We have described biofilm formation, CAS susceptibility, and prevalence of PG among isolates from five medically relevant Candida
species, including C. albicans
, C. tropicalis
, C. parapsilosis
, C. orthopsilosis
, and C. metapsilosis
. Previously, Song and colleagues reported that C. orthopsilosis
and C. metapsilosis
, also known as group II and group III C. parapsilosis
, respectively, did not form biofilms, suggesting that the biofilm-forming phenotype ascribed to C. parapsilosis
was limited to group I isolates (23
). One explanation for the discrepancy in results is the difference in the biofilm growth and quantitation methods between the two studies. In our study, we used the chemically defined RPMI 1640 medium (pH 7.0) containing minimal (0.2%) glucose, whereas Song et al. used a complex medium, Sabouraud dextrose broth, supplemented with 8% glucose (23
). Studies of bacterial biofilms have shown that nutrient density and medium osmolarity are important environmental factors which regulate biofilm attachment, growth, and maturation (24
); and for most microbial species, moderate nutrient limitation favors biofilm growth relative to the growth achieved under nutrient-rich and nutrient-poor conditions (24
). Another important difference that may have contributed to our result was our use of the XTT reduction assay to measure biofilm metabolic activity rather than spectrophotometric measurement of the decreased light transmission caused by biofilm formation on microtiter plate wells. XTT reduction has been widely used to measure biofilm activity and allows the detection of small differences in metabolic activity between strains when the colorimetric reaction is measured during the linear phase of the reaction (8
MIC results indicated that CAS has excellent in vitro activity against all isolates tested in the planktonic growth mode and against many isolates grown as biofilms. Interestingly, C. parapsilosis
biofilms were more resistant to CAS than C. orthopsilosis
and C. metapsilosis
biofilms, even though they had the same MICs in planktonic cell tests. Since CAS acts by disrupting β-1,3-glucan synthesis, the intrinsic ability of a given strain to compensate for the reduction of this polymer may promote reduced caspofungin susceptibility. β-1,3-Glucan has also been shown to be an important constituent of the Candida
biofilm extracellular matrix, where it contributes to substrate and/or cell-cell adherence and overall biofilm stability (3
). The CAS-induced reduction of β-1,3-glucan in the biofilm matrix may increase the fragility of the biofilm and, likewise, the susceptibility to antifungal killing. Furthermore, Candida
strains that have adapted to life with reduced amounts of β-1,3-glucan in the cell wall and biofilm matrix may be less susceptible to the antifungal effects of CAS. Quantitative comparisons of the β-1,3-glucan content in the cell wall and the extracellular matrix among various Candida
species represent an interesting area for further exploration.
PG following exposure to supra-MICs of CAS has previously been described for Candida
sp. isolates grown as planktonic cells but not as biofilms (25
). We observed PG in 40% of the isolates when they were tested with CAS as planktonic cells and twice that (80%) when the same isolates were tested as biofilms, suggesting that PG is not an uncommon phenomenon.
In this study, we used simple light microscopy to visualize the morphological changes in planktonic and biofilm cells associated with CAS exposure and PG. Cell clumping and the appearance of enlarged, globose cells were hallmarks of PG. One explanation is the fungal cell wall changes due to the reduced β-1,3- and β-1,6-glucan contents and increased chitin content. A study by Stevens et al. (26
) reported that cell wall preparations from a C. albicans
isolate capable of PG and grown in the presence of supra-MICs of CAS had 81% and 73% reductions in β-1,3- and β-1,6-glucans contents, respectively, and an 898% increase in chitin content compared to the contents in cells grown in the absence of CAS. Furthermore, Nakai et al. reported that the PG of Candida
spp. in broth microdilution tests with micafungin was dependent on the osmotic conditions of the growth medium; PG was produced only under hyperosmotic conditions (17
). Understanding of the links between PG and (i) the presence of large, rounded cells in biofilms, (ii) the shift in the contents of the key components of the fungal cell wall, and (iii) the dependency on the osmolarity of the medium will be key to understanding the basis of echinocandin-associated PG among Candida
sp. isolates and the clinical significance of PG.
In conclusion, Candida sp. biofilms can display PG in the presence of high concentrations of CAS and do so more readily then planktonic cells of the same strains. The cellular morphological changes associated with PG can be observed microscopically and are likely due to alterations in the fungal cell wall. The clinical significance of PG remains unclear, yet the proposed use of high concentrations of echinocandin antifungals as catheter lock therapy for the treatment and prevention of catheter-associated candidemia may be thwarted by the stimulation of Candida biofilm growth at CAS concentrations above the MIC. On the basis of these findings, further studies to determine the occurrence of PG in vivo with animal models of catheter-associated infection and antifungal lock therapy are warranted.