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Fungi such as Candida albicans exist in biofilm phenotypes, which present as viscoelastic materials; however, a method to measure linear viscoelastic moduli, yield stress, and yield strain is lacking. Characterization methods for fungal materials have been limited to techniques specific to particular industries. Here, we present a method to measure the shear stress, strain amplitude, and creep of C. albicans BWP17 biofilms. Our method includes features tailored to the analysis of fungi including an in vitro growth protocol attuned to the slow growth rates of C. albicans biofilms and a resultant cultured biofilm that has sufficient integrity to be transferred to the rheometer tooling without disrupting its structure. The method's performance is demonstrated by showing that results are insensitive to gap, evaporative sealant, length of experiment, and specimen radius. Multiscale imaging of the fungal biofilm showed complex entanglement networks at the hundred-micrometer scale. For a wild-type strain cultivated for 14 days, using small-amplitude oscillatory rheology, we found that the elastic (G′) and viscous (G″) moduli were nearly independent of frequency over the range 0.1–10 s −1 , with magnitudes of [Formula: see text] and [Formula: see text], respectively. The yield stress was approximately [Formula: see text]. We modeled the linear creep response of the fungal biofilm and found that C. albicans has a characteristic relaxation time of [Formula: see text] and a viscosity of [Formula: see text]. We applied this method to probe the effects of altered chitin deposition in the C. albicans cell wall. Differences between the biofilm's phenotypic cell shape and rheological properties in mutants with altered chitin synthase activity were resolved. Discovering how genotypic, phenotypic, and environmental factors impact the material properties of these microbial communities can have implications for understanding fungal biofilm growth and aid in the development of remediation strategies.
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Both Pseudomonas aeruginosa and Candida albicans Accumulate Greater Biomass in Dual-Species Biofilms under Flow
ABSTRACT Microbe-microbe interactions can strongly influence growth and biofilm formation kinetics. For Pseudomonas aeruginosa and Candida albicans , which are found together in diverse clinical sites, including urinary and intravenous catheters and the lungs of individuals with cystic fibrosis (CF), we compared the kinetics of biofilm formation by each species in dual-species and single-species biofilms. We engineered fluorescent protein constructs for P. aeruginosa (producing mKO-κ ) and C. albicans (producing mKate2 ) that did not alter growth and enabled single-cell resolution imaging by live-sample microscopy. Using these strains in an optically clear derivative of synthetic CF sputum medium, we found that both P. aeruginosa and C. albicans displayed increased biovolume accumulation—by three- and sixfold, respectively—in dual-species biofilms relative to single-species biofilms. This result was specific to the biofilm environment, as enhanced growth was not observed in planktonic cocultures. Stimulation of C. albicans biofilm formation occurred regardless of whether P. aeruginosa was added at the time of fungal inoculation or 24 h after the initiation of biofilm development. P. aeruginosa biofilm increases in cocultures did not require the Pel extracellular polysaccharide, phenazines, and siderophores known to influence C. albicans . P. aeruginosa mutants lacking Anr, LasR, and BapA were not significantly stimulated by C. albicans , but they still promoted a significant enhancement of biofilm development of the fungus, suggesting a fungal response to the presence of bacteria. Last, we showed that a set of P. aeruginosa clinical isolates also prompted an increase of biovolume by C. albicans in coculture. IMPORTANCE There is an abundance of work on both P. aeruginosa and C. albicans in isolation, and quite some work as well on the way these two microbes interact. These studies do not, however, consider biofilm environments under flow, and our results here show that the expected outcome of interaction between these two pathogens can actually be reversed under flow, from pure antagonism to an increase in biomass on the part of both. Our work also highlights the importance of cellular-scale spatial structure in biofilms for understanding multispecies population dynamics.
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- Award ID(s):
- 1817342
- PAR ID:
- 10274907
- Editor(s):
- Mitchell, Aaron P.
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 6
- Issue:
- 3
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/mSphere.00416-21
