijs. 2020. ‘Lactobacilli-Containing Vaginal Probiotics to Cure or Prevent Bacterial or Fungal Vaginal Dysbiosis: A Systematic Review and Recommendations for Future Trial Designs’. BJOG: An International Journal of Obstetrics & Gynaecology 127 (2): 287–99. https://doi.org/10.1111/1471-0528.15870. Witkin et al. 2013. ‘Influence of Vaginal Bacteria and D- and l-Lactic Acid Isomers on Vaginal Extracellular Matrix Metalloproteinase Inducer: Implications for Protection against Upper Genital Tract Infections’. MBio 4 (4). https://doi.org/10.1128/mBio.00460-13. Additional Declarations No competing interests reported. Supplementary Files Table4.pdf Table2.pdf Table3.pdf Table1.pdf Table5.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Feb, 2023 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Major revision 17 Dec, 2022 Reviews received at journal 05 Nov, 2022 Reviewers agreed at journal 28 Oct, 2022 Reviewers invited by journal 27 Oct, 2022 Editor assigned by journal 27 Oct, 2022 Submission checks completed at journal 27 Oct, 2022 First submitted to journal 25 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2201461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":147540514,"identity":"241e3536-a924-4355-bc8d-4aefb79aba01","order_by":0,"name":"Claudia Cappello","email":"","orcid":"","institution":"Free University of Bolzano","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Cappello","suffix":""},{"id":147540516,"identity":"5af6da89-56ed-4df4-a4b1-7a8bdae1e043","order_by":1,"name":"Marta Acin-Albiac","email":"","orcid":"","institution":"Free University of Bolzano","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Acin-Albiac","suffix":""},{"id":147540517,"identity":"46892508-a648-4976-aa63-9aa7223d429a","order_by":2,"name":"Daniela Pinto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDCCA4wNQNKGgY2BueEAA4MEgwFRWg4wpAG1MBKtBYwPA0mwdQyEtfDdbm57/KHivF2fRGLjgZ87LPLM2c8+3fCDwU5OtwG7Fsk7B9sNDpy5ndwmkdhwsPeMRLFlT7rZzR6GZGOzA9i1GNxIbJM42HY7mQ2o5QAvUOOGA2lsN3gYDiRuw6/lHFjLwb8gLeefsd38Q1jLATuQlsNgW26ksd3GZwvQL20SZ84kJ7DxPGw4LNsmUWxw4xnbbRkD3H7hu93+TKKiws5evj358Me3bXV5BufT2G6+qbCTw6UFGHdgkNgA5SdAHYxDOZIWexg/AY/aUTAKRsEoGKEAAOMpa2gAtjPJAAAAAElFTkSuQmCC","orcid":"","institution":"Human Microbiome Advanced Project","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Pinto","suffix":""},{"id":147540520,"identity":"aab509fc-85e6-42ab-9b51-acdb19848cfd","order_by":3,"name":"Andrea Polo","email":"","orcid":"","institution":"Free University of Bolzano","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Polo","suffix":""},{"id":147540522,"identity":"b7e449d6-c345-4f2d-be15-c031fbfdcfd8","order_by":4,"name":"Pasquale Filannino","email":"","orcid":"","institution":"University of Bari Aldo Moro","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pasquale","middleName":"","lastName":"Filannino","suffix":""},{"id":147540525,"identity":"5e38d482-2054-4ce5-a260-4240a81ff8f8","order_by":5,"name":"Fabio Rinaldi","email":"","orcid":"","institution":"Human Microbiome Advanced Project","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fabio","middleName":"","lastName":"Rinaldi","suffix":""},{"id":147540527,"identity":"f2eaf27e-d55a-4148-acd1-468067f6ed7a","order_by":6,"name":"Marco Gobbetti","email":"","orcid":"","institution":"Free University of Bolzano","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Gobbetti","suffix":""},{"id":147540530,"identity":"6db04dbf-8fed-4ec4-9e43-f914dbeef6dc","order_by":7,"name":"Raffaella Di Cagno","email":"","orcid":"","institution":"Free University of Bolzano","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Raffaella","middleName":"Di","lastName":"Cagno","suffix":""}],"badges":[],"createdAt":"2022-10-25 08:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2201461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2201461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-023-02030-4","type":"published","date":"2023-02-11T18:43:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28532136,"identity":"e6884228-2ed0-4264-b4ef-5e14a57cbe35","added_by":"auto","created_at":"2022-11-01 19:35:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":769987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional assays results. \u003c/strong\u003eFunctional assays profiling, categorized by strain, of total lactic acid (Panel A), lactic acid isomer (Panel B), peroxide production (Panel C), and auto-aggregation and hydrophobicity (Panel D) characterization of nomadic lactobacilli strains after 18-24 h at 37 ºC of incubation in MRS. Principal component analysis of data gathered during the first screening\u003cstrong\u003e \u003c/strong\u003e(Panel E). Dunn test significant statistical differences after FDR correction between groups indicated as follows: * (P \u0026lt; 0.05), ** (P \u0026lt; 0.005), *** (P \u0026lt; 0.0005), **** (P \u0026lt; 0.00005).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/2c89fee0c6a394835c289cf7.png"},{"id":28532137,"identity":"ae4025ef-ab6d-4f02-b0a4-3853ac1ffb55","added_by":"auto","created_at":"2022-11-01 19:35:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3397995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree dimensional images from biofilms observed under the confocal laser scanning microscopy\u003c/strong\u003e. Biofilm produced by \u003cem\u003eL. plantarum\u003c/em\u003e C5 (a), \u003cem\u003eL. plantarum\u003c/em\u003e POM1 (b), \u003cem\u003eL. plantarum \u003c/em\u003e1LS16 (c), \u003cem\u003eL. plantarum\u003c/em\u003e D3.15 (d), \u003cem\u003eL. plantarum\u003c/em\u003e OE1 (e), \u003cem\u003eL. plantarum\u003c/em\u003e D6.6 (f), \u003cem\u003eL. plantarum\u003c/em\u003e E3.8 (g), \u003cem\u003eLact. paracasei \u003c/em\u003e41j (h).\u003cem\u003e \u003c/em\u003eMetabolically active cells (green fluorescence) and biofilm exopolysaccharides in the extracellular polymeric matrix (red fluorescence) are shown in the same panel. Bars represent 50 μm; units of the x, y, and z axes are μm. Images are representatives of three biological replicates analyzed in triplicate.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/6172bb829ef4e3df556049a7.png"},{"id":28531703,"identity":"a588c091-5daf-4e47-b456-9d4fa00f8675","added_by":"auto","created_at":"2022-11-01 19:30:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":269959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE-cadherin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Interleukin 8\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (mRNA Relative Quantification) in the Human Vaginal Epithelium\u003c/strong\u003e. Expression rates were calculated as the relative quantification (RQ) data. The data represent mean ± standard deviation. (a–c Means within the same treatment with different letters are significantly different (P \u0026lt; 0.05)).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/5b3f76f4326888d12ded2999.png"},{"id":44719200,"identity":"a856f600-52c6-438a-bbe2-3102d6a1b24f","added_by":"auto","created_at":"2023-10-16 18:53:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2653694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/1b62b1cb-34b3-4be1-8ec8-87d3e20cd6dc.pdf"},{"id":28531697,"identity":"70327543-a18e-4cda-86a2-096eeb6782ff","added_by":"auto","created_at":"2022-11-01 19:30:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10491,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/84329f1f7d8523e85864e5b3.pdf"},{"id":28531698,"identity":"e65fcc19-b355-48a0-a13c-6e54472e0024","added_by":"auto","created_at":"2022-11-01 19:30:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14705,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/501c556afe3859da84625d67.pdf"},{"id":28531702,"identity":"f4e24476-36f0-4ed9-894b-9b0ebe836d11","added_by":"auto","created_at":"2022-11-01 19:30:08","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17041,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/ac9ebcf2f587a95a5842808b.pdf"},{"id":28531700,"identity":"0c8ccd7b-03e6-4d9a-bd59-eef58bb86d5c","added_by":"auto","created_at":"2022-11-01 19:30:07","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":87870,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/bf73300d9bfdec05d6126615.pdf"},{"id":28531699,"identity":"1b17af98-8aa3-47e9-91ca-1e3ce4e8c281","added_by":"auto","created_at":"2022-11-01 19:30:07","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20135,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2201461/v1/78861d199dbba65711c25727.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach","fulltext":[{"header":"Background","content":"\u003cp\u003eThe human vaginal microbiome is a key determinant of vaginal health. The vagina ecosystem is often dominated by highly adapted lactobacilli. Vaginal microbiota can be clustered into five community state types (CSTs). Four of these CSTs are dominated by \u003cem\u003eLactobacillus crispatus\u003c/em\u003e (CST-I), \u003cem\u003eLactobacillus iners\u003c/em\u003e (CST-III), \u003cem\u003eLactobacillus gasseri\u003c/em\u003e (CST-II) or \u003cem\u003eLactobacillus jensenii\u003c/em\u003e (CST-V), while CST-IV is characterized by strict and facultative anaerobes not belonging to \u003cem\u003eLactobacillus\u003c/em\u003e genus (Smith and Ravel 2017). The presence of \u003cem\u003eLactobacillus\u003c/em\u003e spp. is correlated with a healthy vaginal microbiome, whereas the CTS-IV is associated with a status of dysbiosis and a consequently higher risk of infections and obstetric complications. Functional traits of lactobacilli involve the production of lactic acid and hydrogen peroxide, synthesis of bacteriocins as a response to the imbalance of the vaginal microbiome, host protection against vaginal pathogens, promotion of immunomodulation mechanisms by triggering the innate immunity system, and stimulation of anti-inflammatory mechanisms (Graf et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kovachev \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Borges, Silva, and Teixeira 2014; Han and Ren 2021). Infections of the host such as bacterial vaginosis (BV), urinary tract infections, yeast vaginitis, and sexually transmitted diseases like human immunodeficiency virus are all prevented in large part by the microorganisms that colonize the vaginal environment (Cribby, Taylor, and Reid 2008). Aerobic vaginitis defined by disruption in \u003cem\u003eLactobacillus\u003c/em\u003e dominance is accompanied by more extreme inflammatory changes than BV and the presence of mainly aerobic enteric commensals or pathogens, including \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e (Group B \u003cem\u003eStreptococcus\u003c/em\u003e [GBS]). \u003cem\u003eS. agalactiae\u003c/em\u003e is a member of the commensal microbiota of the human intestinal and genitourinary tracts and rarely causes infections in healthy adults. Infrequently it may cause morbidity in the elderly, pregnant women, and patients with underlying predisposing factors. Pregnancy-related maternal GBS colonization during birth is linked to newborn pneumonia, meningitis, and sepsis (Kaambo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eCandida albicans\u003c/em\u003e is a widespread fungus that lives in human mucosa and a few other environmental reservoirs. The human mouth, vagina, and stomach colonization normally begins in infancy, mostly after vaginal delivery or breastfeeding. The vagina is the primary mucocutaneous surface affected by \u003cem\u003eC. albicans\u003c/em\u003e, resulting in vulvovaginal candidiasis (VVC) (Lopes and Lionakis 2022).\u003c/p\u003e \u003cp\u003eVaginal microbiota transits among different CSTs during a woman's lifespan, being \u003cem\u003eLactobacillus\u003c/em\u003e-dominated, are more common among reproductive age and, particularly, in a pregnant woman. In addition, other lactobacilli may also be found, especially nomadic species like \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e and \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e group (Borges, Silva, and Teixeira 2014; Cribby, Taylor, and Reid 2008). A nomadic lifestyle is formally defined as a dynamic, generalist way of life of a species that involves both environmental and host niches, with no signs of specialization (Duar et al. 2017; Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, van Hijum, et al. 2016). Consequently, nomadic lactobacilli are found in contrasting environments, from vertebrates and invertebrate hosts to fermented vegetable and dairy products (Atassi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ciocia et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Khemariya et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mu\u0026ntilde;oz-Quezada et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These bacteria did not undergo a reductive evolution strategy and thus their large genomes encompass increased metabolic flexibility, which enables them to thrive in diverse environments (Duar et al. 2017; Inglin, Meile, and Stevens 2018; Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, van Hijum, et al. 2016). \u003cem\u003eL. plantarum\u003c/em\u003e exemplifies the paradigm of the nomadic lifestyle and possesses one of the largest genomes among lactobacilli (3.3 Mbp). Strains isolated from different environments could reshape their phenotype expression towards a common direction under the same environmental condition (Acin-Albiac et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the same way, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e and \u003cem\u003eLact. casei\u003c/em\u003e genotypes and phenotypes do not correlate properly with their source of isolation (Broadbent et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ceapa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Due to their ability to thrive under contrasting conditions when compared to host-adapted lactobacilli, nomadic lactobacilli hold a huge potential as probiotics capable of positively modulating several aspects of human health (Fidanza, Panigrahi, and Kollmann \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHence, the aim of this study is the selection of nomadic lactobacilli to be used as vaginal probiotics. A multistep approach was followed, where the first step encompassed a wise panel of assays for functional traits and pathogens inhibition using a custom scoring procedure to select the most promising strains. Then, eight candidate strains were further investigated for biofilm production. Subsequently, the two final promising candidate strains were included in a gel formulation prototype alone or as a binary combination. The ability of these prototypes to reduce the expression of the genes involved in the inflammatory cascade using the reconstituted human vaginal epithelium (HVE) infected with \u003cem\u003eC. albicans\u003c/em\u003e was evaluated.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eLactic acid isomers and hydrogen peroxide quantification\u003c/h2\u003e\n \u003cp\u003eThe capacity of lactobacilli to produce lactic acid highly varied at species and strain levels (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The highest value of total lactic acid was found for \u003cem\u003eL. plantarum\u003c/em\u003e POM1 (1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), followed by \u003cem\u003eL. plantarum\u003c/em\u003e C5 (1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and D3.15 (1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Within the \u003cem\u003eL. pentosus\u003c/em\u003e species, E1.4 strain showed the highest total lactic acid yield (0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0. 06 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eLact. rhamnosus\u003c/em\u003e B4.2 and B6.19 produced almost equal quantities (0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). Overall, the production of total lactic acid from \u003cem\u003eLact. paracasei\u003c/em\u003e strains was quite low, with the highest amount produced from \u003cem\u003eLact. paracasei\u003c/em\u003e 93j (0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003eA significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) was observed between \u003cem\u003eL. pentosus\u003c/em\u003e and \u003cem\u003eLact. paracasei\u003c/em\u003e, and between \u003cem\u003eLact. paracasei\u003c/em\u003e and \u003cem\u003eLact. rhamnosus\u003c/em\u003e. Moreover \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eLact. paracasei\u003c/em\u003e showed a significant difference with P\u0026thinsp;\u0026lt;\u0026thinsp;0.00005. Further investigation of lactic acid production revealed different patterns of lactic acid isomer production (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Among \u003cem\u003eL. plantarum\u003c/em\u003e strains, POM1 produced the highest amount of D-lactate (0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), followed by C5 (0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and P1 (0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The production of D-lactate by \u003cem\u003eL. pentosus\u003c/em\u003e species ranged from 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (strain E1.4) to 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (strain D2.15). The two \u003cem\u003eLact. rhamnosus\u003c/em\u003e strains showed an opposite behavior where \u003cem\u003eLact. rhamnosus\u003c/em\u003e B4.2 produced 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of D-lactate, whereas \u003cem\u003eLact. rhamnosus\u003c/em\u003e B6.19 produced 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Almost no strains of \u003cem\u003eLact. paracasei\u003c/em\u003e produced D-lactate, with eight strains failing to produce both isomers. \u003cem\u003eLact. paracasei\u003c/em\u003e strains produced the highest levels of L-lactate, especially \u003cem\u003eLact. paracasei\u003c/em\u003e 84f (0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The highest L-lactate yields came mostly from the \u003cem\u003eLact. paracasei\u003c/em\u003e group, and in the specific \u003cem\u003eLact. paracasei\u003c/em\u003e 84f showed the highest production (0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). After it came the L-lactate yields from \u003cem\u003eL. plantarum\u003c/em\u003e strains, with the highest yield found for \u003cem\u003eL. plantarum\u003c/em\u003e AFI (0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the lowest for \u003cem\u003eL. plantarum\u003c/em\u003e CB5 (0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eLact. rhamnosus\u003c/em\u003e B6.19 and B4.2 produced 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of L-lactate, respectively. Within the \u003cem\u003eL. pentosus\u003c/em\u003e species, the L-lactate highest yield was found for the E1.4 strain (0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the lowest for \u003cem\u003eL. pentosus\u003c/em\u003e D2.15 (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production also showed high variability at species and strains level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Overall, \u003cem\u003eLact. paracasei\u003c/em\u003e produced the highest amounts of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e ranging from 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 (25h) to 22.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07 (ALII8) \u0026micro;M whereas \u003cem\u003eLact. paracasei\u003c/em\u003e 22e was the only one within this species unable to produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. No H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was produced from the strains belonging to \u003cem\u003eL. pentosus\u003c/em\u003e species. Most of the \u003cem\u003eL. plantarum\u003c/em\u003e examined did not produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, with \u003cem\u003eL. plantarum\u003c/em\u003e D2.6 (6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28 \u0026micro;M) having the highest production within the species. \u003cem\u003eLact. rhamnosus\u003c/em\u003e B4.2 and B6.19 strains produced 2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20 and 1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24 \u0026micro;M, respectively. Based on statistical analysis at the species level, a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed between \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eLact. paracasei\u003c/em\u003e, and between \u003cem\u003eLact. paracasei\u003c/em\u003e and \u003cem\u003eLact. rhamnosus\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eAuto-aggregation capacity and hydrophobicity characteristics\u003c/h2\u003e\n \u003cp\u003eThe results from auto-aggregation and hydrophobicity assays were not species-dependent (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. Approximately 60% of lactobacilli strains isolated from various sources and belonging to different species, showed an auto-aggregation capacity above the average value (63.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18%). The highest aggregation capacity was reached by \u003cem\u003eL. plantarum\u003c/em\u003e D9.46 (81.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01%). A significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed between \u003cem\u003eLact. paracasei\u003c/em\u003e and \u003cem\u003eLact. rhamnosus\u003c/em\u003e species. The capability of the bacteria to potentially adhere to the epithelial cells was further investigated through the characterization of cell wall hydrophobicity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). \u003cem\u003eL. plantarum\u003c/em\u003e S6w5 had the highest hydrophobicity capacity (88.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29%). All the other strains were almost equally distributed around the average value, showing a value lower (41.1% of all the strains) or higher (43.2% of all the strains) than the average (31.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7%) value. Only a few strains (15.8%), belonging to different species and isolated from different sources, did not show any hydrophobicity characteristics. A significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed between \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eLact. paracasei\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003ePathogen growth inhibition screening\u003c/h2\u003e\n \u003cp\u003eGrowth kinetics of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 and \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 were assessed in the presence of cell-free supernatants (CFSs) from all \u003cem\u003eLactobacillus\u003c/em\u003e strains and compared against the control conditions. The inhibitory activity was tested using CFSs at the original pH of ca. 4.0 (o-CFSs) or neutralized CFSs at pH 7 (n-CFSs), to exclude pH-dependent effects. \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 grown at pH 7.0 as a control condition reached a maximum absorbance (\u003cem\u003eA\u003c/em\u003e) of 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, with the lag phase (\u003cem\u003e\u0026lambda;\u003c/em\u003e) 1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 h, and growth rate (\u003cem\u003e\u0026micro;\u003c/em\u003e) 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 h\u003csup\u003e-1\u003c/sup\u003e. At pH ca. 4.0, lower \u003cem\u003eA\u003c/em\u003e (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), longer \u003cem\u003e\u0026lambda;\u003c/em\u003e (8.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.98 h), and smaller \u003cem\u003e\u0026micro;\u003c/em\u003e (0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 h\u003csup\u003e-1\u003c/sup\u003e) were reached. Three common patterns of inhibition of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 were observed with n-CFSs, while no common pattern of inhibition was observed with o-CFSs. n-CFSs from seventy lactobacilli strains caused a statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction of the \u003cem\u003eA\u003c/em\u003e value when compared to the control condition at pH 7.0. n-CFSs from \u003cem\u003eLact. paracasei\u003c/em\u003e HHI10 had the strongest repressive effect on \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 in terms of \u003cem\u003eA\u003c/em\u003e (0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02). n-CFSs from 25 strains most of which belonged to \u003cem\u003eL. plantarum\u003c/em\u003e species, did not inhibit \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 in terms of \u003cem\u003eA.\u003c/em\u003e Most of the n-CFSs were collected from \u003cem\u003eLact. paracasei\u003c/em\u003e significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) extended the lag phase (\u003cem\u003e\u0026lambda;\u003c/em\u003e) of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3, with the highest \u003cem\u003e\u0026lambda;\u003c/em\u003e value detected when \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 was grown with n-CFS from \u003cem\u003eLact. paracasei\u003c/em\u003e 104g (2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 h). When \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 was cultured with n-CFSs from sixteen \u003cem\u003eLact. paracasei\u003c/em\u003e strains no lag phase was observed. Negative values of \u003cem\u003e\u0026lambda;\u003c/em\u003e can be interpreted biologically as the pathogen starting to grow right after inoculation. Significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction of \u003cem\u003e\u0026micro;\u003c/em\u003e at pH 7.0 was found mostly with n-CFSs collected from \u003cem\u003eLact. paracasei\u003c/em\u003e strains, with the lowest (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) value for \u003cem\u003eLact. paracasei\u003c/em\u003e F23 (0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00). By monitoring the \u003cem\u003eA\u003c/em\u003e parameter, complete inhibition of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 growth was found during culturing with o-CFSs from \u003cem\u003eL. plantarum\u003c/em\u003e (36 strains), \u003cem\u003eLact. paracasei\u003c/em\u003e (20), \u003cem\u003eL. pentosus\u003c/em\u003e (3), and \u003cem\u003eLact. rhamnosus\u003c/em\u003e (2). \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 cultured at pH 7.0 as a control condition reached a maximum \u003cem\u003eA\u003c/em\u003e of 1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, with a \u003cem\u003e\u0026lambda;\u003c/em\u003e of 6.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 h, and \u003cem\u003e\u0026micro;\u003c/em\u003e of 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 h\u003csup\u003e-1\u003c/sup\u003e. At pH 4.0 as the control condition, \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 reached lower \u003cem\u003eA\u003c/em\u003e (1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08), longer \u003cem\u003e\u0026lambda;\u003c/em\u003e (8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 h), and smaller \u003cem\u003e\u0026micro;\u003c/em\u003e (0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 h\u003csup\u003e-1\u003c/sup\u003e). Eight n-CFSs that were obtained from \u003cem\u003eL. plantarum\u003c/em\u003e (1 strain) and \u003cem\u003eLact. paracasei\u003c/em\u003e (7 strains), led to a higher \u003cem\u003eA\u003c/em\u003e parameter when compared to the control condition at pH 7.0, while 87 strains had a repressive effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) on the \u003cem\u003eA\u003c/em\u003e parameter. The n-CFS that was obtained from \u003cem\u003eLact. paracasei\u003c/em\u003e wI10 had the strongest inhibition effect in terms of \u003cem\u003eA\u003c/em\u003e at pH 7.0 (1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14). n-CFSs from 19 strains, mostly \u003cem\u003eL. plantarum\u003c/em\u003e, extended the lag phase (\u003cem\u003e\u0026lambda;\u003c/em\u003e parameter), with the longest value reached with \u003cem\u003eL. plantarum\u003c/em\u003e 1LS16 (7.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 h). No significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) reduction of the \u003cem\u003e\u0026micro;\u003c/em\u003e parameter of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 was observed at pH 7.0. Lower significant inhibition of the growth curve of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 was observed with o-CFSs under acidic condition (pH 4). Inhibition was obtained from only 12 o-CFSs collected from \u003cem\u003eLact. paracasei\u003c/em\u003e (5), \u003cem\u003eL. plantarum\u003c/em\u003e (6), and \u003cem\u003eL. pentosus\u003c/em\u003e (1). The lowest value of \u003cem\u003eA\u003c/em\u003e was reached with o-CFSs from \u003cem\u003eLact. paracasei\u003c/em\u003e 41j (0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17). Only five o-CFSs that were collected from 5 strains of \u003cem\u003eL. pentosus\u003c/em\u003e (3) and \u003cem\u003eLact. paracasei\u003c/em\u003e (2) affected the \u003cem\u003e\u0026lambda;\u003c/em\u003e value of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54. The highest value of \u003cem\u003e\u0026lambda;\u003c/em\u003e was detected during incubation with o-CFS from \u003cem\u003eL. plantarum\u003c/em\u003e D2.6 (9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 h). No significant inhibition on the \u003cem\u003e\u0026micro;\u003c/em\u003e parameter for \u003cem\u003eC. albicans\u003c/em\u003e was attributable to the o-CFSs under acidic condition (pH ca. 4.0). To evaluate the differences among all the strains and the overall characteristics investigated, we carried out a principal component analysis (PCA) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). \u003cem\u003eL. plantarum\u003c/em\u003e differ from \u003cem\u003eLact. paracasei\u003c/em\u003e in their functional and pathogen inhibition traits of interest in vaginal health. Furthermore, particularly interesting is the position of \u003cem\u003eL. plantarum\u003c/em\u003e POM1 and C5 in the corner of the PCA. These two strains differentiate also from other standalone \u003cem\u003eL. plantarum\u003c/em\u003e strains.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eCandidate selection approach and scoring procedure\u003c/h2\u003e\n \u003cp\u003eA scoring procedure was set for both the functional and the pathogen inhibition assays for the selection of the best-performing strains through the definition of a cumulative score (see Materials and Methods). Overall, the best performing assays for the selection were the D-lactic acid production and hydrophobicity characteristics, where a total of 45 CFSs showed a relative score of 1 or 2, meaning that the value is either between Q2 and Q3, or higher than Q3. For the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production and auto-aggregation evaluation, a total of 44 CFSs showed a relative score equal to 1 or 2, and lastly total lactic acid production, where only 9 strains showed positive results (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eSingle or double hit of inhibition\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eS. agalactiae\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSingle hit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eDouble hit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSingle hit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDouble hit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eo-CFS (original acidic pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-CFSs (neutral pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eo-CFS (original acidic pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-CFSs (neutral pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eo-CFS (original acidic pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003en-CFSs (neutral pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eo-CFS (original acidic pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-CFSs (neutral pH)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLact. paracasei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. pentosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLact. rhamnosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrand Total\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe total number of strains categorized per species whose cell-free supernatants at the original acidic pH (o-CFSs) and CFSs at neutral pH (n-CFSs) showed a single and double hit of inhibition on the growth of each pathogen. o-CFS and n-CFS had a significant effect on \u003cem\u003eA\u003c/em\u003e or \u003cem\u003e\u0026lambda;\u003c/em\u003e of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, but never on its \u003cem\u003e\u0026micro;\u003c/em\u003e, thus resulting in a single hit of inhibition (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other side, some n-CFS had a significant effect on more than one growth parameter of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 at pH 7.0, giving a double hit of inhibition (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, some o-CFS had a remarkable effect on \u003cem\u003eA\u003c/em\u003e of \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 at acidic pH (pH ca. 4.0) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on their pathogen inhibition and functional assays score (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), eight best-performing \u003cem\u003eLactobacillus\u003c/em\u003e strains were selected: \u003cem\u003eL. plantarum\u003c/em\u003e OE1, C5, D3.15, 1LS16, POM1, E3.8 and D2.6, and \u003cem\u003eLact. paracasei\u003c/em\u003e41j.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eScoring overview for the first eight best-performing lactobacilli\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecie\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSub-source of isolation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCode\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePathogen inhibition scoring\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFunctional scoring\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCumulative scoring\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarrot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOlives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTomato\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePOM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLact. paracasei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePineapple\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1LS16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eBiofilm imaging by confocal laser scanning microscopy\u003c/h2\u003e\n \u003cp\u003eThree-dimensional images of the biofilm colonies grown on membrane filters were collected through CLSM (confocal laser scanning microscopy) observations of the mature biofilms resulting from the 8 selected strains after 48 h of incubation. All biofilms appeared as widespread agglomerates of sessile cells (green fluorescence) embedded in exopolysaccharides (EPS) (red fluorescence) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Biofilm architecture was strain-dependent, with a lower quantity of red fluorescence visible in the images captured from \u003cem\u003eL. plantarum\u003c/em\u003e E3.8 and \u003cem\u003eL. plantarum\u003c/em\u003e POM1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eBiofilm growth and extracellular matrix characterization\u003c/h2\u003e\n \u003cp\u003eBiomass, cell density, and extracellular matrix (ECM) (protein, extracellular DNA (eDNA), and total saccharides) content from the mature biofilms of the eight selected strains formed after 48 h of incubation are shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest biofilm formation in terms of biomass was achieved by \u003cem\u003eL. plantarum\u003c/em\u003e POM1 (36.20\u0026thinsp;\u0026plusmn;\u0026thinsp;12.02 mg), whereas \u003cem\u003eL. plantarum\u003c/em\u003e C5 led to the lowest one (11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 mg). In terms of cell density and ECM protein content, there was no statistically significant difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among the strains. The eDNA concentration ranged between a minimum for \u003cem\u003eLact. paracasei\u003c/em\u003e 41j (0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to a maximum for \u003cem\u003eL. plantarum\u003c/em\u003e C5 (1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest production of EPS was found in ECMs extracted from the biofilm of \u003cem\u003eL. plantarum\u003c/em\u003e C5 (80.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), being significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher than EPS extracted from \u003cem\u003eL. plantarum\u003c/em\u003e D2.6 (35.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eL. plantarum\u003c/em\u003e POM1 (26.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eL. plantarum\u003c/em\u003e E3.8 (25.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eL. plantarum\u003c/em\u003e 1LS16 (22.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and \u003cem\u003eLact. paracasei\u003c/em\u003e 41j (17.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Taking into consideration all these results, two strains, \u003cem\u003eL. plantarum\u003c/em\u003e C5 and \u003cem\u003ePOM1\u003c/em\u003e, were further investigated for more specific analysis to understand their behavior in the vaginal environment.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiofilm characterization\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBiomass\u003c/p\u003e\n \u003cp\u003e(mg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCell density (Log CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eeDNA\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProteins\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEPS\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e C5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.11\u0026thinsp;\u0026plusmn;\u0026thinsp;16.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e POM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.20\u0026thinsp;\u0026plusmn;\u0026thinsp;12.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e 1LS16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.70\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e D3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.07\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e OE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.35\u0026thinsp;\u0026plusmn;\u0026thinsp;9.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.11\u0026thinsp;\u0026plusmn;\u0026thinsp;10.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e D2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e E3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLact. paracasei\u003c/em\u003e 41j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.91\u0026thinsp;\u0026plusmn;\u0026thinsp;20.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eBiomass (mg), cell density (Log CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and ECMs (eDNA, proteins, and exopolysaccharide concentration, expressed as \u0026micro;g/mg of the total biomass) characterization of the newly formed biofilm, and reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. (a\u0026ndash;b Means within the columns with different letters are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEvaluation of CFSs preservation of HVE metabolic activity after infection with\u003cem\u003e\u0026nbsp;C. albicans\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eCompared to the negative control, \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 without the addition of CFS (positive control) sharply decreased the metabolic activity of the HVE (25.91 \u0026plusmn; 0.78 %) according to the MTT assay (\u003cstrong\u003eTable 4\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of metabolic activity of Human Vaginal Epithelium model\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eViability (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e C5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.78\u0026thinsp;\u0026plusmn;\u0026thinsp;11.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e POM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCombination\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eL. plantarum\u003c/em\u003e C5\u0026thinsp;+\u0026thinsp;POM1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.55\u0026thinsp;\u0026plusmn;\u0026thinsp;9.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003eNegative control is the untreated epithelium, positive control is the epithelium infected with \u003cem\u003eCandida albicans\u003c/em\u003e. \u003cem\u003eL. plantarum\u003c/em\u003e C5, \u003cem\u003eL. plantarum\u003c/em\u003e POM1, and C5\u0026thinsp;+\u0026thinsp;POM1 are the epithelium models infected with the pathogen and then treated with the gel containing the respective strain or the combination of both. Data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. (a\u0026ndash;c Means with different letters are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eGenerally, both \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1 strains were able to prevent HVE metabolic activity loss either when cultured in single or in combination. In detail, the metabolic activity of the infected HVE treated with a gel containing \u003cem\u003eL. plantarum\u003c/em\u003e C5 and \u003cem\u003eL. plantarum\u003c/em\u003e POM1 reached 56.78\u0026thinsp;\u0026plusmn;\u0026thinsp;11.09% and 64.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24%, respectively, with a statistically (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) significant difference from the viability of cells of positive control. The combination of the two strains showed a metabolic activity of 71.55\u0026thinsp;\u0026plusmn;\u0026thinsp;9.85%, demonstrating a synergistic effect of nomadic lactobacilli, still significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the positive control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eGene expression profiling\u003c/h2\u003e\n \u003cp\u003eHVEs showed a significant decrease (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in interleukin 8 (IL-8) expression, compared to the positive control when HVE models were treated with gels containing the single lactobacilli strains. In addition, a synergistic effect was found when HVEs were treated with the gel containing both lactobacilli strains, also showing a significant difference from the control. A positive effect of the treatment with the gels containing the lactobacilli was visible also from the analysis of the \u003cem\u003eE-cadherin\u003c/em\u003e expression. The \u003cem\u003eE-cadherin\u003c/em\u003e expression, which sharply decreased in the HVEs infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, increased when the epithelium model was treated with a gel containing lactobacilli, both in single and in combination (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results of the gene expression analyses of HVE infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and treated with a gel containing selected lactobacilli were shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The negative control represents the gene expression in the untreated epithelium model, and it is standardized as equal to 1. Under-expression or over-expression of the genes are represented by lower or higher values, respectively. The blank sample is the epithelium model treated with blank gel, positive control is the epithelium infected with \u003cem\u003eC. albicans\u003c/em\u003e, while the last three are epithelium models infected with the pathogen and treated with the gel containing the respective strains (\u003cem\u003eL. plantarum\u003c/em\u003e C5 or \u003cem\u003eL. plantarum\u003c/em\u003e POM1), or combination of the two (C5\u0026thinsp;+\u0026thinsp;POM1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA healthy human vaginal microbiome is dominated by homofermentative \u003cem\u003eLactobacillus\u003c/em\u003e spp., which reflects vaginal community states (De Seta et al. 2019; Vaneechoutte \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The choice of optimum probiotic strains for therapeutic use is crucial since their use can lessen the possibility of the rapid growth of non-native lactobacilli in the urogenital tract in the vaginal environment (He et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The lactobacilli group is the major source of probiotic strains because of their professed benefits on human health (Vitali et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and because many of them have been granted the status of qualified presumption of safety (QPS) by the European food safety authority (EFSA) (EFSA Panel on Biological Hazards - BIOHAZ, 2013). Until now, many commercial vaginal probiotics have been focusing on using exogenous lactobacilli neglecting the fact of their lifestyle and inherent ability to thrive in contrasting environments (van de Wijgert and Verwijs 2020). However, nomadic lactobacilli (\u003cem\u003eLactiplantibacillus\u003c/em\u003e spp. and \u003cem\u003eLacticaseibacillus\u003c/em\u003e spp.) may also thrive and colonize successfully vaginal niches (Martino, Bayjanov, Caffrey, Wels, Joncour, Hughes, Gillet, Kleerebezem, Hijum, et al. 2016). Moreover, their upstream processing scalability for product development is more feasible when compared to native vaginal lactobacilli, such as \u003cem\u003eLb. crispatus\u003c/em\u003e (Fidanza, Panigrahi, and Kollmann \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The vaginal epithelial barrier is shielded from pathogen colonization and invasion by lactobacilli, which produce antibacterial chemicals including lactic acid and hydrogen peroxide, stick to vaginal epithelial cells to form a protective film, and block pathogen adhesion (He et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, we screened 95 nomadic lactobacilli isolated from different sources using a multistep approach, for desirable metabolic traits to maintain and restore vaginal health. \u003cem\u003eL. plantarum\u003c/em\u003e POM1 isolated from tomatoes was the highest producer of lactic acid and D-lactate. \u003cem\u003eLact. paracasei\u003c/em\u003e spp. produced significantly lower amounts of lactic acid. This might be because \u003cem\u003eLact. paracasei\u003c/em\u003e spp. only synthesizes L- lactic acid isomer since it lacks the gene coding for D-lactate dehydrogenase (Liu et al., 2021). The D-lactate production within the vaginal microbiome suppresses the vaginal extracellular matrix metalloproteinase inducer, adding another level of protection against upper genital tract infections (Witkin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tozetto-Mendoza et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Analogously, most of the native vaginal lactobacilli (\u003cem\u003eLb. gasseri, Lb. crispatus\u003c/em\u003e, and \u003cem\u003eLb. jensenii\u003c/em\u003e) produce both isomers of lactic acid. Therefore, we favored total and D-lactic acid producers in our scoring procedure. The highest production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a key factor in maintaining the balance of a healthy vaginal environment, was observed for \u003cem\u003eLact. paracasei\u003c/em\u003e spp., all isolated from dairy, fruits, and vegetables. Auto-aggregation is suggested to be necessary for the adhesion of probiotic microorganisms to the intestinal epithelium. Both auto-aggregation and hydrophobicity characteristics can be considered a pre-test for selecting probiotic strains which potentially adhere to epithelial cells (Krausova et al., 2019). \u003cem\u003eL. plantarum\u003c/em\u003e D9.46 showed the highest auto-aggregation capacity, while \u003cem\u003eL. plantarum\u003c/em\u003e DM was the only strain showing none. No common characteristics among the strains were observed for both auto-aggregation and hydrophobicity. \u003cem\u003eS. agalactiae\u003c/em\u003e (GBS) causes an important life-threatening infection in infants, which is transmitted during pregnancy (Rosen et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kaambo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, \u003cem\u003eC. albicans\u003c/em\u003e is an opportunistic pathogen, which can overgrow in the vaginal ecosystem causing VVC (Mendling \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given the importance and prevalence of these vaginal pathogens, we assessed the capacity of nomadic lactobacilli CFSs to inhibit them. To exclude pH-dependent inhibition we also included neutralized CFSs. The pathogens' growth inhibition differed substantially across the lactobacilli and pathogens examined (Osset, Bartolome, and Garcı 2014). Our findings suggest that the studied \u003cem\u003eLactobacillus\u003c/em\u003e strains showed inter-strain differences in anti-\u003cem\u003eCandida\u003c/em\u003e activity. \u003cem\u003eLactobacillus\u003c/em\u003e strains that produce the highest quantities of lactic acid have the strongest antagonistic effect on GBS (Marziali et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These findings imply that \u003cem\u003eS. agalactiae\u003c/em\u003e is sensitive to organic acids and that individual lactobacilli's capacity to acidify is critical for anti-GBS action. Microecological preparations, when administered orally or vaginally, have been shown in numerous clinical studies to significantly lower incidence and recurrence rates, prolong the time between recurrences, increase recovery rates, relieve symptoms, and enhance the vaginal microecological patterns of BV and VVC (He et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). More in general, some lactobacilli adhere to the vaginal epithelial cells, while others can prevent uropathogens attachment to these cells and suppress uropathogens\u0026rsquo; growth. Indeed, certain \u003cem\u003eLactobacillus\u003c/em\u003e strains can restrict the growth of vaginal pathogens and excrete chemicals that prevent them from multiplying, which are two crucial phases in the pathogenesis of urinary infections (Osset, Bartolome, and Garcı 2014). Biofilm formation by lactobacilli is responsible for their stable maintenance of a stable ecosystem, as it grants long-term permanence to the host\u0026rsquo;s vaginal mucosa (Ventolini, Mitchell, and Salazar \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The accessory genome of \u003cem\u003eLb. crispatus\u003c/em\u003e contains genomic islands that encode enzymes involved in EPS biosynthesis (Ojala et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In fact, this species is associated with the healthiest vaginal community state (De Seta et al. 2019). Hence, we determined the relative composition of protein, eDNA, and total saccharides biofilm components. As a result, all the eight previously selected strains were able to form biofilms with distinct macrostructure and composition, which highly diverged between \u003cem\u003eL. plantarum\u003c/em\u003e strains and \u003cem\u003eLact. paracasei\u003c/em\u003e 41j. The main component of ECM is EPS, for all the eight strains, with significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) observed among the strains. We further selected \u003cem\u003eL. plantarum\u003c/em\u003e POM1 and C5 due to their differentiation from other \u003cem\u003eL. plantarum\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and also due to their high biomass production and quantity of EPS (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), respectively, for further investigation. Topical gel formulations containing the two selected strains, in single and in combination, were formulated as a prototype method to be applied to the vaginal surface, using the HVE \u003cem\u003ein vitro\u003c/em\u003e model previously infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54. \u003cem\u003eC. albicans\u003c/em\u003e endocytosis has been linked to the proteolytic breakdown of E-cadherin which is the major protein in vaginal epithelial cell junctions (Naglik et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Compared to negative control, infection with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 decreased the HVEs cell viability up to 25%, while significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher metabolic activity was found when treating HVEs with the topical gel. HVE treated with the gel showed an improved cell viability compared to the untreated one, due to an antagonistic effect of lactobacilli against \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, as already demonstrated by Allonsius et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The positive role of the two selected strains was confirmed by the expression analysis of genes encoding for anti-inflammatory cytokines in HVEs. Common vaginal pathogens, including \u003cem\u003eC. albicans\u003c/em\u003e have been previously associated with elevated expression of IL-8, a proinflammatory chemokine capable of attracting neutrophils to sites of infection (Agace et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Spear et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). IL-8 gene was overexpressed in cells infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, while the cells treated with the gels containing lactobacilli showed a statistically significant decrease in IL-8 gene expression. A synergistic effect was observed when the cells were treated with the gel containing the combination of the two \u003cem\u003eL. plantarum\u003c/em\u003e strains. On the other hand, the treatment with the gels containing the two \u003cem\u003eL. plantarum\u003c/em\u003e strains, both in single and in combination, prevented the reduction of \u003cem\u003eE-cadherin\u003c/em\u003e gene expression normally caused by the pathogen.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo summarize, we highlighted the potential of nomadic lactobacilli isolated from different foods, animal, and human sources as suggested probiotic strains for the vaginal environment, thanks to their functional properties and their inhibitory activity against vaginal pathogens (\u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3). This investigation was carried out to find the isolates with most of the qualities required for \u003cem\u003eLactobacillus\u003c/em\u003e to operate as biotherapeutic microorganisms. By using a cumulative scoring-based approach, the pool of potential probiotic candidates was narrowed down. In the end, the ability to form biofilms led to the selection of two \u003cem\u003eL. plantarum\u003c/em\u003e strains. We provided \u003cem\u003ein vitro\u003c/em\u003e evidence on the use of a topic gel containing the two selected potential probiotic strains that can reduce the inflammation caused by \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 infection in the HVE model, either alone or in combination, and thus appear to be promising probiotic strains for vaginal health. Nonetheless, comprehensive clinical investigations will be required to confirm their actual therapeutic advantages.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eBacterial cultures and growth conditions\u003c/h2\u003e\n \u003cp\u003eFor this study, a total of 95 lactobacilli were investigated; 48 strains were isolated from dairy products, 31 from fruits and vegetables, 15 from sourdough, and 1 from other sources (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). These strains, together with the pathogenic strain \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 and \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, belong to the Micro4Food collection from the University of Bolzano-Bozen. All the cultures were maintained as frozen stocks at -20\u0026deg;C in their specific broth medium with 20% glycerol for subsequent analysis. Before their use, the lactobacilli were propagated twice in MRS broth (Sigma Aldrich) at 37\u0026deg;C (body temperature) for 24 h. \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 was refreshed in Sabouraud broth (Scharlau, Spain) with 5% Tween\u003csup\u003e\u0026reg;\u003c/sup\u003e80 (Sigma Aldrich) and incubated overnight at 37\u0026deg;C. \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 was refreshed in Brain-Heart Infusion (BHI) broth and incubated at 37\u0026deg;C until it reached the stationary phase (ca. 24 h).\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab5\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eLactobacilli strains (n\u0026thinsp;=\u0026thinsp;95) used in this study\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of strains\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource and sub-source of isolation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31a, 25h, 100i, 104g, 22e, 25e, 25g, 28g, 41j, 45j,\u003c/p\u003e\n \u003cp\u003e50a, 52i, 76d, 83e, 84f, 93j, 99a, AAI9, AII8, BBII10, bI5, dII1, eI3, GII3, HHI10, iiII4, iiII9, JJI8, kI12,\u003c/p\u003e\n \u003cp\u003eLII1, MMII7, wI10, WWI9, zzI10, zzI3, zzI4, zzI6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDairy (Milk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAFI10, AFI7, AFII5, ALII8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Apple-by-products)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS4d8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Sauerkraut)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF1, F10, F12, F13, F2, F23, F25, F5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDairy (Cheese)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus pentosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD2.15, E1.4, E3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP1, CB5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDairy (Cheese)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDairy (Milk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Carrot)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCIL6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Cherry)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFin6, Fin10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Fennel)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIT1, IT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Grape)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK1, K13, K2, K9, KI-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Kiwi)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Olives)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Papaya)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1LS16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Pineapple)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePR14, PR3, PR6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Prune)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS6w5, AFI5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Sauerkraut)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePOM1, POM20, POM27, POM35, POM42, POM43, POM40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFruits and Vegetables (Tomato)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE3.13, E3.19, D9.30, D9.40, D9.46, D3.15, C5.10, D9.18, E3.8, D2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB6.19, B4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSourdough\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eCell-free supernatants collection\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e strains were grown in MRS broth for 24 h at 37\u0026deg;C, and then the CFSs were recovered by centrifugation (7,500 rpm, 10 min). Further, CFSs were fractionated in two aliquots. One aliquot was neutralized at pH 7.0 (n-CFSs) with NaOH and sterilized by using a 0.22 \u0026micro;m filter. The other aliquot was preserved at the original acidic pH of ca. 4.0 (o-CFSs) and was further divided into two aliquots: one was sterilized by using a 0.22 \u0026micro;m filter, and one was kept not sterile. All the aliquots of CFSs were stored at -20\u0026deg;C until needed. Sterile CFSs were used for pathogen inhibition screening, while non-sterile CFSs were used for functional assays (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and lactic acid quantification).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eLactic acid isomers and hydrogen peroxide quantification\u003c/h2\u003e\n \u003cp\u003eThe quantification of lactic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the CFSs was done using two commercial kits, respectively the Megazyme D-Lactate and L-Lactate Assay Kit (Megazyme International Ireland Ltd., Wicklow) and the Peroxide assay kit (Sigma Aldrich), following the instructions provided by the manufacturers.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eAuto-aggregation capacity and hydrophobicity characterization\u003c/h2\u003e\n \u003cp\u003eThe candidate strains were grown in MRS at 37\u0026deg;C for 18\u0026ndash;22 hours. The pellets were harvested by centrifugation (10,000 g, 10 mins, 5\u0026deg;C), washed with PBS (phosphate buffer solution) (pH 7.0), and re-suspended in the same buffer. Cell suspensions were adjusted to an optical density (OD) of 620 nm of ca. 0.25 and used for both analyses (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e). Cell auto-aggregation was performed according to Gil-Rodr\u0026iacute;guez, Carrascosa, and Requena (2015) as modified by Di Cagno et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Each cell suspension was left to settle at room temperature, then the OD\u003csub\u003e620\u003c/sub\u003e was measured after 2, 4, and 24 h (A\u003csub\u003et\u003c/sub\u003e). The percentage of auto-aggregation (\u003cem\u003eA\u003c/em\u003e) was calculated according to the following formulae:\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equa\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg 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width=\"148\" height=\"45\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eEstimation of hydrophobicity was performed according to Burns et al. (\u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e) as modified by Di Cagno et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Xylene was used to determine the hydrophobicity of the cell surface. A total volume of 0.4 mL of xylene (Sigma Aldrich) was added to the cell suspension (2 mL), and vortexed for 120s. After phase stabilization and separation (1 h, 37\u0026deg;C), the OD of the aqueous phase was measured at 620 nm (Ht). The percentage of hydrophobicity (\u003cem\u003eH\u003c/em\u003e) was calculated according to the following formulae:\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equb\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\u003cimg 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width=\"157\" height=\"46\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003eHigh-throughput pathogen inhibition screening\u003c/h2\u003e\n \u003cp\u003eGrowth inhibitions of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 by CFSs, previously collected and stored, were screened. Briefly, overnight cultures of the pathogens were washed twice with saline solution (0.9% NaCl) and resuspended in saline solution to a final OD\u003csub\u003e600\u003c/sub\u003e of 0.1 for \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, or OD\u003csub\u003e620\u003c/sub\u003e of 0.25 for \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3. The 96-well plates were set using one volume of CFSs and three volumes of each pathogen. The analysis was done in triplicate using CFSs previously neutralized (pH 7.0) and the acidic CFSs (pH ca. 4.0). Control wells were prepared using MRS medium at pH 4.0, and MRS at pH 7.0, and run in triplicates. Control MRS was acidified using a racemic solution of lactic acid to pH 4.0 and adjusted back to pH 7.0 using NaOH 1M to exclude the osmolyte effect. Growth kinetics of the pathogens were recorded for 46 h at 37\u0026deg;C measuring the absorbance (at a wavelength of 600 nm for \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54, and 620 nm for \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3) every 15 min with the Infinite\u003csup\u003e\u0026reg;\u003c/sup\u003e M Nano\u0026thinsp;+\u0026thinsp;Spectrophotometer (TECAN, Austria).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003eCandidate selection approach and scoring procedure\u003c/h2\u003e\n \u003cp\u003eThe best-performing lactobacilli were selected through a scoring procedure based on the results of the previously mentioned tests and considering the measure of central tendency along with quartiles. Strains were considered \u0026ldquo;positive\u0026rdquo; if the value of each strain was higher than the third quartile (e.g., a value\u0026thinsp;\u0026ge;\u0026thinsp;25% of the highest values in the dataset for one assay).\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equc\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e$$Score= \\frac{\\sum positive}{total number of essays} \\times 100$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eStrains were given a score according to their relative performance on each functional assay: score 1 if Q2\u0026thinsp;\u0026lt;\u0026thinsp;X\u0026thinsp;\u0026gt;\u0026thinsp;Q3 and score 2 if X\u0026thinsp;\u0026gt;\u0026thinsp;Q3.\u003c/p\u003e\n \u003cp\u003eDouble hits (DH\u0026thinsp;=\u0026thinsp;1 point) of inhibition by the n-CFSs against \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 (SA) or \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 (CA) corresponded to a significant effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) towards a given pathogen detectable for two or more growth parameters. Strains whose n-CFSs at pH 7.0 showed a double hit (DH) inhibition and o-CFSs that showed a single inhibition effect (H) on \u003cem\u003eA\u003c/em\u003e at pH 4.0 were scored according to the following calculation:\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equd\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e$$Inhibition Score =\\left(4\\times {DH}_{CA}+ {DH}_{SA}\\right)+({4 \\times H}_{CA}+ {H}_{SA})$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eDouble hit (DH) or hit (H) on \u003cem\u003eA\u003c/em\u003e for \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 were multiplied by 4 since the frequency of a double hit of inhibition on \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 was ca. 4 times lower than the one on \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003eBiofilm preparation\u003c/h2\u003e\n \u003cp\u003eOvernight planktonic cultures of the lactobacilli were centrifuged (7,500 rpm, 10 min, 4\u0026deg;C) and the pellets from each strain were collected and washed twice in saline solution. The suspensions were diluted to an OD\u003csub\u003e620\u003c/sub\u003e of ca. 0.25 with saline solution. One 5-\u0026micro;L drop of diluted culture was used to inoculate individual sterile membrane filters (pore size, 0.22 \u0026micro;m, Whatman) resting on MRS agar Petri dishes. The membranes were sterilized by UV exposure (15 min per side) before inoculation. The plates were inverted after the inoculum and incubated at 37\u0026deg;C, with the membrane-supported biofilms transferred to a fresh MRS agar Petri dish every 8 to 10 h (Anderl, Franklin, and Stewart \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e). Five membranes were prepared for each strain.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003eBiofilm imaging by confocal laser scanning microscopy\u003c/h2\u003e\n \u003cp\u003eOne membrane-supported biofilm for each strain culture was visually inspected and photographed by CLSM (Leica SP8LIA, Leica Microsystems). The membrane-supported biofilms were carefully mounted on glass slides. Bacterial cells and the polysaccharide fraction of ECM were stained with 15 \u0026micro;M SYTO\u0026reg; 9 (Invitrogen) and 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Texas Red\u0026reg;-labeled Concanavalin A (ConA, Invitrogen, stock solution, 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0.1 M sodium bicarbonate) solution in PBS (pH 7.5). Samples were incubated under dark conditions for 1 h at room temperature. Then, biofilm images were collected with a CLSM with excitation at 488 nm and emission\u0026thinsp;\u0026gt;\u0026thinsp;552 nm lasers. Fluorescence emission was observed between 500\u0026ndash;565 nm (for SYTO\u0026reg; 9) and 565\u0026ndash;645 nm (for ConA). Images were captured with a 40x lens using immersion oil and analyzed with the software LAS X (Leica).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec21\"\u003e\n \u003ch2\u003eBiofilm growth and ECM characterization\u003c/h2\u003e\n \u003cp\u003eAt the end of the incubation, two membrane-supported biofilms for each strain were inserted in separate falcon tubes containing saline solution (9 mL) to quantify the biomass (mg). Cells were then detached and suspended through the vortex (1 min at maximum speed) and serially diluted. Dilutions were plated on MRS agar Petri dishes and the cell density (Log colony forming unit [CFU] mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was enumerated after 48 h of incubation at 37\u0026deg;C. The ECM components of the biofilm colonies were collected and characterized following the method described by Chiba et al. (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Briefly, the biofilm colonies were scraped from the membrane filters and suspended in NaCl solution (1.5 M). The suspensions were centrifuged (5,000 g, 10 min, 25\u0026deg;C) and the supernatants were collected as ECM fractions for the quantification of proteins, total saccharides, and eDNA. The concentration of protein was measured following the Bradford assay (Bradford, 1976), using bovine serum albumin (BSA) as a standard. The protein concentration was measured at 590 nm with the UV-1800 Spectrophotometer (SHIMADZU). The total saccharide concentration in the ECMs was measured by the phenol sulfuric acid method (Chiba et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e), using glucose as a standard. Briefly, the isolated ECM fractions (20 \u0026micro;L) were mixed with 5% phenol (20 \u0026micro;L) in a 96-well plate, before adding sulfuric acid (100 \u0026micro;L). The plate was then incubated for 10 min at room temperature, and then the absorbance was read at 492 nm with an Infinite\u0026reg; M Nano\u0026thinsp;+\u0026thinsp;Spectrophotometer (TECAN, Austria). The concentration of the eDNA in the ECM fractions was measured with NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec22\"\u003e\n \u003ch2\u003eGel formulation\u003c/h2\u003e\n \u003cp\u003eFrom the results of the second screening, \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1 were selected to be inserted in gel formulations that were developed in collaboration with the R\u0026amp;D Innovation center of Giuliani S.p.A. (Milan, Italy). The cell densities of overnight cultures of the selected lactobacilli were calculated by measuring the OD\u003csub\u003e620\u003c/sub\u003e using the 6715 UV/VIS Spectrophotometer (Jenway, UK). A final pellet of ca. 11 Log CFU mL\u003csup\u003e-1\u003c/sup\u003e was collected, then resuspended in 10 mL of vegetal glycerol (Acef s.p.a., Italy), to a final cell density of ca. 10 Log CFU mL\u003csup\u003e-1\u003c/sup\u003e. The glycerol suspension containing lactobacilli was then inserted in an aqueous-based gel formulation. The final formulation contained deionized water (77%), vegetal glycerol suspension (20%) (Solagum tara, Seppic, France), containing lactobacilli (2%), and EUXYL K712 (1%), a liquid cosmetic preservative, containing sodium benzoate and potassium sorbate. The final cell density of the lactobacilli in the probiotic gel was about 9 Log CFU mL\u003csup\u003e-1\u003c/sup\u003e. The viability of the lactobacilli in the gel was assessed by preparing serial dilutions and by plating these on MRS agar medium before any treatment. A total of four gels were prepared, one for each strain previously selected, \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1, one with the combination of the two strains, and one with no cell suspensions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec23\"\u003e\n \u003ch2\u003eHuman Vaginal Epithelium infection and treatment\u003c/h2\u003e\n \u003cp\u003eThe SkinEthic\u003csup\u003e\u0026trade;\u003c/sup\u003e HVE model was obtained from EpiSkin (Lyon, France). The HVE model is based on the vulvar epidermoid carcinoma cell line A431 cells which form a 3-D tissue like the human \u003cem\u003ein vivo\u003c/em\u003e vaginal mucosa when cultivated \u003cem\u003ein vitro\u003c/em\u003e on a polycarbonate filter in a chemically defined medium (de Brugerolle \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). After the arrival of the HVEs, the inserts were placed in a 24-well plate, containing maintenance medium (SkinEthic, Episkin) (1 mL), and re-equilibrated for 24 h at 37\u0026deg;C, in a humidified, 5% carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) atmosphere. The HVEs were inoculated with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 previously adjusted to an OD\u003csub\u003e600\u003c/sub\u003e ca. 6 Log CFU mL\u003csup\u003e-1\u003c/sup\u003e using a maintenance medium (SkinEthic, Episkin). The HVEs were previously incubated with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 (30 \u0026micro;L) in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, at 37\u0026deg;C for 24 h, to allow the pathogen adhesion and infection. At the end of the incubation period, the HVEs were treated with the gel formula previously prepared (within 24 h from the moment of preparation): two gel formulations containing the single strains of \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1, one gel containing the combination of these two strains, and a gel not containing any strain. Also, some HVEs were left untreated, while HVEs infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 were used as a positive control. After the analysis, the inserts were all incubated for the same time and in the same conditions previously described.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec24\"\u003e\n \u003ch2\u003eThe MTT assay for cell viability determination\u003c/h2\u003e\n \u003cp\u003eThe cell viability of HVEs after infection with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and treatment with the gels were determined by the MTT assay (Grela, Kozłowska, and Grabowiecka 2018). Briefly, the yellow water-soluble salt is reduced by mitochondrial dehydrogenases to purple water-insoluble formazan, according to the viability of cells. A final formazan extraction step is required, using an organic solvent (e.g., isopropanol). A stock solution of MTT (5 mg mL\u003csup\u003e-1\u003c/sup\u003e in PBS) was diluted (1:10) in the cell culture medium to prepare the MTT solution. The MTT assay was assessed using untreated HVEs (negative control), HVEs treated with blank gel (blank), HVEs infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 (positive control), and HVEs infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and treated separately with \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1, and with the gel containing a combination of these two strains. After 24 h of incubation, the inserts were rinsed with PBS and placed in a new 12-well plate containing MTT solution (300 \u0026micro;L). After 2 h of incubation in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, at 37\u0026deg;C, isopropanol (800 \u0026micro;L) was added to each well. The plate was incubated again for 1.5 h, in the same conditions above mentioned. Two aliquots (200 \u0026micro;L) were then taken from each well and placed in 96-well plates. The OD\u003csub\u003e570\u003c/sub\u003e was measured with a BioTek Micro-volume Plate Reader (BioTek Instruments Inc., Bad Friedrichshall, Germany) and elaborated with the ELX808 software (BioTek Instruments Inc., Bad Friedrichshall, Germany) (reference filter: 630 nm). Results were expressed as a percentage of viability compared to the negative control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of triplicate cultures), using the following formula:\u003c/p\u003e\n \u003cp\u003eviability (%) = [OD \u003csub\u003e(570 nm \u0026ndash; 630 nm)\u003c/sub\u003e test product / OD \u003csub\u003e(570 nm \u0026ndash; 630 nm)\u003c/sub\u003e negative control] x 100\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec25\"\u003e\n \u003ch2\u003eGene expression profiling\u003c/h2\u003e\n \u003cp\u003eThe total RNA was extracted from the HVEs, previously infected with \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 and treated with the gels, using the RNeasy mini kit (Qiagen, Valencia, CA, USA), according to De Vuyst (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, the circumference of the polycarbonate filter was dissected from the bottom of the insert using a sharp surgical blade and then transferred into a 12-well culture plate containing RLT buffer (600 \u0026micro;L). After 1 or 2 min, the stratum corneum, detached from the epidermis, was removed using a pair of tweezers and discarded. For disrupting keratinocytes, the epidermis was gently scratched with a micropipette tip in the lysis buffer, provided by the kit. The lysate was homogenized by pipetting and then transferred into a spin column placed in a 2.0 mL collection tube. This procedure allows the recovery of enough RNA from the HVEs for real-time (RT) polymerase chain reaction (PCR) analysis of gene expression. Following the instructions provided by the manufacturer, 2 \u0026micro;g of RNA templates were used to synthesize complementary DNA (cDNA) in a 20 \u0026micro;L reaction volume, using the \u003cem\u003ePrimeScriptTM RT Reagent Kit\u003c/em\u003e (TakaraBioInc., Japan). The cDNA was amplified and detected by the Stratagene Mx3000P RT-PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). PCR conditions were the following: 37\u0026deg;C for 15 min, 85\u0026deg;C for 5 sec, and 25\u0026deg;C for 2 min. Afterward, the TaqMan\u0026reg; Gene Expression Assays were carried out for RT-PCR using the following genes: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1, IL-8 Hs00174103_m1, the cadherin 1 (CDH1) Hs01023894_m1. The GAPDH was used as a housekeeping gene. PCR amplifications were carried out in 20 \u0026micro;L of total volume. The mixture of reaction contained 10 \u0026micro;L of 2X Premix Ex Taq (Takara, Japan), 1 \u0026micro;L of 20\u0026times; TaqMan Gene Expression assay, 0.4 \u0026micro;L of RoX Reference Dye II (Takara, Japan), 4.6 \u0026micro;L of water, and 4 \u0026micro;L of cDNA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec26\"\u003e\n \u003ch2\u003eData and statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll the analyses were performed considering three biological replicates analyzed in triplicate. The growth parameters of each pathogen were determined using the grofit R package (Kahm et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e) for each replicate of CFSs and controls. Growth parameters were determined using the free splines approach and bootstrapping with 100 resamplings. The inhibition effect was assessed through a non-parametric one-way Kruskal-Wallis followed by the Dunn Control post hoc test provided by the PMCMR R package (Pohlert, 2016). The resulting p-values were adjusted for multiple hypothesis testing using Benjamini and Hochberg false discovery rate correction (FDR). Data from the determination of biofilm formation were submitted for analysis of variance by the General Linear Model (GLM) of R statistical package (R, version 1.6.2 rcompanion.org/handbook/). Multi-comparison of treatment means was achieved by a Tukey-adjusted comparison procedure with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Mangiafico \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). For the gene expression profiling, the average value of the target gene was normalized using the GAPDH gene, and the relative quantification of the levels of gene expression was determined by comparing the \u0026Delta; cycle threshold (\u0026Delta;C\u003csub\u003et\u003c/sub\u003e) value (Vigetti et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). The statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc). Data are expressed as the mean, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), or mean fold change\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBHI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBrain Heart Infusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBacterial vaginosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCDH1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecadherin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ecDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplementary DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCFS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell-free supernatant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCFU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColony Forming Unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCLSM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConfocal laser scanning microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbon Dioxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eConA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConcanavalin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCST\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCommunity state type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eECM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eeDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEFSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEuropean Food Safety Authority\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEPS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eexopolysaccharides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFDA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and drug administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFDR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFalse discovery rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGroup B \u003cem\u003eStreptococcus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGRAS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGenerally recognized as safe\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydrogen peroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHVE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman vaginal epithelium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIL-8\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMRS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDe-Man-Rogosa-Sharpe\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMTT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffer solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eQPS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQualified Presumption of Safety\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-Time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard error of mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVVC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evulvovaginal candidiasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Open Access Publishing Fund of the Free University of Bozen-Bolzano.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRDC, MG, and FR conceived the idea. MAA designed the experiments. RDC and MG were the supervisors and coordinators of the research units. MAA, CC, DP, and AP carried out the experiments. MAA and CC analyzed the data and wrote the original manuscript draft. RDC and PF reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAcin-Albiac, Marta, Pasquale Filannino, Rossana Coda, Carlo G. Rizzello, Marco Gobbetti, and Raffaella Di Cagno. 2021. \u0026lsquo;How Water-Soluble Saccharides Drives the Metabolism of Lactic Acid Bacteria during Fermentation of Brewers\u0026acute; Spent Grain\u0026rsquo;. \u003cem\u003eMicrobial Biotechnology\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eAgace, W W, S R Hedges, M Ceska, and C Svanborg. 1993. \u0026lsquo;Interleukin-8 and the Neutrophil Response to Mucosal Gram-Negative Infection.\u0026rsquo; \u003cem\u003eJournal of Clinical Investigation\u003c/em\u003e 92 (2): 780\u0026ndash;85. https://doi.org/10.1172/JCI116650.\u003c/li\u003e\n \u003cli\u003eAllonsius, Camille N., Marianne F.L. van den Broek, Ilke De Boeck, Shari Kiekens, Eline F.M. Oerlemans, Filip Kiekens, Kenn Foubert, et al. 2017. \u0026lsquo;Interplay between Lactobacillus Rhamnosus GG and Candida and the Involvement of Exopolysaccharides\u0026rsquo;. \u003cem\u003eMicrobial Biotechnology\u003c/em\u003e 10 (6): 1753\u0026ndash;63. https://doi.org/10.1111/1751-7915.12799.\u003c/li\u003e\n \u003cli\u003eAnderl, Jeff N., Michael J. Franklin, and Philip S. Stewart. 2000. \u0026lsquo;Role of Antibiotic Penetration Limitation in \u003cem\u003eKlebsiella Pneumoniae\u003c/em\u003e Biofilm Resistance to Ampicillin and Ciprofloxacin\u0026rsquo;. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e 44 (7): 1818\u0026ndash;24. https://doi.org/10.1128/AAC.44.7.1818-1824.2000.\u003c/li\u003e\n \u003cli\u003eAtassi, Fabrice, Dominique Brassart, Philipp Grob, Federico Graf, and Alain L. Servin. 2006. \u0026lsquo;Lactobacillus Strains Isolated from the Vaginal Microbiota of Healthy Women Inhibit Prevotella Bivia and Gardnerella Vaginalis in Coculture and Cell Culture\u0026rsquo;. \u003cem\u003eFEMS Immunology and Medical Microbiology\u003c/em\u003e 48 (3): 424\u0026ndash;32. https://doi.org/10.1111/j.1574-695X.2006.00162.x.\u003c/li\u003e\n \u003cli\u003eBorges, Sandra, Joana Silva, and Paula Teixeira. 2014. \u0026lsquo;The Role of Lactobacilli and Probiotics in Maintaining Vaginal Health\u0026rsquo;. \u003cem\u003eArchives of Gynecology and Obstetrics\u003c/em\u003e 289 (3): 479\u0026ndash;89. https://doi.org/10.1007/s00404-013-3064-9.\u003c/li\u003e\n \u003cli\u003eBradford, Marion M. n.d. \u0026lsquo;A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding\u0026rsquo;, 7.\u003c/li\u003e\n \u003cli\u003eBroadbent, Jeff R., Eric C. Neeno-Eckwall, Buffy Stahl, Kanokwan Tandee, Hui Cai, Wesley Morovic, Philippe Horvath, et al. 2012. \u0026lsquo;Analysis of the Lactobacillus Casei Supragenome and Its Influence in Species Evolution and Lifestyle Adaptation\u0026rsquo;. \u003cem\u003eBMC Genomics\u003c/em\u003e 13 (1): 533. https://doi.org/10.1186/1471-2164-13-533.\u003c/li\u003e\n \u003cli\u003eBrugerolle, Anne de. 2007. \u0026lsquo;SkinEthic Laboratories, a Company Devoted to Develop and Produce In Vitro Alternative Methods to Animal Use\u0026rsquo;. \u003cem\u003eALTEX\u003c/em\u003e, 167\u0026ndash;71. https://doi.org/10.14573/altex.2007.3.167.\u003c/li\u003e\n \u003cli\u003eBurns, P., G. Vinderola, A. Binetti, A. Quiberoni, C.G. de los Reyes-Gavil\u0026aacute;n, and J. Reinheimer. 2008. \u0026lsquo;Bile-Resistant Derivatives Obtained from Non-Intestinal Dairy Lactobacilli\u0026rsquo;. \u003cem\u003eInternational Dairy Journal\u003c/em\u003e 18 (4): 377\u0026ndash;85. https://doi.org/10.1016/j.idairyj.2007.10.012.\u003c/li\u003e\n \u003cli\u003eCai, Hui, Rebecca Thompson, Mateo F. Budinich, Jeff R. Broadbent, and James L. Steele. 2009. \u0026lsquo;Genome Sequence and Comparative Genome Analysis of Lactobacillus Casei: Insights into Their Niche-Associated Evolution\u0026rsquo;. \u003cem\u003eGenome Biology and Evolution\u003c/em\u003e 1: 239\u0026ndash;57. https://doi.org/10.1093/gbe/evp019.\u003c/li\u003e\n \u003cli\u003eCeapa, Corina, Mark Davids, Jarmo Ritari, Jolanda Lambert, Michiel Wels, Tamara Smokvina, Willem M de Vos, Jan Knol, and Michiel Kleerebezem. 2016. \u0026lsquo;The Variable Regions of Lactobacillus Rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires\u0026rsquo;. \u003cem\u003eGenome Biology and Evolution\u003c/em\u003e 8 (6): 1889\u0026ndash;1905.\u003c/li\u003e\n \u003cli\u003eCeapa, Corina, Jolanda Lambert, Kees van Limpt, Michiel Wels, Tamara Smokvina, Jan Knol, and Michiel Kleerebezem. 2015. \u0026lsquo;Correlation of Lactobacillus Rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling\u0026rsquo;. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e 81 (16): 5458\u0026ndash;70. https://doi.org/10.1128/AEM.00851-15.\u003c/li\u003e\n \u003cli\u003eChiba, Akio, Shinya Sugimoto, Fumiya Sato, Seiji Hori, and Yoshimitsu Mizunoe. 2015. \u0026lsquo;A Refined Technique for Extraction of Extracellular Matrices from Bacterial Biofilms and Its Applicability\u0026rsquo;. \u003cem\u003eMicrobial Biotechnology\u003c/em\u003e 8 (3): 392\u0026ndash;403. https://doi.org/10.1111/1751-7915.12155.\u003c/li\u003e\n \u003cli\u003eCiocia, Felicia, Paul L. H. McSweeney, Paolo Piraino, and Eugenio Parente.\u0026nbsp;2013. \u0026lsquo;Use of Dairy and Non-Dairy Lactobacillus Plantarum , Lactobacillus Paraplantarum and Lactobacillus Pentosus Strains as Adjuncts in Cheddar Cheese\u0026rsquo;. \u003cem\u003eDairy Science \u0026amp; Technology\u003c/em\u003e 93 (6): 623\u0026ndash;40. https://doi.org/10.1007/s13594-013-0131-8.\u003c/li\u003e\n \u003cli\u003eCribby, Sarah, Michelle Taylor, and Gregor Reid. 2008. \u0026lsquo;Vaginal Microbiota and the Use of Probiotics\u0026rsquo;. \u003cem\u003eInterdisciplinary Perspectives on Infectious Diseases\u003c/em\u003e 2008: 1\u0026ndash;9. https://doi.org/10.1155/2008/256490.\u003c/li\u003e\n \u003cli\u003eDe Seta, Francesco, Giuseppina Campisciano, Nunzia Zanotta, Giuseppe Ricci, and Manola Comar.\u0026nbsp;2019. \u0026lsquo;The Vaginal Community State Types Microbiome-Immune Network as Key Factor for Bacterial Vaginosis and Aerobic Vaginitis\u0026rsquo;. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e 10 (October): 2451. https://doi.org/10.3389/fmicb.2019.02451.\u003c/li\u003e\n \u003cli\u003eDe Vuyst, Evelyn. 2014. \u0026lsquo;The Reconstructed Human Epidermis Models in Fundamental Research\u0026rsquo;. In \u003cem\u003eFundamentals of Tissue Engineering and Regenerative Medicine\u003c/em\u003e, edited by Ulrich Meyer, J\u0026ouml;rg Handschel, Hans Peter Wiesmann, Thomas Meyer, and Yves Poumay, 967\u0026ndash;76. Berlin, Heidelberg: Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-540-77755-7_67.\u003c/li\u003e\n \u003cli\u003eDi Cagno, Raffaella, Pasquale Filannino, Vincenzo Cantatore, Andrea Polo, Giuseppe Celano, Anđela Martinovic, Ivana Cavoski, and Marco Gobbetti.\u0026nbsp;2020. \u0026lsquo;Design of Potential Probiotic Yeast Starters Tailored for Making a Cornelian Cherry (Cornus Mas L.) Functional Beverage\u0026rsquo;. \u003cem\u003eInternational Journal of Food Microbiology\u003c/em\u003e 323 (June): 108591. https://doi.org/10.1016/j.ijfoodmicro.2020.108591.\u003c/li\u003e\n \u003cli\u003eDuar, Rebbeca M., Xiaoxi B. Lin, Jinshui Zheng, Maria Elena Martino, Th\u0026eacute;odore Grenier, Mar\u0026iacute;a Elisa P\u0026eacute;rez-Mu\u0026ntilde;oz, Fran\u0026ccedil;ois Leulier, Michael G\u0026auml;nzle, and Jens Walter. 2017. \u0026lsquo;Lifestyles in Transition: Evolution and Natural History of the Genus Lactobacillus\u0026rsquo;. \u003cem\u003eFEMS Microbiology Reviews\u003c/em\u003e 41 (1): S27\u0026ndash;48. https://doi.org/10.1093/femsre/fux030.\u003c/li\u003e\n \u003cli\u003eEFSA Panel on Biological Hazards (BIOHAZ). 2013. \u0026lsquo;Scientific Opinion on the Maintenance of the List of QPS Biological Agents Intentionally Added to Food and Feed (2013 Update)\u0026rsquo;. \u003cem\u003eEFSA Journal\u003c/em\u003e 11 (11). https://doi.org/10.2903/j.efsa.2013.3449.\u003c/li\u003e\n \u003cli\u003eFidanza, Mario, Pinaki Panigrahi, and Tobias R. 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In \u003cem\u003eMicrobiota of the Human Body: Implications in Health and Disease\u003c/em\u003e, edited by Andreas Schwiertz, 83\u0026ndash;93. Advances in Experimental Medicine and Biology. Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-31248-4_6.\u003c/li\u003e\n \u003cli\u003eMu\u0026ntilde;oz-Quezada, Sergio, Empar Chenoll, Jos\u0026eacute; Mar\u0026iacute;a Vieites, Salvador Genov\u0026eacute;s, Jos\u0026eacute; Maldonado, Miriam Berm\u0026uacute;dez-Brito, Carolina Gomez-Llorente, et al. 2013. \u0026lsquo;Isolation, Identification and Characterisation of Three Novel Probiotic Strains (Lactobacillus Paracasei CNCM I-4034, Bifidobacterium Breve CNCM I-4035 and Lactobacillus Rhamnosus CNCM I-4036) from the Faeces of Exclusively Breast-Fed Infants\u0026rsquo;. \u003cem\u003eBritish Journal of Nutrition\u003c/em\u003e 109 (S2): S51\u0026ndash;62. https://doi.org/10.1017/S0007114512005211.\u003c/li\u003e\n \u003cli\u003eNaglik, Julian R., David L. 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Reza Zariffard, Mardge H. Cohen, and Beverly E. Sha. 2008. \u0026lsquo;Vaginal IL-8 Levels Are Positively Associated with Candida Albicans and Inversely with Lactobacilli in HIV-Infected Women\u0026rsquo;. \u003cem\u003eJournal of Reproductive Immunology\u003c/em\u003e 78 (1): 76\u0026ndash;79. https://doi.org/10.1016/j.jri.2007.11.001.\u003c/li\u003e\n \u003cli\u003eTozetto-Mendoza, Tania R., Ann Marie Bongiovanni, Evelyn Minis, Iara M. Linhares, Allison Boester, Wilton S. Freire, Silvia H. Lima, et al. 2020. \u0026lsquo;Torquetenovirus Titer in Vaginal Secretions from Pregnant and Postpartum Women: Association with Absence of Lactobacillus Crispatus and Levels of Lactic Acid and Matrix Metalloproteinase-8\u0026rsquo;. \u003cem\u003eReproductive Sciences\u003c/em\u003e 27 (11): 2075\u0026ndash;81. https://doi.org/10.1007/s43032-020-00227-1.\u003c/li\u003e\n \u003cli\u003eVaneechoutte, Mario. 2017. \u0026lsquo;The Human Vaginal Microbial Community\u0026rsquo;. \u003cem\u003eResearch in Microbiology\u003c/em\u003e 168 (9\u0026ndash;10): 811\u0026ndash;25. https://doi.org/10.1016/j.resmic.2017.08.001.\u003c/li\u003e\n \u003cli\u003eVentolini, G., E. Mitchell, and M. Salazar. 2015. \u0026lsquo;Biofilm Formation by Vaginal Lactobacillus in Vivo\u0026rsquo;. \u003cem\u003eMedical Hypotheses\u003c/em\u003e 84 (5): 417\u0026ndash;20. https://doi.org/10.1016/j.mehy.2014.12.020.\u003c/li\u003e\n \u003cli\u003eVigetti, Davide, Manuela Viola, Eugenia Karousou, Manuela Rizzi, Paola Moretto, Anna Genasetti, Moira Clerici, Vincent C. Hascall, Giancarlo De Luca, and Alberto Passi.\u0026nbsp;2008. \u0026lsquo;Hyaluronan-CD44-ERK1/2 Regulate Human Aortic Smooth Muscle Cell Motility during Aging\u0026rsquo;. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e 283 (7): 4448\u0026ndash;58. https://doi.org/10.1074/jbc.M709051200.\u003c/li\u003e\n \u003cli\u003eVitali, Beatrice, Giovanna Minervini, Carlo Giuseppe Rizzello, Enzo Spisni, Simone Maccaferri, Patrizia Brigidi, Marco Gobbetti, and Raffaella Di Cagno.\u0026nbsp;2012. \u0026lsquo;Novel Probiotic Candidates for Humans Isolated from Raw Fruits and Vegetables\u0026rsquo;. \u003cem\u003eFood Microbiology\u003c/em\u003e 31 (1): 116\u0026ndash;25. https://doi.org/10.1016/j.fm.2011.12.027.\u003c/li\u003e\n \u003cli\u003eWijgert, Jhhm van de, and Mc Verwijs. 2020. \u0026lsquo;Lactobacilli-Containing Vaginal Probiotics to Cure or Prevent Bacterial or Fungal Vaginal Dysbiosis: A Systematic Review and Recommendations for Future Trial Designs\u0026rsquo;. \u003cem\u003eBJOG: An International Journal of Obstetrics \u0026amp; Gynaecology\u003c/em\u003e 127 (2): 287\u0026ndash;99. https://doi.org/10.1111/1471-0528.15870.\u003c/li\u003e\n \u003cli\u003eWitkin et al. 2013. \u0026lsquo;Influence of Vaginal Bacteria and D- and l-Lactic Acid Isomers on Vaginal Extracellular Matrix Metalloproteinase Inducer: Implications for Protection against Upper Genital Tract Infections\u0026rsquo;. \u003cem\u003eMBio\u003c/em\u003e 4 (4). https://doi.org/10.1128/mBio.00460-13.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vaginal microbiota, vaginal ecosystem, nomadic lactobacilli, probiotics, pathogens inhibition screening","lastPublishedDoi":"10.21203/rs.3.rs-2201461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2201461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e, which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed \u003cem\u003ein vitro\u003c/em\u003e for their ability to reduce the inflammation caused by \u003cem\u003eC. albicans\u003c/em\u003e infection on the reconstituted human vaginal epithelium (HVE).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLactiplantibacillu\u003c/em\u003es \u003cem\u003eplantarum\u003c/em\u003e produced both isomers of lactic acid, while \u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within \u003cem\u003eLact. paracasei\u003c/em\u003e strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. \u003cem\u003eS. agalactiae\u003c/em\u003e 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of \u003cem\u003eC. albicans\u003c/em\u003e UNIBZ54 was less frequent. Overall, \u003cem\u003eL. plantarum\u003c/em\u003e strains had the highest pathogen inhibition and functional scoring. \u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by \u003cem\u003eC. albicans\u003c/em\u003e infection in the HVE model.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains (\u003cem\u003eL. plantarum\u003c/em\u003e C5 and POM1), being applicable to preserve and improve human vaginal health.\u003c/p\u003e","manuscriptTitle":"Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-01 19:30:05","doi":"10.21203/rs.3.rs-2201461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-17T19:15:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-11-05T13:29:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"749fec83-1752-42ac-b06c-e20c1f0a77fe","date":"2022-10-28T19:43:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-27T19:30:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-27T15:52:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-27T15:52:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2022-10-25T08:36:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12446686-26c1-4075-80cb-8cfa739619c9","owner":[],"postedDate":"November 1st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:52:09+00:00","versionOfRecord":{"articleIdentity":"rs-2201461","link":"https://doi.org/10.1186/s12934-023-02030-4","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2023-02-11 18:43:15","publishedOnDateReadable":"February 11th, 2023"},"versionCreatedAt":"2022-11-01 19:30:05","video":"","vorDoi":"10.1186/s12934-023-02030-4","vorDoiUrl":"https://doi.org/10.1186/s12934-023-02030-4","workflowStages":[]},"version":"v1","identity":"rs-2201461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2201461","identity":"rs-2201461","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}