Jose L. Lopez-Ribot

Candida Biofilms: an Update

Our classical perception of microorganisms as unicellular life forms is almost entirely based on the pure-culture mode of growth; since microbial suspensions can be diluted to a single cell and studied in liquid culture, this mode of growth has traditionally predominated in the study of microbial

Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms

ABSTRACT Candida albicans is implicated in many biomaterial-related infections. Typically, these infections are associated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents. Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of severalC. albicans strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of C. albicans biofilms against these antifungal agents was demonstrated. Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms.

Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection

ABSTRACT It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.

Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule.

ABSTRACT Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.

Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in Candida albicans Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients

ABSTRACT Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles inC. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.

Our current understanding of fungal biofilms.

Fungal biofilms are an escalating clinical problem associated with significant rates of mortality. Candida albicans is the most notorious of all fungal biofilm formers. However, non-Candida species, yeasts such as Cryptococcus neoformans, and filamentous moulds such as Aspergillus fumigatus, have been shown to be implicated in biofilm-associated infections. Fungal biofilms have distinct developmental phases, including adhesion, colonisation, maturation and dispersal, which are governed by complex molecular events. Recalcitrance to antifungal therapy remains the greatest threat to patients with fungal biofilms. This review discusses our current understanding of the basic biology and clinical implications associated with fungal biofilms.

In Vitro Activity of Caspofungin against Candida albicans Biofilms

ABSTRACT Most manifestations of candidiasis are associated with biofilm formation on biological or inanimate surfaces. Candida albicans biofilms are recalcitrant to treatment with conventional antifungal therapies. Here we report on the activity of caspofungin, a new semisynthetic echinocandin, against C. albicans biofilms. Caspofungin displayed potent in vitro activity against sessile C. albicans cells within biofilms, with MICs at which 50% of the sessile cells were inhibited well within the drugs therapeutic range. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the effects of caspofungin on preformed C. albicans biofilms, and the results indicated that caspofungin affected the cellular morphology and the metabolic status of cells within the biofilms. The coating of biomaterials with caspofungin had an inhibitory effect on subsequent biofilm development by C. albicans. Together these findings indicate that caspofungin displays potent activity against C. albicans biofilms in vitro and merits further investigation for the treatment of biofilm-associated infections.

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

In Vitro Pharmacodynamic Properties of Three Antifungal Agents against Preformed Candida albicans Biofilms Determined by Time-Kill Studies

ABSTRACT We have examined the in vitro activities of fluconazole, amphotericin B, and caspofungin against Candida albicans biofilms by time-kill methodology. Fluconazole was ineffective against biofilms. Killing of biofilm cells was suboptimal at therapeutic concentrations of amphotericin B. Caspofungin displayed the most effective pharmacokinetic properties, with ≥99% killing at physiological concentrations.