Biofilm formation in clinical Candida isolates and its association with virulence

Abstract

Biofilm formation, an important virulence trait of Candida species was measured in 107 Candida isolates from 32 candidemic patients by XTT [2,3-bis (2-methoxy-4nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] activity and compared to biofilm formation of Candida isolates from oropharyngeal lesions of 19 AIDS patients. Biofilm formation by XTT varied among species and C. albicans; C. lusitaniae and C. krusei produced more biofilm than the other Candida species. C. tropicalis was the most dominant species isolated from blood followed by C. albicans, and other non-albicans species whereas only C. albicans was recovered from oral lesions. Importantly, though Biofilm formation was variable within a species it was stable in sequential isolates during chronic infection. Sequential isolates exhibited identical Karyotype pattern or RAPD patterns unless patients were co-infected with more than one strain. High biofilm formation was associated with slow growth rate but not with adherence. Murine infection studies demonstrated that, degree of in-vitro biofilm formation was associated with virulence in mice, as mice infected both with no and low biofilm formers survived longer than mice infected with high biofilm former C. albicans (p< or =0.001). We conclude that biofilm formation is a stable but strain specific characteristic that can greatly vary among C. albicans and non-albicans strains, and plays an important role in persistence of infection.

Fig. 1

BF is dependent on Candida species. (A) BF is highly variable among clinical Candida species (B) BF was highly variable among C. albicans isolates from both candidemic (invasive-black bar) and oropharyngeal lesion of HIV positive patients (non-invasive-gray bar) (C) Like C. albicans, BF was variable among C. parapsilosis but was relatively stable among C. tropicalis isolates from candidemic patients.

Fig. 2

A) BF was comparable among serial isolates isolated up to 85 days apart (C. tropicalis-P9). In some cases co-infection with different strains of the same species (C. parapsilosis-P20) resulted in variation of BF whereas others exhibited comparable BF (C. glabrata-P22, P26). Black and gray bar indicates serial isolates from individual patients. (B) Differences in BF of serial C. parapsilosis were associated with karyotype variability and infection with two strains. Serial C. glabrata isolates exhibited in karyotypic variability but no differences in BF unless infection with different strains of same species (P20). (C) Seven different karyotype patterns were found among C. albicans OPC isolates from HIV patients. (D) RAPD profile of sequential isolates by M13 primer are shown for i) C. albicans, P21, P23, P12, P17, P13, P7, P8 and ii) C. tropicalis, P27, P29, P30, P32. Note that this method demonstrated persistence of original strain for C. albicans isolates and more heterogenecity than karyotyping. For C. tropicals RAPD patterns were very heterogenous analogous to C. glabrata.

Fig. 3

Variability in BF was associated with variability in growth. Doubling time for most Candida strains were comparable. Among C. albicans strains slow growers were found.

Fig. 4

(A) Survival of BALB/c mice (n=5 per group) after intravenous challenge with 3×10⁶ blastospore of high, low and no biofilm formers (HBF, LBF, NBF) C. albicans differs significantly (p<0.001, log rank) (B) Histopathological examination of kidney. (a) HBF; massive accumulation of yeast cells, hyphal element and few leukocytes in calyx (b) HBF; kidney cortex showing infiltration of yeast and hyphal elements and degraded glomerulus. (c) LBF; papilla showing leukocytosis and fungal infiltration in collecting duct (d) LBF; little fungal infiltration in cortex and showing intact gromerulus. (Periodic acid Schiff stain was used).