Biosurfactant from vaginal Lactobacillus crispatus BC1 as a promising agent to interfere with Candida adhesion

Abstract

Background: Lactobacillus spp. dominating the vaginal microbiota of healthy women contribute to the prevention of urogenital and sexually transmitted infections. Their protective role in the vagina can be mediated by Lactobacillus cells themselves, metabolites or bacterial components, able to interfere with pathogen adhesion and infectivity. Vulvovaginal candidiasis (VVC) is a common genital infection, caused by the overgrowth of opportunistic Candida spp. including C. albicans, C. glabrata, C. krusei and C. tropicalis. Azole antifungal drugs are not always efficient in resolving VVC and preventing recurrent infections, thus alternative anti-Candida agents based on vaginal probiotics have gained more importance. The present work aims to chemically characterize the biosurfactant (BS) isolated from a vaginal Lactobacillus crispatus strain, L. crispatus BC1, and to investigate its safety and antiadhesive/antimicrobial activity against Candida spp., employing in vitro and in vivo assays.

Results: BS isolated from vaginal L. crispatus BC1 was characterised as non-homogeneous lipopeptide molecules with a critical micellar concentration value of 2 mg/mL, and good emulsification and mucoadhesive properties. At 1.25 mg/mL, the BS was not cytotoxic and reduced Candida strains' ability to adhere to human cervical epithelial cells, mainly by exclusion mechanism. Moreover, intravaginal (i.va.) inoculation of BS in a murine experimental model was safe and did not perturb vaginal cytology, histology and cultivable vaginal microbiota. In the case of i.va. challenge of mice with C. albicans, BS was able to reduce leukocyte influx.

Conclusions: These results indicate that BS from vaginal L. crispatus BC1 is able to interfere with Candida adhesion in vitro and in vivo, and suggest its potential as a preventive agent to reduce mucosal damage occasioned by Candida during VVC.

Keywords: Anti-Candida activity; Biosurfactant; Candida spp.; HeLa cells; Lactobacillus crispatus; Murine model; Vagina; Vulvovaginal candidiasis.

Fig. 1

Murine experimental models. a Safety assessment of biosurfactant (BS) from L. crispatus BC1 in the murine vaginal tract. b Preventive, simultaneous and preventive–simultaneous effect of BS against C. albicans 2 infection. H: Subcutaneous injection of 0.02 mg β-Estradiol 17-valerate (↑). Arrow correspond to one intravaginal (i.va.) inoculation of: saline (S, ), BS [20 µL containing 1.25 mg/mL of BS ( )], or C. albicans 2 [20 µL containing 10⁶ CFU (C.a., )] into BALB/c mice. Sd ( ): sampling day

Fig. 2

FT-IR spectrum of biosurfactant isolated from L. crispatus BC1

Fig. 3

Surface tension versus biosurfactant (BS) concentration (mg/mL). Data are plotted as mean values of surface tension (dyne/cm) ± standard deviation (n = 3)

Fig. 4

Emulsification properties of biosurfactant (BS) from L. crispatus BC1. Emulsification index at 24 h (EI24%) of BS and Tween 80 calculated on different substrates: olive oil, sunflower oil and wheat germ oil. Data are plotted as mean values of EI24% ± standard deviation (n = 3)

Fig. 5

Viability of cervix epithelial cells (HeLa) in presence of biosurfactant (BS) from L. crispatus BC1. Data are plotted as average values of cell viability (%) ± standard error. Statistically significant differences in mean values of cell viability (%) are indicated by different letters (P < 0.05)

Fig. 6

Interference of biosurfactant (BS) from L. crispatus BC1 with Candida adhesion to HeLa cells. C. albicans 1, 2 and 4 (a–c), C. tropicalis (d) C. krusei (e) and C. glabrata (f). Exclusion, competition and displacement mechanisms were studied. The results were expresses as percentages of adherent yeasts per HeLa cell and compared to adhesion without BS (control value), taken as 100%. Data are plotted as average values of number of Candida/HeLa cells (%) ± standard error. Statistically significant differences in mean values in each test (exclusion, competition, displacement) are indicated by different letters (P < 0.05)

Fig. 7

Anti-Candida activity of biosurfactant (BS) from L. crispatus BC1 in a murine experimental model. aC. albicans 2 (C.a.) viable cells and b leukocytes in vaginal samples of mice receiving preventive (P), preventive–simultaneous (P–S) or simultaneous (S) intravaginally (i.va.) administration of BS against i.va. challenge with C.a. C and BS correspond to mice inoculated only with C.a. and BS, respectively. Data are plotted as average values of C.a. viable cell or leukocyte numbers ± standard error. Statistically significant differences between the values of experimental groups on the same sampling day are indicated by different letters (P < 0.05). c May Grunwald–Giemsa-stained vaginal smears and d Hematoxylin–Eosin-stained vaginal slides of the different experimental group at day 1 of sampling. Influx of leukocytes in the vaginal washing and lumen of C.a.-challenged mice is indicated with black arrows. Results are representative of two independent experiments