In vitro characterization of Lactobacillus crispatus IP174178 and its live biotherapeutic product for prevention of vaginal infections

Background : Probiotics enhance human well-being. They can be used either as a preventive approach to maintain the microbiota or as an add-on treatment for some diseases. The lactobacilli constitute the predominant vaginal microorganism and have been associated with a beneficial effect in vaginal health by protecting and controlling the vaginal microbiota against bacterial vaginosis, vaginal candidiasis, and urinary tract infection. Results: We investigated the probiotic properties of the new vaginal Lactobacillus crispatus IP174178 strain isolated from healthy women. Lactobacillus crispatus IP174178 strain was identified by 16S rRNA sequencing and by biochemical characterization. L. crispatus IP174178 is Gram positive, Catalase negative, ADH negative, and an H 2 0 2 (5 mM) and lactic acid (D-lactate 6 g/l, L-lactate 3 g/l) producer, with a strong adherence capacity. Its sensitivity to antibiotics was evaluated. Conclusions: The native strain L. crispatus IP174178 showed natural antagonistic properties against vaginal pathogens ( Gardenerella vaginalis , Candida spp.) which is potentiated in its commercial live biotherapeutic product Physioflor ® . The L. crispatus IP174178 strain and its live biotherapeutic product Physioflor ® can thus be considered a good probiotic candidate for the prevention of vaginal infections. Results of ADH activity and H2O2 and lactic acid production were expressed by mean ± standard deviation between triplicate experiment and negative control.


Background
The Food and Agriculture Organization of the United Nations (FAO) define probiotics as "live microorganisms which, when administered in adequate amount, confer a health benefit on the host" [1]. These microorganisms exert health promoting properties for both prevention and treatment of a number of human diseases [2,3]. Interest in probiotic bacteria is increasing with the development of adjunct or alternative therapies to 3 traditional treatments. However, the isolation and characterization of new probiotic strains are required for the development of new therapies [4]. Lactobacilli have been commonly used as probiotics for many years since they are easy to "select", cultivate and produce [5,6]. Moreover, the use of selected lactobacilli may be effective in restoring the vaginal microbiota and preventing infections [7].
Lactobacilli are typically the prevalent microorganisms in the vaginal microbiota of healthy women of reproductive age. Lactobacillus spp. are commonly identified as the biomarker of a healthy vagina which protect the mucosa against the establishment of pathogenic microorganisms [8].This protection is mainly due to the ability to adhere to vaginal cells and the secretion of antimicrobial compounds such as H 2 O 2 , organic acids [9,10], and bacteriocins [11,12]. The vaginal microbiota has been characterized by the dominance of a single species most closely related to the Lactobacillus spp. (70 %) mainly L. crispatus (26 %), L. gasseri (6 %), L. jensenii (5 %) or L. iners (34 %) [13][14][15]. The identification of L. crispatus as the most frequently occurring species in white and Asian women is associated with a healthy and stable community [14,16]. Moreover, the vaginal colonization by this species has been reported to decrease the risk of bacterial vaginosis [17].
Vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and urinary tract infection (UTI) are the most common urogenital pathologies. These pathologies negatively impact women's quality of life and represent a significant healthcare cost [18,19]. Lactobacillus spp., and in particular L. crispatus, are commonly used as probiotics to prevent the development of pathogens, including those responsible for VVC and BV [20,21]. Importantly, the use of probiotics for the development of new therapies requires evaluation of quality of products (linked to the manufacturing process) and a standardization of the characterization techniques used. 4 In this study, we characterized the L. crispatus IP174178 strain isolated from the vaginal tract of healthy women. The genetic, phenotypic and antimicrobial properties were investigated to determine probiotic potential and safety evaluation of L.    Antimicrobial molecule detection Three antimicrobial activities were studied for the native strain (Table 5). No L. crispatus IP174178 alcohol dehydrogenase (ADH) activity was detected. Quantitative analysis by titration showed that the L. crispatus IP174178 produced 5.02 mM ±0.55 of H2O2. The level of D-Lactate and L-Lactate produced by L. crispatus IP174178 ranged from 3.10 g/l to 5.90 g/l. The ability to use glucose or glycogen as a carbon source was equivalent for both. Table 5. Physiological activity of L. crispatus IP174178 after 48 hours culture. Activity L. crispatus IP174178 ADH activity -H²O² 5.02 mM ±0.55 Lactic acid Glucose D-Lactate : 5.90 g/l ± 1.62 (66 %) L-Lactate : 3.10 g/l ± 1.01 (34 %) Glycogen D-Lactate : 5.65 g/l ± 1.96 (64 %) L-Lactate : 3.16 g/l ± 1.04 (36 %) Results of ADH activity and H2O2 and lactic acid production were expressed by mean ± standard deviation between triplicate experiment and negative control.
In vitro adhesion to epithelial cells The adhesion of the microorganisms to epithelial cells was quantified. L. crispatus IP174178 showed a significant ability to adhere to Caco-2 and CRL-2616 vaginal cells at MOI 100 to MOI 0.1: > 20 % after 3 hours of incubation. No significant difference in adhesion properties was detected between the two epithelial cell 7 types (Fig 1A).

IP174178 immunomodulation The in vitro immunomodulation of PBMCs induced by
IP174178 revealed low secretion of anti-inflammatory IL-10 compared to Bifidobacterium longum IPL, the reference anti-inflammatory strain of the Lille Pasteur Institute (Fig. 1B).
IP174178 induced a slight increase in TNF-α secretion but no significant production of IL-12p70 or IFN-γ cytokines compared to the reference pro-inflammatory Lactococcus lactis IPL.
Acid and bile tolerance The native strain and its LBP Physioflor® showed a good resistance to pH 2.5, pH 3.0 and pH 4.0 for 90 min (Fig 2A-2B) and tolerance to exposure to 3% bile salt for 240 min (Fig 2C-2D) with no impairment of viability.
Antimicrobial activity of Lactobacillus crispatus IP174178 and Physioflor® During coculture the native strain induced a loss of viability of 2.02 log and 2.29 log to C. albicans ATCC® 10231TM after respectively 24 h and 48h ( Fig 3A). Similarly, a loss of viability of 1.12 log and 1.66 log to clinical C. albicans was observed after 24 and 48 hours of coculture ( Fig 3B). No significant growth inhibition of C. glabrata was detected after 24 hours of co-culture but after 48 hours a loss of viability of 1.87 log was observed ( Fig 3C).
Physioflor® induced complete loss of viability of C. albicans ATCC® 10231TM (7.1 log), clinical C. albicans (7.23 log) and C. glabrata (7.38 log) after 24 hours of contact ( are essential to guarantee their identity and consequently their safety and proclaimed health benefits [22].
Vaginal microbiota of healthy women is dominated by Lactobacillus spp., which play a key role against pathogen infections [23]. If the microbiota balance is disturbed, pathologies can develop such as bacterial vaginosis. This pathology usually induces dysbiosis with a decreased abundance of lactobacilli. LBP supplementation could restore healthy communities [24]. This is the case for recurrent vaginal infections. The usual drugs are relatively effective to treat recurrent BV and VVC, but probiotic supplementation provides restoration of vaginal communities and thus reduces the risk of infection recurrences [25,26].
The safety assessment including antimicrobial susceptibility is a necessary evaluation to be considered for probiotic application. The immune response induced by L. crispatus IP174178 was evaluated by flow cytometry analysis of cytokine production. IP174178 induced a slight increase in IL-10 and TNF-α secretion and no significant production of the immune mediators IL-12p70 and INF-y cytokines compared to reference strains. The level of INF-γ was correlated with the level of IL-12p70 as generally observed for lactobacilli [27]. The cytokine profile of IP174178 on human PBMCs tends to be neutral (cytokine ratio <5) according to Pot et al [28]. So, L. crispatus IP174178 was not associated with inflammatory response, consistently with in vitro and in vivo studies which demonstrate that vaginal lactobacilli are generally non-inflammatory [29,30]. Commensal and probiotic bacteria play a role in maintaining vaginal homeostasis through several mechanisms to promote antimicrobial defense while not inducing immunomodulation [31].
According to EFSA recommendations, IP174178 antimicrobial susceptibility was determined against 14 antibiotics.L. crispatus IP174178 strain demonstrate a sensitivity to 8 antibiotics tested. As majority of Lactobacillus, IP174178 strain is susceptible to ampicillin, tetracycline, erythromycin and chloramphenicol [32]. Conversely, L. crispatus IP174178 could be associated with metronidazole treatment to eliminate vaginal infections.
The safety assessment of L. crispatus IP174178 strain, isolated from healthy vaginal microbiota, reveals an interesting probiotic potential which induced an in vitro characterization of its probiotic properties. The adherence capability and ability to survive under high acidic conditions are key properties to be considered during probiotic potential evaluation. As several Lactobacillus; L. crispatus IP174178 showed acid and bile salt tolerance which suggest appropriate probiotic application. Moreover, adhesion of probiotic strains to the vaginal epithelium is key event order to prevent the colonization of pathogenic microorganisms by occupying binding sites [33]. Our study showed favorable adhesion to human vaginal and intestinal cells suggesting that IP174178 has capability to adhere to the human epithelium. Similarly, high adherence capability to Caco-2 cells was reported for Lactobacillus strains [34] Herein, we show that the strain isolated from healthy vaginal microbiota, IP174178, produces both hydrogen peroxide and lactic acid which could contribute to create a hostile environment for the growth and development of pathogenic microorganisms [35]. Indeed, lactic acid has previously been shown to maintain vaginal pH below 4.5 [12,36].
Moreover, in vitro,L. crispatus IP1714178 induced a significant loss of viability of the pathogenic strains. L. crispatus IP174178 was selected for its probiotic potential, taxonomy and its anti-Candida properties for a vaginal application. As shown in previous studies, manufacturing processes and large scale production influence the strain properties and have sometimes improving potency [37][38][39]. Because the industrial process impact the intrinsic properties of bacteria, same parameters need to be reevaluated using final product containing probiotic bacteria [38,39]. In order to evaluate tolerance and efficacy of Physioflor ® , a clinical trial needs to be carry-out. However, we previously aimed to select and optimize the industrial formulation.
The optimization parameters were thus performed by analyzing the bacterial viability, antimicrobial properties and probiotic use. crispatus IP174178 need to be investigated and will be addressed in other publication.

Genetic identification
L. crispatus IP174178 specific primers were designed with a home-made BioPerl software using Primer3 and their specificity checked using Fuzznuc [42,43]. Their specificities were further checked using Blast software [44,45]  al. [47]. For undefined antibiotics, the sensitive profile was defined by an inhibition zone > 0 mm.
In vitro adhesion to epithelial cells 15 Adhesion capacity of L. crispatus IP174178 strain was determined according to Nivoliez et al. approach [38]. Two human cell lines were used: colon carcinoma cell line Caco-2 [48] and normal vaginal epithelial cells (VK2/E6E7 ATCC-CRL-2616) [49]. Acid and bile tolerance According to Nivoliez et al. protocol [38] the L. crispatus IP174178 acid tolerance was evaluated using gastric juice at pH 3.0 and pH 2.5. Bile tolerance was assed in MRS medium containing 3% of bile salts. Gastric juice and bile solutions were inoculated with 1.10 8 CFU/ml of a 48-h culture of IP174178 in a final volume of 10 ml. The residual viability was determined by plating serial dilutions onto MRS agar plates at T0, T45 and T90 for acid tolerance test and at T0, T60, T120, T180, and T240 min for bile tolerance test.
All tolerance tests were performed in triplicate.

Detection of antimicrobial molecules
To investigate the antimicrobial molecules secreted by L. crispatus IP174178 the approach described previously by Nivoliez et al. [38] was applied with some modifications.
The bacterial arginine deaminase activity was determined using Diatab kit (EUROBIO, Courtaboeuf, France) according to the manufacturer's instructions.
Hydrogen peroxide production was quantified by colorimetry using an enzymatic peroxidase reaction. After 48 h of anaerobic incubation in BHI broth (MRS interacts with reagents) at 37 °C, L. crispatus IP174178 cells were exposed one hour to aerobic conditions. One mililitre of supernatant was added to a solution containing 100 µl of 4aminoantipyrine (4 mg.mL-1 solution of 4-amino-2, 3-dimethyl-1-phenyl-3-pyrazolin-5- To measure the lactic acid production, L. crispatus IP174178 was inoculated at 1.10 6 CFU/ml in MRS broth containing a only glucose or glycogen carbon source at 20 g/l and incubated at 37 °C for 48 h. The culture was then centrifuged at 9.000 g for 15 mins. The lactic acid production was measured in the supernatant using a specific enzymatic kit (Enzytec TM d/l-Lactic acid kit, BIO-CONTROL SYSTEM, Nieuwerkerk aan den IJssel, Netherlands).

Vaginal pathogen growth inhibition
The pathogens C. albicans and G.vaginalis [38], and the probiotic strain were subcultured for 48 h in their respective media and then mixed in similar ratios (1.10 8 CFU/ml each).
During the 48 h of co-incubation, viable pathogen cells were enumerated by plating serial dilutions and pH was measured in sample aliquots (numeric probe Mettler Toledo MA235 -InLab413). The controls corresponded to pathogens incubated with the additive of Physioflor ® and without L. crispatus IP174178 in culture medium with adjusted pH.
Because G. vaginalis is sensitive to pH, a culture medium pH adjusted before inoculation 18 with HCl 0.1 M (DUTSCHER, Brumath, France) was used as growth control. Medium adjusted at pH 4 and pH 5 were evaluated for all pathogens, and medium at pH 4.5 was also tested with G. vaginalis. Each assay was performed in triplicate.

Statistical analysis
Statistical analyses were performed using a one-way ANOVA test followed by Bonferroni correction for growth experiments and were carried out using GraphPad Prism 5 software (www.graphpad.com/prism) at several significance levels (* p < 0.05; ** p < 0.01; *** p < 0.001).

Ethics approval and consent to participate
Not applicable Consent for publication Not applicable Availability of data and materials All data generated and analyzed during this study are included in the published article.

Competing interest
Adrien NIVOLIEZ and Caroline DAUSSET have an institutional affiliation with the company which manufactures the product.
Christelle DANIEL and Gilles BRAMI have an institutional affiliation with IPRAD which markets the product.
Funding IPRAD company provided financial support in the form of salaries and analysis, this commercial affiliation didn't influence the study.

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AN analyzed data managed the project and contribute in writing the manuscript. GB was a contributor of project management and in reviewing the manuscript. ChD was a contributor in writing the manuscript. CaD performed several experimentations and was a major contributor in writing the manuscript. experiments were performed in triplicate and the mean of results are expressed with standard deviations.