Recurrent Vulvovaginal Candidiasis: a Dynamic Interkingdom Biofilm Disease of Candida and Lactobacillus

Abstract

Despite the strikingly high worldwide prevalence of vulvovaginal candidiasis (VVC), treatment options for recurrent VVC (RVVC) remain limited, with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown, with few studies identifying potential mechanisms of treatment failure. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, the vaginal microbiome, and how their interaction influences disease pathology. 16S rRNA sequencing characterized disease by a reduction in specific health-associated Lactobacillus species, such as Lactobacillus crispatus, coupled with an increase in Lactobacillus iners. In vitro analysis showed that Candida albicans clinical isolates are capable of heterogeneous biofilm formation, and we found the presence of hyphae and C. albicans aggregates in vaginal lavage fluid. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to identify a possible mechanism by which L. crispatus may contribute to re-establishing a healthy vaginal environment through amino acid acquisition from C. albicans. This study highlights the potential formation and impact of Candida biofilms in RVVC. Additionally, it suggests that RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and that understanding interactions between this yeast and vaginal Lactobacillus species may be crucial to elucidating the cause of RVVC and developing appropriate therapies. IMPORTANCE RVVC is a significant burden, both economically and for women's health, but its prevalence is poorly documented globally due to the levels of self-treatment. Identifying triggers for development and recurrence of VVC and the pathogenesis of the microbes involved could considerably improve prevention and treatment options for women with recurrent, azole-resistant cases. This study therefore aimed to examine the interkingdom dynamics from healthy women and those with RVVC using next-generation sequencing techniques and to further investigate the molecular interactions between C. albicans and L. crispatus in a relevant biofilm coculture system.

Keywords: Candida; Lactobacillus; antifungal resistance; biofilm; clinical; interkingdom; microbiome; vulvovaginal candidiasis.

FIG 1

Fungal burden is elevated in women with RVVC while bacterial load remains unchanged. To assess fungal load, patient lavage was incubated on Candida Chromogenic agar plates and colonies counted after 48 h (a). For calculation of CFE/ml, the ITS region of Candida was amplified using genus-specific primers (b). Levels of bacteria DNA were also assessed molecularly by amplification of the 16S rRNA region (c). Correlation between bacterial and fungal burden was also observed (d). Data are means ± standard deviations (SD). Statistical significance was calculated using unpaired t tests with Welch’s correction, as data did not share equal standard deviations (*, P < 0.05; **, P < 0.01).

FIG 2

Bacterial genera present in healthy women and those with RVVC. DNA extracted from swab samples was used for 16S rRNA Illumina sequencing (n = 100). Bacterial diversity measured by Shannon index (a) and genus-level taxa identification and percentage abundance of microbial populations present (b) in health and RVVC.

FIG 3

Hydrogen peroxide-producing lactobacillus strains are reduced during RVVC infection, resulting in an L. iners-dominated microbiome. Species-level identification of bacterial taxa present in healthy women (a) and those with RVVC (b). Random forest plot showing the most distinct species-level taxa present between healthy women and those with RVVC (c).

FIG 4

Species-level taxon abundance relative to patient metadata. Species-level bacterial taxa based on presence/absence of Candida (a) and length of time with disease (b). Random forest plots showing distinct bacterial taxa present in each analysis (c and d).

FIG 5

Vaginal Candida isolates from patients with RVVC are capable of heterogeneous biofilm formation. A total of 33 Candida clinical isolates were isolated from lavage samples. MALDI-TOF was used to identify each isolate and assess species distribution (“Others” comprises 1 isolate each of C. dubliniensis, C. parapsilosis, and C. krusei) (a). Candida isolates from healthy women (n = 9) and those with RVVC (n = 24) were assessed for biofilm-forming capabilities by crystal violet staining (b). Vaginal lavage was stained with calcofluor white (CFW) for 1 h at 37°C before imaging (c). Images are representative of C. albicans aggregates and hyphae observed in lavage fluid from a patient with an HBF isolate. White arrows represent pseudohyphal/hyphal formation, and red arrows depict cell aggregates. Data are means ± SD. Statistical analysis was performed using unpaired t-tests (****, P < 0.0001).

FIG 6

Lactobacillus species display antagonism with C. albicans in vitro. To observe inhibitory effects of Lactobacillus against C. albicans biofilm formation, C. albicans and a panel of Lactobacillus species were cocultured in THB/RPMI (1:1) medium in 5% CO₂ for 24 h (a), or C. albicans was grown for 4 h prior to addition of Lactobacillus species for 20 h (b). C. albicans biofilm-associated gene expression was measured in the presence of L. rhamnosus, which is associated with health, and L. iners, which is hypothesized to indicate dysbiosis. The mean log fold change relative to single species C. albicans biofilms is shown (c). Data are means and SD.

FIG 7

Differential expression analysis of C. albicans single-species and C. albicans–L. crispatus dual-species biofilms. Heat map displaying the top 50 significantly differentially expressed genes in C. albicans between single- and dual-species 24-h biofilms (P < 0.05).

FIG 8

Gene networks of gene ontology (GO) terms and upregulated genes in dual-species biofilms. Constructed gene networks of GO terms in 24-h biofilms with L. crispatus (a). Important biological, cellular, and molecular functions in dual-species biofilms with L. crispatus (b). Log₂ fold change of key gene expression in C. albicans from single- to dual-species biofilms with L. crispatus (c). Nodes are colored by significance. All GO terms have an adjusted P value of <0.05. Networks were created using ClueGO.

FIG 9

Twice-daily addition of L. crispatus reduces C. albicans load within a complex biofilm model in vitro. The potential probiotic properties of L. crispatus against C. albicans were assessed in a 11-species biofilm model treated twice daily with L. crispatus. Live/dead qPCR allowed quantification of percentage composition of total (a) and live (b) C. albicans DNA within the biofilm. Average fold change in the C. albicans percentage composition from the untreated 11-species biofilm is also shown (c). Data are means and SD. Statistical analysis was performed using paired t tests comparing raw CFE values (*, P < 0.05).