Evaluation of antipathogenic activity and adherence properties of human Lactobacillus strains for vaginal formulations.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Evaluation of antipathogenic activity and adherence properties of human Lactobacillus strains for vaginal formulations M.C. Verdenelli1,2, M.M. Coman2,3, C. Cecchini1,2, S. Silvi1,2, C. Orpianesi1,2 and A. Cresci1,2 1 School of Biosciences and Biotechnologies, University of Camerino, Camerino, Italy 2 Synbiotec S.r.l., Spin-off of UNICAM, Camerino, Italy 3 School of Advanced Studies, University of Camerino, Camerino, Italy

Keywords adhesion, antipathogenic activity, Candida, Lactobacillus, vaginal ovules. Correspondence Maria C. Verdenelli, School of Biosciences and Biotechnologies, Via Gentile III da Varano, Camerino 62032, Italy. E-mail: [email protected] 2013/2243: received 8 November 2013, revised 21 January 2014 and accepted 22 January 2014 doi:10.1111/jam.12459

Abstract Aims: To test different Lactobacillus strains for their antipathogenic activity towards Candida strains and their adhesion properties for the preparation of vaginal ovules and douches to be used in vaginal candidiasis prevention. Methods and Results: Five strains of lactobacilli were tested for their antimicrobial potential against different clinically isolated Candida strains. They were also screened for their ability to produce hydrogen peroxide and to coaggregate with pathogens. Adhesion properties of the five different Lactobacillus strains to HeLa cells and the presence of arcA gene were also assessed. The in vitro experiments demonstrated that all the five Lactobacillus strains tested possessed inhibitory action against the Candida strains using the radial streak method, but the effect is strain dependent. The same situation arises with regard to the ability of coaggregation that is present in all the strains into different degrees. Only Lactobacillus rhamnosus IMC 501â and Lactobacillus paracasei IMC 502â were able to produce H2O2 and none of the strains possess arcA gene. The most adherent strains to HeLa cells were Lact. rhamnosus IMC 501â, Lact. paracasei IMC 502â and also their combination SYNBIOâ. This latter was selected for the preparation of ovules and douches using different matrix. Witepsolâ ovules have proved the best formulation in terms of probiotic viability. Conclusions: Lactobacillus rhamnosus IMC 501â, Lact. paracasei IMC 502â and SYNBIOâ were able to produce H2O2, to coaggregate and to exert antimicrobial activity against pathogenic Candida strains and to strongly adhere to HeLa cells. All these properties together with those technological make these strains good candidates for the realization of formulations suitable for vaginal health. Significance and Impact of the Study: To develop new vaginal formulations taking into account the impact of probiotic strains on pathogens as well as the technological properties of the strains to validate their effectiveness in human health.

Introduction Lactobacilli are the dominant bacteria of a healthy human vagina (Keane et al. 1997). An important role of vaginal lactobacilli is related to the maintenance of an environ-

ment that limits the growth of pathogenic microorganisms (Mastromarino et al. 2002). Certain factors that increase the risk of bacterial vaginosis (BV), yeast vaginitis and urinary tract infections (UTIs) are associated with decreased vaginal Lactobacillus populations (Klebanoff

Journal of Applied Microbiology 116, 1297--1307 © 2014 The Society for Applied Microbiology

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Lactobacillus against Candida

M.C. Verdenelli et al.

et al. 1991; Hawes et al. 1996). Moreover, recent studies have demonstrated a significant association between infection with HIV-1 and the depletion of vaginal lactobacilli, particularly among women with severe bacterial vaginosis (Atashili et al. 2008). These observations have led to research on the strains and properties of vaginal lactobacilli, which may be responsible for the maintenance of a pathogen-free environment in the urogenital tract. The protective role of lactobacilli seems to be based upon two mechanisms, namely specific adherence by selected Lactobacillus species to vaginal epithelium, leading to intensive colonization of this surface by formation of microcolonies and biofilm, and control of the remaining vaginal microflora by production of active metabolites, including acidic products, bacteriocin-like substances and hydrogen peroxide (H2O2; Strus et al. 2005). There is a growing interest in the use of lactobacilli of human origin as probiotics against urogenital tract infections (Mastromarino et al. 2002). Moreover, antimicrobial treatments are not always effective, and problems remain due to bacterial and yeast resistance, recurrent infections, as well as side effects. Alternative remedies are of interest to patients and their caregivers. The concept of restoring the Lactobacillus content of the vaginal microbiota as a barrier to prevent infection was first conceived by Canadian urologist Andrew Bruce in the early 1970s. Extensive research since then has shown that certain Lactobacillus strains are able to colonize the vagina, following vaginal suppository use and reduce the risk of urinary tract infection, yeast vaginitis and bacterial vaginosis. Strain selection at that time, and even recently, has been based upon in vitro tests and source of the strains, with human studies providing the definitive answer to whether or not strains can function as probiotics (Reid et al. 2003). Moreover, it is important to note that to confer healthy function, Lactobacillus to be used as probiotics should be viable upon administration, that’s why to evaluate the feasibility of the industrial production of probiotic formulation is as important as demonstrating the functional characteristics. In this study, several Lactobacillus strains were analysed for properties related to vaginal mucosal colonization and antagonism towards Candida species (adhesion to epithelial cells, hydrogen peroxide production, antimicrobial activity against different Candida species and coaggregation with pathogens). The aim of the study was to confirm the efficacy of these strains and to use them for the design of a product for local application to the vaginal tract. Vaginal ovules and douches containing selected Lactobacillus strains were produced and tested for the maintenance of lactobacilli viability over time. 1298

Materials and methods Microbial strains, cell lines and growth conditions The Lactobacillus strains used in this study (Table 1) were isolated from Italian elderly subjects during an EU project named Crownalife (Silvi et al. 2003). Lactobacillus rhamnosus IMC 501â and Lactobacillus paracasei IMC 502â were characterized as probiotics in previous studies (Verdenelli et al. 2009, 2011). Candida strains (Table 1) were clinically isolated from human vagina and supplied by Istituto Superiore di Sanita in Rome. Lactobacilli were grown in de Man, Rogosa, Sharpe (MRS) broth (Oxoid, Basingstoke, UK), whereas Candida strains were grown in Sabouraud (SAB) dextrose medium (Oxoid). All the strains were grown aerobically at 37°C. The HeLa cell line was grown in Dulbecco’s minimal essential medium (DMEM; PAA Laboratories GmbH, Pasching, Austria), supplemented with 10% foetal bovine serum (FBS), 1% L-glutamine and 1% antibiotic/antimycotic. Cells were cultured at 37°C in a humidified (95%) atmosphere 5% CO2 and sub-cultured twice per week, and the medium changed every 3 days. Detection of antifungal inhibitory activity by agar well diffusion assay Cell-free supernatant (CFS) of the Lactobacillus strains (15 9 108 CFU ml1) was obtained by centrifuging 1 ml of 05 McFarland suspension of each strain at 11 000 g for 20 min at 4°C (BIOFUGE pico; KENDRO Laboratory Products GmbH, Hanau, Germany). The pH of the final CFS was adjusted using 20 mol l1 NaOH or 01 mol l1 HCl and measured by pH meter (JENWAY 3510 pH meter; Stone Staffordshire, Stone, UK) to a value near Table 1 Lactobacillus and Candida strains used in this study Strain

Origin

Lactobacillus paracasei subsp. paracasei 303 Lactobacillus plantarum 319 Lactobacillus fermentum 404 Lactobacillus rhamnosus IMC 501â Lactobacillus paracasei IMC 502â SYNBIOâ* Candida albicans ISS1 Candida albicans ISS2 Candida glabrata ISS3 Candida krusei ISS4 Candida parapsilosis ISS5 Candida tropicalis ISS6 Candida albicans ISS7

Intestinal Intestinal Intestinal Intestinal Intestinal Clinical Clinical Clinical Clinical Clinical Clinical Clinical

isolate isolate isolate isolate isolate

isolate isolate isolate isolate isolate isolate isolate

*Combination 1:1 of Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â.




neutral pH from 55 to 65 to rule out possible inhibition effects due to organic acids. A part of neutralized CFS was then treated with 05 g l1 of catalase (Delgado et al. 2001) to eliminate the inhibitory activity of hydrogen peroxide. In this case, both H2O2 and organic acids action were ruled out so that inhibition zone, if present, will indicate the presence of bacteriocins. For the agar well diffusion assay, Petri dishes (9 cm) were filled with 20 ml molten SAB agar and allowed to solidify. The surface was dried in sterile working cabinet. 100 ll of pathogen (05 McFarland, 15 9 108 CFU ml1) suspension was seeded in the SAB plates. Wells (5 mm in diameter) were cut into these agar plates. The cut wells were filled with 100 ll of cell free supernatant of the Lactobacillus strain and labelled accordingly. The plates were incubated aerobically for 24 h at 37°C and later examined for zones of inhibition. The diameter of the zones of inhibition produced by the test strain was measured with a caliper. Determination of H2O2 production The qualitative plate assay for hydrogen peroxide by method of Tomas et al. (2004), in which a chromogen (TMB: 3,30 ,5,50 -tetramethyl-benzidine) and peroxidase enzyme (EC 1.11.1.7/horseradish peroxidase type II) are mixed with MRS agar, was used with some modifications (Tomas et al. 2004). The modification regards the addition of corn starch 2%, magnesium sulphate anhydrous 006% and manganese sulphate monohydrate 0012%, which were assay components proposed by Rabe and Hillier (2003) that improved the intensity of blue colour formed by the hydrogen peroxide producers. Active lactobacilli generated by sub-culturing the strains twice in MRS broth at 37°C for 24 h were serial diluted in saline solution. The diluted culture (01 ml) at dilution factor of 106–107 was spread on 15 ml of modified TMB-MRS agar supplemented with 13 ml suspension of the assay components. The plates were incubated at 37°C for 48 h in anaerobic jar. After the incubation the plates were exposed to air for 30 min to allow the development of colours. Every colony that was able to produce H2O2 developed a blue colour. Evaluation of antipathogenic activity by radial method To evaluate antipathogenic activity of Lactobacillus strains towards Candida spp. radial method was performed. Lactobacillus strains with a concentration corresponding to 05 McFarland (15 9 108 CFU ml1) were inoculated by a means of a wire loop on MRS+SAB agar by covering a circle area in the centre of a Petri dish. After 48 h of incubation at 37°C, the plates were seeded, by a means of a wire loop, with pathogen indicator strains (05

McFarland) by radial lines of inoculum from the border to the centre of the plate. Microbial interactions were analysed after 24 h of incubation at 37°C, observing the inhibition zone size. The growth inhibitory activity (GI) was calculated, subtracting the circle diameter (CD, mm) of the Lactobacillus spreading zone from the inhibition zone diameter observed (IZD, mm) as follows GI = (IZD-CD)/2 (Bosch et al. 2012). Coaggregation assay Lactobacillus strains were tested for their ability to coaggregate with the pathogens. The assay was performed as reported by Mastromarino et al. (2002) where a modified version of the previously reported method by Reid et al. (1990) was performed. 1 ml of each Lactobacillus suspension (109 CFU ml1 of phosphate-buffered saline —PBS) was mixed with 1 ml of Candida suspension (109 CFU ml1 of PBS) on a vortex mixer for at least 10 seconds and then incubated in a 12-well tissue culture tray for 4 h at 37°C, under agitation. The suspensions were then observed by inversion light microscopy to evaluate the aggregation degree and scored according to a scale from 0 (no aggregation) to 4 (maximum aggregation). A droplet of each suspension was then put on a glass slide and gram-stained for visual observation of aggregates. Adhesion test Adhesion of microorganisms to epithelial cells was studied as reported by Mastromarino et al. (2002) with some modifications. HeLa cells were grown in 75-cm2 flasks to confluent monolayer in 5% CO2 at 37°C in DMEM containing 10% FBS, 1% L-glutamine, 1% antibiotic/antimycotic. After the passage of HeLa cells by washing them with PBS and the detachment using Trypsin, manual count of HeLa cells was performed using Trypan blue. Lactobacillus strains were incubated in MRS broth at 37°C in aerobic condition for 48 hours. Candida strains were grown in SAB liquid medium at 37°C for 18 h. Microorganisms were then refreshed in newly made medium and incubated overnight in the same experimental conditions. After the incubation, cultures of microorganisms were washed twice at 2200 g for 10 min in PBS, pH 72. A bacterial suspension was prepared in DMEM with a concentration of 15 9 109 CFU ml1, corresponding to 5 McFarland standard solution. The concentration was also checked with absorbance reading. The initial bacteria viability was evaluated through serial decimal dilutions performed on the initial bacterial suspension prepared to treat the cells. 50 ll aliquots of each dilution were plated


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onto MRS agar and spread over the surface. After an aerobic incubation at 37°C for 24–48 h, the count was performed. Adhesion reaction was performed using 6-well culture plate containing a sterile coverslip in each well. Then, to each well 2 ml of HeLa cell suspension was added at a concentration of 4 9 105 cells ml1 and incubated in a 5% CO2 atmosphere at 37°C. After 48 hours, when the cells were grown to approximately 60% confluence, they were washed twice with PBS and inoculated with 1 ml of bacterial suspension at a concentration of 15 9 109 CFU ml1. The plates were then incubated for 1 h at 37°C in a 5% CO2 atmosphere to allow the adhesion of bacteria on cells. After 1 hour, cell monolayers were washed twice in PBS and fixed with 08 ml of MayGr€ unwald solution for microscopy (E. Merck, Darmstadt, Germany) per each well for 4 min, washed twice again with water and stained with Giemsa solution for microscopy (E. Merck) dye for 30 minutes under constant shaking. Then cells were washed twice, and coverslips were attached on slides. The slide was analysed with oil immersion microscopy (100X), and each HeLa cell was scored for the presence and number of bacteria attached. Each adherence assay was conducted in duplicate, and 50 randomly chosen cells were evaluated for microorganism adhesion. Bacterial counting after adhesion assay was performed through the use of microbiological method. After 1 h of incubation at 37°C, the cells were washed three times with PBS to remove non-adhering bacteria, while the adherent bacteria were released by applying a solution of Triton X100 (Sigma-Aldrich, Steinheim, Germany) and incubated for 5 min at 37°C. Cells were then suspended in 5 ml of saline solution, centrifuged at 2200 g for 5 min and resuspended in 1 ml of saline solution. Tenfold dilutions of microorganisms suspension were spread in triplicate onto MRS agar plates. Colonies were enumerated after 24–48 h of incubation at 37°C. arcA gene detection To detect arcA gene (key enzyme of the arginine deaminase pathway; Amer et al. 2013) the DNA has been isolated from Lactobacillus cells using the method of benzyl chloride (Zhu et al. 1993; Fujimoto et al. 2008). After the Lactobacillus strains were cultured on MRS agar from MRS broth culture and 48 hours of incubation at 37°C aerobically, one colony of pure culture of each strain was suspended in 1 ml of physiological solution. The pellet obtained after a centrifugation of 7 minutes at 11 000 g was resuspended with 250 ll of extraction buffer (100 mmol l1 TRIS-HCL, 40 mmol l1 EDTA, pH 9). 50 ll of SDS 10% and 150 ll of benzyl chloride were added, and the samples were incubated at 50°C for 30 min in a thermomixer at maximum speed. Following 1300

the addition of 150 ll of 3 mol l1sodium acetate, samples were kept on ice for 5 min and then centrifuged for 12 min at 11 000 g. Supernatants were collected and 06 volumes (v/v) of isopropanol were added at room temperature. Samples were maintained at room temperature for 2 min and centrifuged for 15 min at 11 000 g. After the elimination of the supernatant, 1 ml of 70% ethanol was added and after a centrifugation of 5 min at 11 000 g, supernatant was discarded and the eppendorf were kept open at room temperature to allow ethanol to evaporate. DNA concentrations were quantified using a BioPhotometer (Hamburg, Germany). The arcA gene was amplified by PCR using primers (forward 50 GTAAAGTGTAGCATAAGTGC-30 ; reverse 50 -CCTAAGCTATCAATGAACTTACG-30 ) based on the arcA gene (NCBI Accession No.AE0064331) of Lactococcus lactis ssp. lactis IL1403. PCR amplifications were performed in a Tpersonal thermal cycler (Biometra, G€ ottingen, Germany). The reaction mixtures contained 1 ll of genomic DNA, 125 ll of 2X Master Mix (Fermentas, Burlington, Canada), 05 ll (500 pmol ll1) of each primer and sterile ultrapure water in a final volume of 25 ll. PCR conditions were set as follows: 30 cycles of denaturing at 94°C for 1 min, annealing at 50°C for 2 min and elongating at 72°C for 3 min (Kim et al. 2007). Following amplification, PCR product sizes (approx. 1400 bp) were controlled on a 2% (wt/vol) agarose gel. Lactobacillus lactis 202, a strain available in our laboratory collection, was used as positive control. Because Lact. paracasei genomes are not harbouring arcA gene, they were used as negative controls. Vaginal formulations and productions Two different formulation including vaginal ovules and vaginal douches were prepared. To prepare ovules, two different matrices were used: polyethylene glycol 1500 (PEG; A.C.E.F., Piacenza, Italia) and Witepsolâ H 15 (A.C.E.F.). PEG 400 (A.C.E.F.) was used for douches preparation. The ovules were prepared by melting 80 g of PEG at 50°C and cooling down until it starts to solidify. A 08 g of lyophilized powder of the two bacterial strains, Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â, was added. After mixing well to obtain an homogeneous product, the product was poured into plastic stencils and cooled down at room temperature. The stencils were maintained in fridge until solidification, then they were opened and every vaginal ovules were taken with gloves and closed in aluminium package. For the Witepsolâ vaginal ovules production, the same procedure of PEG vaginal ovules was followed. The douche was prepared by transferring 40 ml of PEG 400 in a 50 ml Falcon. A 04 g of lyophilized powder was added. After




mixing well to obtain a homogeneous solution, the product was distributed in plastic packages. Vaginal ovules and douches were stored at room temperature. Viability test The viability of SYNBIOâ (Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â) added to the vaginal ovules and douches stored at room temperature was determined at the production date and every month up to 6 months. The serial dilution method, by plating onto MRS agar medium, was used for counting total lactobacilli. Data analysis, calculations and statistical analysis Results for the adhesion assay were expressed as the average of three independent experiments. Each experiment was performed with two parallels to correct for intra-assay variations. Statistical analysis was made by the program â GRAPHPAD PRISM 5.1 (GraphPad Software, San Diego, CA, USA). Data were subjected to one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Results Detection of antifungal inhibitory activity by agar well diffusion assay Lactobacilli were tested for their ability to produce inhibitory substances against the growth of Candida species. No inhibition against all the Candida species tested was detected by well diffusion assay (data not shown). Determination of H2O2 production Some of the strains tested for H2O2 production were found to be able to produce this antimicrobial compound. Following the exposure of the plates to atmospheric oxygen for 30 minutes, Lactobacillus strains able to produce H2O2 developed a blue colour. Lactobacillus rhamnosus IMC 501â and Lact. paracasei IMC 502â are able to produce H2O2 with a higher production in the case of Lact. paracasei IMC 502â. On the other hand, Lactobacillus fermentum 404, Lactobacillus plantarum 319, Lactobacillus paracasei subsp. paracasei 303 did not develop a blue colour, meaning that they are not H2O2 producing strains. Evaluation of antipathogenic activity by radial method Antipathogenic activity of Lactobacillus strains was evaluated through a novel method that takes into account the

ability of the whole cells of Lactobacillus strains to inhibit Candida strains while growing on the same plate with a mixed agar medium containing both media suitable for Lactobacillus strains (MRS) and medium suitable for Candida (SAB). Radial experiments showed that the capacity of lactobacilli to inhibit different Candida species varies according to the single Lactobacillus strain and the pathogen involved. As reported in Table 2, Lact. plantarum 319 showed a very good inhibitory activity towards all Candida species in particular against Candida albicans ISS1 and ISS7. Also Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â showed good inhibiting activity against Candida strains, in particular towards Candida krusei ISS4 and C. albicans ISS7, respectively. A higher antimicrobial activity of the mixture SYNBIOâ compared to individual strains towards Candida ISS1, ISS4, ISS5 and ISS6 has been highlighted. Coaggregation assay Coaggregation assay gave the possibility to obtain a measurement of the interaction between the several Lactobacillus strains and Candida species. Coaggregation ability appeared to be strains specific and to vary according to the Candida species involved (Table 3). Lactobacillus plantarum 319 showed the highest degree of coaggregation among the different Lactobacillus strains tested followed by SYNBIOâ. By the way, the coaggregation activity was observed also for the other tested strains. Figure 1 shows an example of coaggregation observed with a light microscope. Adhesion test Because the adhesion of microorganisms to epithelial cells represents an essential step for colonization and persistence in a specific site, Lactobacillus strains were examined for their ability to adhere to human HeLa cells, a cell line that originated from a human carcinoma of the cervix. The adhesion of the different strains to HeLa cells was expressed both as percentage and average number of adherent microorganisms per cell. A Number of adherent microorganisms per cell were evaluated by light microscope, examining 50 random cells. The percentage of cells with adherent microorganisms was also evaluated by microscopic examination, considering adherent cells only the cells with a number of microorganisms major to 5. In the case of bacterial adhesion (Fig. 2), the majority of the strains showed a high capacity to adhere to HeLa cells (Table 4). Only Lact. fermentum 404 showed a low adhesion ability with a mean of 785 049 bacteria per cell. Regarding the percentage of adhesion (Table 4), all the bacterial strains had a high percentage of adhesion


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Table 2 Growth inhibitory activity (GI) of Lactobacillus strains towards Candida species (mm) Candida species

Lactobacillus strains Lact. paracasei subsp. paracasei 303 Lact. plantarum 319 Lact. fermentum 404 Lact. rhamnosus IMC 501â Lact. paracasei IMC 502â SYNBIOâ†

C. albicans ISS1 223 144* 1440 345 648 335 730

070 171 088 007 028

C. albicans ISS2 688 010 1030 791 897 773 788

106 020 166 116 137

C. glabrata ISS3 298 060 368 302 518 470 355

053 081 060 106 014

C. krusei ISS4 930 141 1295 850 1065 530 1420

014 098 183 162 176

C. parapsilosis ISS5

C. tropicalis ISS6

415 099

448 194

508 502 618 393 633

081 119 102 060 173

705 531 632 693 830

049 145 144 060 176

C. albicans ISS7 642 010 1435 751 847 1743 1310

028 025 053 123 113

*Expressed as mean SD. †SYNBIOâ, mixture 1:1 of Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â.

Table 3 Coaggregation between Lactobacillus strains and Candida species Coaggregation score

Lactobacillus strains Lact. paracasei subsp. paracasei 303 Lact. plantarum 319 Lact. salivarius 404 Lact. rhamnosus IMC 501â Lact. paracasei IMC 502â SYNBIOâ

C. albicans ISS1

C. albicans ISS2

C. glabrata ISS3

C. krusei ISS4

C. parapsilosis ISS5

C. tropicalis ISS6

C. albicans ISS7

1*

1

2

2

1

2

3

2 1 1 1 2

3 1 2 2 2

4 2 3 3 3

3 2 2 2 2

3 2 2 2 2

3 2 2 2 2

4 3 3 3 3

*Score from 0 (no aggregation) to 4 (maximum aggregation; Reid et al. 1990).

Lact. paracasei IMC 502â and SYNBIOâ showed values of adhered cells significantly higher than those of the other two strains (P < 005, by ANOVA and Tukey’s Test). Moreover, the percentage of the adhesion was comparable to the results obtained with the viable count performed by light microscope observation. arcA gene detection No arcA gene was detected on DNA extracted from Lactobacillus strains, apart from the positive control Lactococcus lactis 202.

Figure 1 Coaggregation between Lactobacillus paracasei IMC 502â and Candida glabrata ISS3 (by light microscope, 1009).

around 100%, with the only exception of Lact. fermentum 404 (1932%). The viable count of adherent bacteria is represented in Table 5. It is possible to observe that all the Lactobacillus strains tested showed the ability to adhere to HeLa cells preserving their viability, particularly Lact. plantarum 319, Lact. rhamnosus IMC 501â, 1302

Vaginal ovules and douches production and probiotic strains viability On the basis of the antipathogenic properties, H2O2 production and especially the excellent adhesive properties, SYNBIOâ was chosen for the final formulation of vaginal ovules and douches. Figure 3 shows the viability of the strains of Lactobacillus was tested during 6 months of storage at +4°C. Witepsolâ formulation showed the highest bacterial concentration viability during the evaluated




(a)

(b)

(c)

(d)

(e)

(f)

Figure 2 Adherence of bacteria to vaginal epithelial cells (HeLa cells) as observed by light microscope after May-Grunwald/Giemsa stain (9100). (a) Lactobacillus paracasei subsp. paracasei 303; (b) Lact. plantarum 319; (c) Lact. fermentum 404; (d) Lact. rhamnosus IMC 501â; (e) Lact. paracasei IMC 502â; (f) SYNBIOâ.

Table 4 Adhesion of Lactobacillus strains to HeLa cells

Lactobacillus strains Lact. paracasei subsp. paracasei 303 Lact. plantarum 319 Lact. fermentum 404 Lact. rhamnosus IMC 501â Lact. paracasei IMC 502â SYNBIOâ

Cells with adherent bacteria*

% cells with adherent bacteria

Number of adherent bacteria per cell*

5000 000

100

3900 230

5000 000

100

3104 112

966 150

1932

785 049

5000 000

100

4710 266

5000 000

100

4140 208

4900 100

98

4503 143

Calculated evaluating 50 cells randomly. *Expressed as mean SD.

time, whereas PEG formulation both for ovules and for douches resulted to be less suitable for the preservation of bacteria. Discussion It is generally believed that vaginal Lactobacillus strains control the vaginal microflora, including Candida albicans (McGroarty, 1993), by colonizing the vaginal epithelium and inhibiting the growth of the other microorganisms. Therefore, Lactobacillus strains, as candidates for vaginal probiotics, are usually tested in vitro for their ability to adhere to the vaginal epithelium; their antimicrobial activity towards different pathogens, and their production of antimicrobial substances such as H2O2 which, according to

several authors, may be mainly responsible for inhibitory activity (Klebanoff et al. 1991; McGroarty et al. 1992). In our study, we evaluated bacteriocin production of all lactobacilli isolates against Candida strains by agar antagonism method, but no inhibitory activity was detected while the use of the radial method gave significant antipathogenic results. This is a direct antagonism procedure based on diffusion of inhibitory substances in agar medium. Antimicrobial properties of Lactobacillus strains tested are probably connected with production of extracellular, diffusible inhibitory substances, mainly lactic acid. It is remarkable to note that the SYNBIOâ combination is able to increase the antipathogenic activity of the two components, Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â, against C. albicans ISS1, C. krusei ISS4, C. parapsilosis ISS5 and C. tropicalis ISS6. These results can support the use of probiotic combination to improve their efficacy (Verdenelli et al. 2011). Lactobacillus plantarum 319 has also given very good results as inhibitory activity. The Presence of typical intestinal lactobacilli, such as Lact. plantarum, in vaginal environment has been reported previously and related to the decreased risk of bacterial vaginosis (Martın et al. 2012). Lactobacillus strains that produce hydrogen peroxide have been isolated from 79% to 96% of women with a health vaginal ecosystem (Silva et al. 1987). To select the most suitable strain, hydrogen peroxide production, an intrinsic protective mechanism in the vaginal compartment, was measured for Lact. rhamnosus IMC 501â, Lact. paracasei IMC 502â, Lact. plantarum 319, Lact. paracasei subsp. paracasei 303 and Lact. fermentum 404. Results from this study indicated that Lact. rhamnosus IMC 501â and Lact. paracasei IMC 502â were the only two strains able to produce hydrogen peroxide. The


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Table 5 Number of adherent bacteria determined by microbial viable count before and after the treatment of HeLa cells Viable cell count CFU ml1* Initial cell count

Lactobacillus strains Lact. paracasei subsp. paracasei 303 Lact. plantarum 319 Lact. fermentum 404 Lact. rhamnosus IMC 501â Lact. paracasei IMC 502â SYNBIOâ

(500 (115 (300 (400 (421 (200

012) 001) 021) 033) 004) 006)

9 9 9 9 9 9

Final cell count† 8

10 109 108 109 108 109

(853 (304 (350 (175 (127 (855

133) 004) 010) 021) 052) 024)

9 9 9 9 9 9

% of adhesion‡ 7a

10 108a,b 107a 109b 108a,b,c 108b

171 264 116 437 302 428

Means with different letters are significantly different from each other (Tukey’s test, P < 005). *Expressed as mean SD. †After the treatment of HeLa cells with the Lactobacillus strains. ‡(CFU ml1 (final) 9 100)/(CFU ml1(initial)).

Log CFU g–1 of product

10 8 6 4 2 0 0

1

4 2 3 Time (months)

5

6

Figure 3 Viable cell count of SYNBIOâ contained in vaginal ovules and douches during 6 months of storage. (▲) Polyethylene glycol (PEG) douches, (■) Witepsolâ ovules, (●) PEG ovules.

production of H2O2 by lactobacilli may represent an important no specific antimicrobial defence mechanism in the vaginal ecosystem. Hydrogen peroxide is toxic to many microorganisms at concentrations that are typical in the vaginal fluid, and thus providing an intrinsic protective mechanism in the vagina. In vaginal exudates, H2O2 is converted to reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide and hydroxylfree radicals that are highly toxic against several microorganisms (Kulisaar et al. 2002). Besides that lactobacilli keep a high oxireduction potential in the vaginal environment, which inhibits multiplication of strictly anaerobic bacteria (Aroutcheva et al. 2001). The absence of hydrogen peroxide-producing lactobacilli has been related to a higher risk of BV, recurrent urinary tract infection by Escherichia coli and increased susceptibility to the infection by human immunodeficiency virus (HIV-1; Reid et al. 1990; Tomas et al. 2003; Gil et al. 2010). In our studies, only some of the Lactobacillus strains showing inhibitory properties against Candida produced H2O2 in amounts detectable by the screening plate method. This does not support the hypothesis that for the direct 1304

inhibition of several Candida strains H2O2 is solely responsible. In the light of our experiments, it seems that anticandidal activity of Lactobacillus isolates is related to various overlapping mechanisms. Production of H2O2 does not seem to be a major mechanism in the direct inhibition caused by lactobacilli, and it is certainly not the sole active inhibitory product. When lactobacilli in the vagina form coaggregates and bind to pathogens, this results in a return to homoeostasis, as coaggregation creates a hostile biochemical micro-environment around a pathogen and prevents it from continuation of growth and domination of the niche (Younes et al. 2012). The co-aggregation can create so a micro-environment around the pathogen with a higher concentration of inhibitory substances and it can also block the dissemination of pathogens to tissue receptors (Drutz 1992) so that coaggregation of lactobacilli with Candida may also be important for the prophylaxis against vaginal infections by preventing the binding of Candida to the receptors of the vaginal epithelium (Boris and Barbes 2000). Indeed, the coaggregation may well impede the access of pathogens to tissue receptors and be an alternative explanation for the decreasing adherence of pathogens to vaginal epithelial cells in the presence of lactobacilli (Boris and Barbes 2000). In the present study, all the tested Lactobacillus strains showed ability to coaggregate even if with different extent. Lact plantarum 319 appeared to be the one with the highest coaggregation activity towards all Candida species studied followed by SYNBIOâ. This result can so be linked to the capacity of Lactobacillus strains to counteract Candida invasion and adhesion and so to be a very important mechanism related to the protective role against Candida vaginal infection demonstrated by several Lactobacillus strains. One of the tests considered as crucial by the FAO/ WHO for the in vitro evaluation of potential probiotic




candidates is their capacity to adhere to mucin and human epithelial cells (WHO 2002). Adhesion of lactobacilli to the vaginal epithelium has been described as the first step in the formation of a barrier to prevent undesiderable pathogen colonization (Oca~ na and NaderMacıas 2004). Adhesion of lactobacilli results in the formation of a bacterial film on the vaginal epithelium and may contribute to the exclusion of pathogens from the vaginal mucosa. In addition, lactobacilli may promote a healthy vaginal epithelial environment. In other words, vaginal epithelial cells may be protected by the adherence of lactobacilli, and the vaginal ecosystem may be more resistant to pathogens because of the presence of lactobacilli (Woojin et al. 2011). Horosova et al. (2006) proved, by in vitro tests, that the adhesion of lactobacilli reduced the number of adhering pathogens and was a pre-condition for the Lactobacillus health benefits. However, the behaviour of the probiotics in the vaginal tract seems to be strain-specific, and so it is important to determine the relevant strain characteristics to be used as a therapeutic agent. So, due to the fact that adhesive properties vary considerably between Lactobacillus strains (Mastromarino et al. 2002), in this study the adhesion capacity of each strains of tested lactobacilli to HeLa cells was investigated. Results indicate that adherence to epithelial cells varied greatly between the Lactobacillus strains studied. Lactobacillus rhamnosus IMC 501â, Lact. paracasei IMC 502â and SYNBIOâ were highly adhesive (>40 adherent bacteria per cell), whereas Lact. para paracasei 303 and Lact. plantarum 319 showed an intermediate adhesiveness (30-40 adherent bacteria per cell). Moreover, results of the viable cell count of Lactobacillus strains after adhesion, demonstrate the ability of all lactobacilli to preserve their viability. This is a very important aspect related to the effectiveness of the strains. In addition to the investigation of probiotic characteristics, Lactobacillus strains tested in this study were investigated for arcA gene presence. arcA gene encodes arginine deaminase (ADI) that is a key enzyme of the ADI pathway. In addition to the Lactobacillus strains, Lactococcus lactis isolated in the laboratory was investigated due to the fact that previous studies revealed it to be able to hydrolyse arginine possessing ADI pathway (Kim et al. 2007). Arginine deaminase catalyses the irreversible conversion of arginine to citrulline and ammonia, thereby decreasing the availability of medium arginine and consequently of ornithine, the starting material for the polyamine biosynthetic pathway. Polyamines are commonly found in increased concentrations in vaginal discharges of women with bacterial vaginosis (Chen et al. 1979, 1982) and contribute to the increased pH of the vaginal microenvironment and also to the clinical symptoms of bacterial vaginosis, in particular the ‘fishy’ odour that is

characteristic of vaginal discharges from affected women (Chen et al. 1979; Clay 1982). Moreover, arginine is involved in several biosynthetic pathways that significantly influence carcinogenesis and tumour biology (e.g. nitric oxide (NO) generation, creatine production and polyamine synthesis; Kim et al. 2007). Therefore, ADI-degraded arginine has been regarded as a potential anticancer agent. This study permitted to detect arcA gene only on positive control Lactococcus lactis 202 that appears so to possess metabolic properties able to inhibit opportunistic pathogens through the deprivation of arginine, an important source of carbon, nitrogen and energy for bacteria, to reduce biochemical markers (polyamine synthesis and elevated pH) observed in bacterial vaginosis, and to act as anticancer agent. In the light of our investigation, Lactococcus lactis appear to be very interesting as an addition value of our probiotic product due to the important characteristics related to the presence of arginine deaminase activity. The previous described tests were important to characterize and select Lactobacillus strains for properties that would make them a good alternative to the use of antibiotics to treat human vaginal infections by Candida. But to use the lactobacilli selected in the maintenance of a healthy state in the human female urogenital system, it is necessary to ensure that the viable lactobacilli remain producing these antagonistic compounds upon manipulations, storage and administration. Vaginal ovules and douches were produced using SYNBIOâ as preliminary evaluation of different matrix and cell viability during 6 months. The combination SYNBIOâ was chosen for the vaginal formulation on the basis of its promising antipathogenic properties and also for the synergistic activity of the two bacteria present in this combination (Cresci et al. 2005; Verdenelli et al. 2011). During 6 months of storage at room temperature, Witepsolâ ovules were the formulation that showed the highest suitability to preserve viable microorganism during time. Douches were not suitable formulation for bacteria viability due to the high activity water that hinders the survival of bacteria during storage at room temperature. In the light of results achieved in this study, some of the strains analysed were found to have good capacities to be considered as possible candidates to be used in the production of vaginal preparations (ovules or douches) alone or in combination with other lactobacilli (Martın et al. 2012). So, thanks to this study, it would be possible to design a novel probiotic preparation containing a mixture of strains of lactobacilli with antimicrobial, adhesive and biochemical characteristics effective as a valuable option to help restore and maintain urogenital health and also in preventing and/or treating the clinical signs


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and symptoms of vaginal infections by Candida. Further researches are needed to elucidate the mechanisms of action of probiotics in the vagina but the potential remain that the health of many women can be improved by probiotic intervention.

Conflict of Interest No conflict of interest declared. References Amer, M.N., Mansour, N.M., El-Diwany, A.I., Dawoud, I.E. and Rashad, F.M. (2013) Isolation of probiotic lactobacilli strains harboring L-asparaginase and arginine deiminase genes from human infant feces for their potential application in cancer prevention. Ann Microbiol 63, 1121–1129. Aroutcheva, A., Gariti, D., Simon, M., Shott, S., Faro, J., Simoes, J.A., Gurguis, A. and Faro, S. (2001) Defense factors of vaginal lactobacilli. Am J Obstet Gynecol 185, 375–379. Atashili, J., Poole, C., Ndumbe, P.M., Adimora, A.A. and Smith, J.S. (2008) Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies. AIDS 22, 1493–1501. Boris, S. and Barbes, C. (2000) Role played by Lactobacillus in controlling the population of vaginal pathogens. Microbes Infect 2, 543–546. Bosch, M., Nart, J., Audivert, S., Bonachera, M.A., Alemany, A.S., Fuentes, M.C. and Cu~ ne, J. (2012) Isolation and characterization of probiotic strains for improving oral health. Arch Oral Biol 57, 539–549. Chen, K.C.S., Forsyth, P.S., Buchanan, T.M. and Holmes, K.K. (1979) Amine content of vaginal fluid from untreated and treated patients with nonspecific vaginitis. J Clin Invest 63, 828–835. Chen, K.C.S., Amsel, R., Eschenbach, D.A. and Holmes, K.K. (1982) Biochemical diagnosis of vaginitis: determination of diamines in vaginal fluid. J Infect Dis 145, 337–345. Clay, J.C. (1982) The odour of non-specific vaginitis: a review. Eur J Clin Microbiol 1, 317–319. Cresci, A., Orpianesi, C., Silvi, S. and Verdenelli, M.C. (2005). Lactic acid bacteria strains exhibiting probiotic properties and compositions comprising the same. European Patent EP 1743042 B. Delgado, A., Brito, D. and Fevereiro, P. (2001) Antimicrobial activity of L. plantarum, isolated from a traditional lactic acid fermentation of table olives. Lait 81, 202–215. Drutz, D.J. (1992) Lactobacillus prophylaxis for Candida vaginitis. Ann Intern Med 116, 419–420. Fujimoto, J., Matsuki, T., Sasamoto, M., Tomii, Y. and Watanabe, K. (2008) Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA. Int J Food Microbiol 126, 210–215.

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Gil, N.F., Martinez, R.C.R., Gomes, B.C., Nomizo, A. and De Martinis, A.C.P. (2010) Vaginal Lactobacilli as potential probiotics against Candida spp. Braz J Microbiol 41, 6–14. Hawes, S.E., Hillier, S.L., Benedetti, J., Stevens, C.E., Koutsky, L.A., Wolner-Hanssen, P. and Holmes, K.K. (1996) Hydrogen peroxide producing lactobacilli and acquisition of vaginal infections. J Infect Dis 174, 1058–1063. Horosova, K., Bujnakova, D. and Kmet, V. (2006) Effect of lactobacilli on E. coli adhesion to Caco-2 cells in vitro. Folia Microbiol 51, 281–282. Keane, F.E.A., Ison, C.A. and Taylor-Robinson, D. (1997) A longitudinal study of the vaginal flora over a menstrual cycle. Int J STD AIDS 8, 489–494. Kim, J.E., Jeong, D.W. and Lee, H.J. (2007) Expression, purification, and characterization of arginine deiminase from Lactococcus lactis spp. lactis ATCC 7962 in Escherichia coli BL21. Protein Expr Purif 53, 9–15. Klebanoff, S.J., Hillier, S.L., Eschenbach, D.A. and Waltersdorph, A.M. (1991) Control of the microbial flora of the vagina by H2O2-generating lactobacilli. J Infect Dis 164, 94–100. Kulisaar, T., Zilmer, M., Mikelsaar, M., Vihalemm, T., Annuk, H., Kairane, C. and Kilk, A. (2002) Two antioxidative lactobacilli strains as promising probiotics. Int J Food Microbiol 72, 215–224. Martın, R., Sanchez, B., Suarez, J.E. and Urdaci, M.C. (2012) Characterization of the adherence properties of human Lactobacilli strains to be used as vaginal probiotics. FEMS Microbiol Lett 328, 166–173. Mastromarino, P., Brigidi, P., Macchia, S., Maggi, L., Pirovano, F., Trinchieri, V., Conte, U. and Matteuzzi, D. (2002) Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets. J Appl Microbiol 93, 884–893. McGroarty, J.A. (1993) Probiotic use of lactobacilli in the human female urogenital tract. FEMS Immunol Med Microbiol 6, 251–264. McGroarty, J.A., Tomczek, L., Pond, D.G., Reid, G., Bruce, A.W. (1992) Hydrogen peroxide production by Lactobacillus species: correlation with susceptibility to the spermicidal compound nonoxynol-9. J Infect Dis 165, 1142–1144. Oca~ na, V.S. and Nader-Macıas, M.E. (2004) Adhesion ability of Lactobacillus to vaginal epithelial cells. Methods Mol Biol 268, 441–445. Rabe, L.K. and Hillier, S.L. (2003) Optimization of media for detection of hydrogen peroxide production by Lactobacillus species. J Clin Microbiol 41, 3260–3264. Reid, G., MacGroarty, J.A., Domingue, P.A.G., Chow, A.W., Bruce, A.W., Eisen, A. and Costerton, J.W. (1990) Coaggregation of urogenital bacteria in vitro and in vivo. Curr Microbiol 20, 47–52. Reid, G., Jass, J., Sebulsky, M.T. and McCormick, J.K. (2003) Potential uses of probiotics in clinical practice. Clin Microbiol Rev 16, 658–672.




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Evaluation of vaginal antifungal formulations in vivo.

Characterisation of probiotic properties in human vaginal lactobacilli strains.

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains.

Characterization of indigenous lactobacillus strains for probiotic properties.

Antimicrobial activity of lactobacillus strains against uropathogens.

Probiotic potential of Lactobacillus strains with antimicrobial activity against some human pathogenic strains.

In vitro evaluation of immunological properties of extracellular polysaccharides produced by Lactobacillus delbrueckii strains.

Preliminary evaluation of probiotic properties of Lactobacillus strains isolated from Sardinian dairy products.

Evaluation of Physical Properties of Generic and Branded Travoprost Formulations.

Evaluation of chitosan based vaginal bioadhesive gel formulations for antifungal drugs.

Effect of two probiotic strains of Lactobacillus on in vitro adherence of Listeria monocytogenes, Streptococcus agalactiae, and Staphylococcus aureus to vaginal epithelial cells.

Evaluation of biochemical and molecular methods for Lactobacillus reuteri strains differentiation.

In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine.

Evaluation of 3 approaches for assessing adherence to vaginal gel application in clinical trials.

Characterization of surface layer proteins and its role in probiotic properties of three Lactobacillus strains.

Chitosan and Kappa-Carrageenan Vaginal Acyclovir Formulations for Prevention of Genital Herpes. In Vitro and Ex Vivo Evaluation.

Probiotic screening and safety evaluation of Lactobacillus strains from plants, artisanal goat cheese, human stools, and breast milk.

Characterization, phylogenetic affiliation and probiotic properties of high cell density Lactobacillus strains recovered from silage.

Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough.

Determination of Bacteriocin Genes and Antibacterial Activity of Lactobacillus Strains Isolated from Fecal of Healthy Individuals.

Probiotic properties of Lactobacillus strains isolated from gizzard of local poultry.

Evaluation of dengue virus strains for human challenge studies.

Safety Assessment of Two New Lactobacillus Strains as Probiotic for Human Using a Rat Model.

Surface structures and adherence properties of diverse strains of Haemophilus influenzae biogroup aegyptius.