Associations between the vaginal microbiome and Candida colonization in women of reproductive age

Abstract

Background: The composition of bacteria within the vaginal microbiome has garnered a lot of recent attention and has been associated with reproductive health and disease. Despite the common occurrence of yeast (primarily Candida) within the vaginal microbiome, there is still an incomplete picture of relationships between yeast and bacteria (especially lactobacilli), as well as how such associations are governed. Such relationships could be important to a more holistic understanding of the vaginal microbiome and its connection to reproductive health.

Objective: The objective of the study was to perform molecular characterization of clinical specimens to define associations between vaginal bacteria (especially Lactobacillus species) and Candida colonization. In vitro studies were conducted to test the 2 most common dominant Lactobacillus species (Lactobacillus crispatus and Lactobacillus iners) in their ability to inhibit Candida growth and to examine the basis for such inhibition.

Study design: A nested cross-sectional study of reproductive-age women from the Contraceptive CHOICE Project was conducted. Vaginal swabs from 299 women were selected to balance race and bacterial vaginosis status, resulting in a similar representation of black and white women in each of the 3 Nugent score categories (normal [0-3], intermediate [4-6], and bacterial vaginosis [7-10]). Sequencing of the 16S ribosomal gene (V4 region) was used to determine the dominant Lactobacillus species present (primarily Lactobacillus iners and Lactobacillus crispatus), defined as >50% of the community. Subjects without dominance by a single Lactobacillus species were classified as Diverse. A Candida-specific quantitative polymerase chain reaction targeting the internally transcribed spacer 1 was validated using vaginal samples collected from a second cohort of women and used to assess Candida colonization. Two hundred fifty-five nonpregnant women with sufficient bacterial biomass for analysis were included in the final analysis. Generalized linear models were used to evaluate associations between Lactobacillus dominance, sociodemographic and risk characteristics, and vaginal Candida colonization. In separate in vitro studies, the potential of cell-free supernatants from Lactobacillus crispatus and Lactobacillus iners cultures to inhibit Candida growth was evaluated.

Results: Forty-two women (16%) were vaginally colonized with Candida. Microbiomes characterized as Diverse (38%), Lactobacillus iners-dominant (39%), and Lactobacillus crispatus-dominant (20%) were the most common. The microbiome, race, and Candida colonization co-varied with a higher prevalence of Candida among black women and Lactobacillus iners-dominant communities compared with white women and Lactobacillus crispatus-dominant communities. Lactobacillus iners-dominant communities were more likely to harbor Candida than Lactobacillus crispatus-dominant communities (odds ratio, 2.85, 95% confidence interval, 1.03-7.21; Fisher exact test, P = .048). In vitro, Lactobacillus crispatus produced greater concentrations of lactic acid and exhibited significantly more pH-dependent growth inhibition of Candida albicans, suggesting a potential mechanism for the clinical observations.

Conclusion: In nonpregnant women, Lactobacillus iners-dominant communities were significantly more likely to harbor Candida than Lactobacillus crispatus-dominant communities, suggesting that Lactobacillus species have different relationships with Candida. In vitro experiments indicate that Lactobacillus crispatus may impede Candida colonization more effectively than Lactobacillus iners through a greater production of lactic acid.

Keywords: Candida; Lactobacillus; pH; race; vaginal microbiome.

Figure 1:. Heatmap of all samples in the cohort clustered by community type.

Heat map of samples clustered by community type showing the top 25 taxa observed across the cohort. The bars above the heatmap indicate community type, BV status by Nugent score, race and Candida status. In the heat map, light blue indicates the highest abundance, darker blues indicate lower abundance and black indicates very low abundance or not present. Black race (p = 0.037) and L. iners-dominant communities (p = 0.045) were associated with Candida colonization.

Figure 2:. In vitro inhibition of Candida by Lactobacillus CFS and lactic acid.

A-B, Characterization of Candida growth medium supplemented with Lactobacillus CFS (YPD-CFS) in native and buffered states from four L. crispatus and four L. iners strains, prior to Candida inoculation. A, pH of YPD-CFS; B, Concentration of protonated lactic acid in YPD-CFS; C, Growth inhibition of Candida laboratory strain SC5314, showing three technical replicates for each Lactobacillus YPD-CFS. Analysis by one-way ANOVA with Tukey’s correction for multiple comparisons. D-F, Characterization of the inhibitory effect of lactic acid supplemented medium on Candida growth. Three technical replicates from two biological experiments are shown. D, Growth inhibition of SC5314 by lactic acid showing Mann-Whitney test comparison of 11 mM to 49 mM protonated lactic acid; E, Lactic acid growth inhibition of 6 vaginal C. albicans isolates; F, Lactic acid growth inhibition of 2 vaginal C. glabrata isolates. Data points in panel D reflect 6 replicates from two experiments for each condition. Error bars in E-F show the standard deviation from the mean of three replicates for each isolate. Approximate starting inoculum for growth assays is indicated by a dashed line. Statistical significance: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001.