biofilm formation proceeds in an organized fashion through the early, intermediate, and maturation phases of development. Similar distinct developmental phases have been reported for biofilm formation by many bacterial species (16
). Thus, microorganisms appear to share common basic steps during biofilm formation. Development of biofilm is closely associated with the generation of matrix, the majority of which is extracellular material. Microscopy strongly suggests that extracellular material is predominantly composed of cell-wall-like polysaccharides containing mannose and glucose residues, based on staining with dyes that specifically bind these carbohydrates. Mature C. albicans
biofilms have a highly heterogeneous architecture in terms of distribution of fungal cells and extracellular material. In addition, compared to biofilms grown on the irregular surface of polymethylmethacrylate, those grown on flat hydrophobic surfaces such as silicone elastomer have a distinct biphasic structure composed of an adherent blastospore layer covered by sparser hyphal elements embedded in a deep layer of extracellular material. A similar biphasic distribution was suggested for C. albicans
biofilms grown on polyvinyl chloride disks (6
). Formation of this biphasic architecture could be in response to environmental conditions, such as differences in pH, oxygen availability, and redox potential, prevailing within the biofilm (32
). Heterogeneity in the biofilms is another characteristic shared among microorganisms. A “heterogeneous mosaic model” for biofilms has been described, containing stacks of bacterial microcolonies held together by extracellular polymeric substances. Below the stacks is an underlying layer of cells (≈5 μm thick) attached to the substratum (44
Like their bacterial counterparts, biofilm-grown C. albicans
cells are highly resistant to antimicrobials. Although drug resistance has been shown in C. albicans
) and bacterial biofilms (7
), this is the first report correlating the emergence of antifungal resistance with the development of biofilms. Developing C. albicans
biofilms are associated with an increasing presence of extracellular material. Extracellular polymeric substance in bacterial biofilms is known to physically interact with antibiotics and is believed to contribute to resistance against these drugs (19
). It is unclear if the increase in drug resistance in C. albicans
biofilms is due to production of extracellular material or due to genetic and biochemical alterations in fungal cells; this is an area for future study. An alternative explanation proposed for antifungal resistance in biofilms is metabolic quiescence of cells. However, this possibility is not likely since biofilm-embedded cells actively metabolize substrates, including XTT and FUN-1.
The biofilm-forming ability of the pathogen C. albicans
is markedly different from that of S. cerevisiae.
While the latter is capable of adherence, it fails to progress to a mature biofilm characterized by the presence of extracellular material. A recent report suggested that S. cerevisiae
forms biofilms in vitro (38
). However, the putative S. cerevisiae
biofilms described in that study do not resemble those formed by C. albicans
in our standardized model. We believe that these results are in agreement with ours and indicate that, although S. cerevisiae
adheres to surfaces in a limited way, it fails to form extracellular material-encased biofilms similar to those formed by C. albicans
. Additionally, our results show that antifungal resistance of adherent S. cerevisiae
cells did not increase with time. This result was in contrast to C. albicans
biofilms, where a significant jump in the MICs of drugs was observed between early and mature phase biofilms.
gene expression is differentially regulated during the transition of Candida
from a planktonic to biofilm-associated organism. This documentation of differential gene expression between the two growth forms represents a small number of the transcriptional changes that are likely to occur during biofilm formation. The formation of extracellular material associated with C. albicans
biofilms suggests that genes encoding enzymes involved in carbohydrate synthesis are differentially regulated during biofilm growth. It is also possible that increased expression of drug resistance genes is responsible for the increased MICs observed for C. albicans
biofilms. Finally, the appearance of a well-defined basal layer of yeast followed by extracellular material production also suggests that genes involved in quorum sensing are important, as is the case with Pseudomonas
). There may also be increased expression of drug resistance genes such as CDR1, CDR2
, and MDR
. Relevant gene regulation can likely only be determined using pathogenic organisms. The well-characterized genome of C. albicans
and the availability of deletion mutants should allow for rapid evaluation of such possibilities.
Fungal biofilm formation is a complex phenomenon distinct from adhesion. It is best studied using pathogenic species grown on relevant bioprosthetic materials under near-physiologic conditions. Study of such systems will reveal the true nature of fungal biofilms and their biology. Demonstration of common biofilm features (distinct developmental phases, heterogeneous architecture, and drug resistance phenotypes) across different taxa extends the implication of this study beyond fungi to other organized cellular communities. The impact of this information will be widespread, ranging from new environmental microbiology insights to the development of antimicrobials specifically targeted against biofilm-associated infections.