Interactions between Candida albicans and vaginal lactobacilli

The aim of this project was to determine the effect of lactobacilli on Candida adhesion to vaginal epithelial cells in vitro and on the pathogenesis of vaginitis in experimental animals. Pretreatment of exfoliated human, mouse or rat vaginal epithelial cells with Lactobacillus acidopilus or L. fermentum inhibited subsequent adhesion of Candida albicans to the cells. Increasing the pretreatment concentration of lactobacilli increased inhibition of Candida adhesion to mouse vaginal cells, up to a maximum of 60%. Fresh vaginal isolates of Lactobacillus species showed greater adhesion to vaginal cells than culture collection strains. They also showed greater inhibition of Candida adhesion. It is possible that subculture of a Lactobacillus isolate may cause it to lose some ability to adhere. A fresh Lactobacillus isolate was able to inhibit four different strains of C. albicans to varying extents. Inhibition was greatest with the yeast strains which had shown least adhesion when pretreatment with lactobacilli was omitted. It is postulated that in the human vagina a C. albicans strain which is poorly adherent may be inhibited by lactobacilli and thus be unable to cause disease. A 20-fold concentrate of Lactobacillus culture supernatant had no effect on yeast adhesion to epithelial cells. It would appear that inhibition of Candida adhesion may be due to steric hindrance by the lactobacilli of the Candida binding sites. Further work was carried out to investigate the effect of vaginal pH, diabetes and steroid hormones upon Candida adhesion. It was observed that there was greater Candida adhesion when assays were carried out at pH 7.2 rather than pH 4.5 (vaginal pH). There was also more inhibition of Candida adhesion by lactobacilli when adherence assays were carried out at pH 7.2. Diabetes is often cited as a predisposing factor in Candida infections and women with diabetes certainly have a greater incidence of vaginal candidosis. In the present study, it was found that yeasts adhered in greater numbers to cells taken from diabetic mice, with the exception of the C. albicans strains NCPF 3153 and 'outbreak'. Growth of yeasts in the presence of oestradiol and progesterone had no significant effect on Candida adhesion. A slight decrease in Candida adherence was observed after hormone pretreatment of either epithelial cells or yeasts. This may have been due to non-specific inhibition. Experiments were carried out to investigate whether lactobacilli prevented Candida infection by inhibiting Candida growth and colonization. Lactobacillus and Candida strains tested were able to grow over a similar range of pH values. This suggests that they are likely to grow in the same environmental conditions and therefore interact. Lactobacilli did not inhibit Candida when the two organisms were grown together in mixed broth cultures. However lactobacilli did inhibit Candida growth in the sandwich plate assay when the plates were preincubated to allow bacterial growth prior to inoculation with Candida. It was found that greater numbers of lactobacilli in the base of the sandwich plate and longer preincubation of the lactobacilli gave greater yeast inhibition. Viable cells were not necessary to give inhibition of Candida growth. Inhibition was not due to nutrient depletion. The antimicrobial activity of lactobacilli was released into the culture supernatant. The active substance(s) passed through a cellulose membrane indicating a molecular mass of less than 10,000 Da. Concentrated culture supernatant of L. acidophilus and L. fermentum showed activity against C. albicans, Cryptococcus neoformans, Aspergillus niger and Trichopyton mejtagrophytes. Butanol-extracted culture supernatants of L. acidophilus and L. fermentum were also tested against a range of bacteria; antibacterial activity was seen against Staphylococcus aureus, Escherichia coll. Bacillus subtilis, B. megaterium, and Proteus vulgaris. After purification, low resolution mass spectrometry identified three compounds produced by the lactobacilli which inhibited Candida growth; they had molecular masses of 419, 433 and 447 Da. The course of Candida infection was followed in mice after treatment of the vagina with different concentrations of C. albicans. For diabetic mice the infection was seen to be much greater than for normal mice. In vivo interactions between C. albicans and lactobacilli were investigated using a rat model of Candida vaginitis. Establishment of a Lactobacillus population in the rat vagina, or pretreatment with concentrated Lactobacillus culture supernatant (which had shown antimicrobial activity against C. albicans and a range of other fungi and bacteria in plate tests) provided no significant protection against infection with C. albicans. However, this may reflect inadequacies of the rat model and does not preclude a role for Lactobacillus-Candida interactions in the pathogenesis of human infections.