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In Vitro Activity of Lactobacillus fermentum LF5 Against Different Candida Species and Gardnerella vaginalis

A New Perspective to Approach Mixed Vaginal Infections?

Deidda, Francesca BS; Amoruso, Angela PhD; Allesina, Serena PhD; Pane, Marco BS; Graziano, Teresa BS; Del Piano, Mario MD; Mogna, Luca PhD

doi: 10.1097/MCG.0000000000000692
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Goals: This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and Gardnerella vaginalis to weigh its potential effectiveness even in mixed vaginal infections.

Background: A wide female population is suffering from various vulvovaginal infections. These diseases are often associated with a decrease in the concentration of Lactobacilli in the vagina. Mixed vaginal infections represent >20% of women with vulvovaginal infection.

Study: LF5 strain was cocultured in De Man, Rogosa and Sharpe with Candida according to a 1:100 ratio in favor of the yeast. Each culture was sampled after 24 hours of incubation for the selective enumeration of the yeasts performed on yeast extract glucose chloramphenicol agar medium.

The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants of L. fermentum LF5 ranging from 5% to 20% was quantified by means of optical density at 600 nm (OD600).

Results: L. fermentum LF5 demonstrated the ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms.

Furthermore, L. fermentum LF5 showed a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth.

Conclusions: The biotherapeutic LF5 could be the only documented strain effective in mixed forms. For this purpose, a human clinical trial is in progress.

*Biolab Research Ltd., Novara

Gastroenterology Department, Santa Rita Hospital—Policlinico di Monza, Vercelli, Italy

The study has been funded by Probiotical S.p.A.

F.D., A.A., S.A., M.P., T.G., and L.M. are employees of Biolab Research Ltd. M.D.P. is the Chief of Gastroenterology Department, Santa Rita Hospital—Policlinico di Monza.

Address correspondence to: Luca Mogna, PhD, Research & Development, Biolab Research Ltd., Via E. Mattei, 3, Novara 28100, Italy (e-mail: l.mogna@mofinalce.it).

A wide female population is suffering from various vulvovaginal infections (VVIs). These diseases are often associated with the decrease in the concentration of Lactobacilli in the vaginal milieu.

Abnormal vaginal discharge is a characteristic feature of VVI. Most common documented causes of symptomatic vaginal discharge includes bacterial vaginosis (BV), followed by vulvovaginal candidiasis and trichomoniasis.1

The incidence of Candida infections has substantially increased in recent years: individual susceptibility, pregnancy, antibiotic therapy, the use of contraceptives and spermicide, diabetes, and immunosuppression are factors that increase the risk for development of candidiasis.1 Use of broad-spectrum antibiotics and intravascular catheters in the intensive care units are also associated with a high risk of candidiasis among immunocompromised patients.

Each Candida strain has its own precise responsiveness to drugs, whereas Candida albicans, Candida dubliniensis, and Candida tropicalis are normally susceptible to all antifungals used for fungemia, Candida glabrata is less susceptible and Candida krusei intrinsically resistant to fluconazole.1 In addition, Candida parapsilosis is less susceptible to the echinocandins.2

Among Candida species, C. albicans accounts for the majority of superficial and systemic infections, usually associated with high morbidity and mortality due to increased antimicrobial resistance and restricted number of antifungal drugs.1,3

On the other side, BV is the most common cause of anomalous vaginal discharge in reproductive age women and one of its causative agents is the gram-variable microbe Gardnerella vaginalis in 80% of the reported cases.4 BV is not associated with a substantial local inflammation and sporadically there may be no symptoms, whereas the “fishy odor” test is always positive. It is not clear whether the primary event eliciting BV is the loss of key lactobacilli or the acquirement of a complex bacterial community typically found in this syndrome. However, these may be concurrent processes leading to a significant imbalance. BV is vastly prevalent, affecting on average from 10% to 30% of women.5

This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and G. vaginalis to weigh its potential effectiveness even in mixed vaginal infections.

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MATERIALS AND METHODS

Yeast Strains and Growth Conditions

Candida strains used in the present study were purchased from the American type culture collection (ATCC). In particular, the following biotypes were used: C. albicans (ATCC 10231), C. parapsilosis (ATCC 22019), C. krusei (ATCC 6258), C. glabrata (ATCC 2001), and C. tropicalis (ATCC 750).6

The Lactobacillus strain used in this study, isolated from vaginal swabs of a healthy female subject, was classified based on its peculiar phenotypic and genotypic features: L. fermentum LF5 (DSM 32277).

The Lactobacillus strain was grown overnight in De Man, Rogosa and Sharpe (MRS) broth (Difco, BD, MD). In all experiments, fresh broth cultures were used with all Candida strains. Before being employed in the experiment, individual Candida strains were grown in Sabouraud Dextrose Broth (Difco, BD, MD), an optimal medium for yeasts, for 48 hours in aerobic conditions.6

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Assessment of the Antagonistic Activity of LF5 Toward Candida Species

LF5 strain was cocultured in the same broth (MRS) with one of the above-mentioned Candida microorganisms. The ratio between the inocula of Lactobacillus and yeast was 1:100 in favor of the latter. Incubation was performed in aerobiosis at 37°C for 24 hours. Each culture (1 mL) was sampled after 24 hours of incubation for the selective enumeration of the yeasts, performed on yeast extract glucose chloramphenicol agar medium (Sigma-Aldrich, Milan, Italy). Yeast extract glucose chloramphenicol agar plates were incubated in aerobic conditions at 37°C for 4 days to allow the growth of any yeast present. Colonies were then counted and results expressed as number of colony forming units/mL.6

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Gardnerella Strain and Growth Conditions

The Gardnerella vaginalis strain employed was purchased from the American type culture collection (ATCC). Before being used in the experiment, the G. vaginalis ATCC 14018 strain was grown in brain-heart infusion broth (BHI) (Oxoid, Milan, Italy) containing 2% (wt/wt) gelatin, 0.5% yeast extract, 0.1% starch, and 0.1% glucose.7

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Assessment of the Antagonistic Activity of LF5 Toward G. vaginalis

Culture supernatants of LF5 were prepared as follows: an overnight culture in MRS broth was centrifuged at 5000×g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1 N, sterilized by filtration through syringe filters (Ministart pore size: 0.22 mm), and assayed for the presence of any inhibitory molecule in the broth. Neutralized supernatants of LF5 were then added in different percentages to fresh BHI broth, prepared as described above, inoculated with G. vaginalis. The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants ranging from 5% to 20%, after 24 and 48 hours of incubation at 37°C in microaerophilic conditions, was quantified by means of optical density at 600 nm (OD600). The growth of positive control (Gardnerella alone) was assessed in BHI broth added to an amount of fresh MRS broth ranging from 5% to 20%. Thus, the positive controls were as similar as possible to test tubes inoculated with LF5. This test was repeated 3 times to ensure reliability and reproducibility.

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RESULTS

Antagonistic Effects of L. Fermentum LF5 on Candida

As shown in Figure 1, L. fermentum LF5 demonstrated a marked ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms. The strongest effect was recorded with C. parapsilosis ATCC 22019 (5-log inhibition).

FIGURE 1

FIGURE 1

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Inhibitory Activity of L. Fermentum LF5 on Gardnerella vaginalis

The in vitro inhibitory results of LF5 toward G. vaginalis ATCC 10231 are shown in Table 1. L. fermentum LF5 demonstrated a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded with the neutralized supernatants in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth (Table 1).

TABLE 1

TABLE 1

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DISCUSSION

Until now, fluconazole and miconazole have been the most widely used molecules to treat vulvovaginal candidiasis.8 Anyway, their most important limitation is the inability to counteract infections carried by G. vaginalis that needs specific antibiotic therapy.

Our in vitro study has demonstrated the ability of the biotherapeutic L. fermentum LF5 to efficiently antagonize different Candida species and G. vaginalis at the same time.

The biotherapeutic LF5 could be the only documented strain effective in mixed forms that represent >20% of women with symptoms of VVIs.9 For this purpose, a human clinical trial is in progress.

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REFERENCES

1. Arendrup MC. Candida and candidaemia. Susceptibility and epidemiology. Dan Med J. 2013;60:B4698 (1-32).
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3. Nasrollahi Z, Yadegari MH, Roudbar Mohammadi S, et al.. Fluconazole resistance Candida albicans in females with recurrent vaginitis and pir1 overexpression. Jundishapur J Microbiol. 2015;8:e21468 (1-3).
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6. Vicariotto F, Del Piano M, Mogna L, et al.. Effectiveness of the association of 2 probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal candidiasis: a pilot study. J Clin Gastroenterol. 2012;46(suppl):S73–S80.
7. Castro J, Henriques A, Machado A, et al.. Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci. 2013;10:1193–1198.
8. Nagashima M, Yamagishi Y, Mikamo H. Antifungal susceptibilities of Candida species isolated from the patients with vaginal candidiasis. J Infect Chemother. 2016;22:124–126.
9. Kalia N, Singh J, Sharma S, et al.. Prevalence of vulvovaginal infections and species specific distribution of vulvovaginal candidiasis in married women of North India. Int J Curr Microbiol App Sci. 2015;4:253–266.
Keywords:

Gardnerella vaginalis; bacterial vaginosis; Lactobacillus fermentum; antimicrobial activity

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