Relative Abundances of Candida albicans and Candida glabrata in In Vitro Coculture Biofilms Impact Biofilm Structure and Formation

Abstract

Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species-Candida albicans and Candida glabrata-are often coisolated from infection sites, suggesting the importance of Candida coculture biofilms. In this work, we report that biofilm formation of the coculture population depends on the relative ratio of starting cell concentrations of C. albicans and C. glabrata When using a starting ratio of C. albicans to C. glabrata of 1:3, ∼6.5- and ∼2.5-fold increases in biofilm biomass were observed relative to those of a C. albicans monoculture and a C. albicans/C. glabrata ratio of 1:1, respectively. Confocal microscopy analysis revealed the heterogeneity and complex structures composed of long C. albicans hyphae and C. glabrata cell clusters in the coculture biofilms, and reverse transcription-quantitative PCR (qRT-PCR) studies showed increases in the relative expression of the HWP1 and ALS3 adhesion genes in the C. albicans/C. glabrata 1:3 biofilm compared to that in the C. albicans monoculture biofilm. Additionally, only the 1:3 C. albicans/C. glabrata biofilm demonstrated an increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a coculture composition-dependent manner.IMPORTANCECandida albicans and Candida glabrata are often coisolated during infection, and the occurrence of coisolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in pathogenesis. During the course of an infection, the prevalence of each Candida species may change over time due to differences in metabolism and in the resistance of each species to antifungal therapies. Therefore, it is necessary to understand the dynamics between C. albicans and C. glabrata in coculture to develop better therapeutic strategies against Candida infections. Existing in vitro work has focused on understanding how an equal-part culture of C. albicans and C. glabrata impacts biofilm formation and pathogenesis. What is not understood, and what is investigated in this work, is how the composition of Candida species in coculture impacts overall biofilm formation, virulence gene expression, and the therapeutic treatment of biofilms.

Keywords: CLSM; Candida; biofilm; biofilms; coculture; mycobiota.

FIG 1

Effect of starting culture composition on Candida coculture biofilm formation. Biofilms formed on glass coverslips (A) or polystyrene microtiter plates (B) for 48 h. Four biological replicates. Ca, C. albicans; Cg, C. glabrata; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with C. albicans monoculture by Student's t tests. Error bars are standard errors of samples.

FIG 2

Confocal microscopy analysis of Candida coculture biofilm thickness. (A) Representative 6-h 3D Candida biofilm cross-sectional views. Red, C. albicans; green, C. glabrata; A, C. albicans; B, C. albicans/C. glabrata 3:1; C, C. albicans/C. glabrata 1:1; D, C. albicans/C. glabrata 1:3; E, C. glabrata. (B) Biofilm thickness at 6, 12, and 24 h. Student's t test, *, P < 0.05; ***, P < 0.001 compared with C. albicans monoculture. Error bars are standard errors from 3 biological replicates.

FIG 3

Effect of culture composition on biofilm structure. (A) Representative 12-h 3D biofilm. Scale bars in microns. (B) Average C. albicans hyphal length in biofilms. (C) Representative 3D biofilm images of C. albicans/C. glabrata 1:3 at 6, 12, and 24 h. Scale bar in microns. Student's t test, *, P < 0.05; ***, P < 0.001 compared with C. albicans monoculture. Error bars are standard errors from 3 biological replicates.

FIG 4

Physical association of C. glabrata with C. albicans hyphae in biofilms. (A) C. albicans/C. glabrata 3:1. (B) C. albicans/C. glabrata 1:1. (C) C. albicans/C. glabrata 1:3. (D) Percent C. glabrata attached to C. albicans hyphae in Candida coculture biofilms. Student's t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with C. albicans monoculture. Error bars are standard errors from 3 biological replicates.

FIG 5

Candida biofilm viability after caspofungin treatment. Student's t test, *, P ≤ 0.05 compared to C. albicans monoculture. Error bars are standard errors from 3 biological replicates.