Candidemia is usually treated with antifungal medications, called azoles (miconazole, terconazole, fluconazole). Usually administered topically (gels, creams, ointments, sprays), or orally (capsules and liquids), injections and suppositories may are also used in more severe cases.
So, it’s no wonder that over 700,000 cases of invasive candidiasis occur across the world each year.
Candidiasis that affect our externals, like mouth, groin, or vagina, is known as superficial candidiasis, whereas those that infect our innards are called systemic or invasive candidiasis, which is often accompanied by fever and chills. Candidemia, a type of invasive candidiasis, commonly seen among hospitalized patients, occurs when candida infects the bloodstream.
Unlike ringworms (also a group of fungi), candida causes yeast infections that aren’t restricted to the skin, nails, and hair, but can spread to several internal body parts, including organs, such as the heart and brain, and soft tissues in the eyes and bones.
Conventional Treatments vs. Cannabidiol for Candidemia
In most cases, a short course of 5 – 10 days of azoles alleviates the symptoms. At times, though, even a 6-month-long course with varying doses may not be enough. In such azole-resistant strains of candida, doctors often prescribe boric acid capsules as an alternative therapy. These are used as suppositories (inserted into the vagina or rectum), as they’re unfit for oral consumption.
Unfortunately, not only do these anti-fungal treatments offer little relief to patients, these medications may do more harm to the human body. For instance, fluconazole is known for adverse effects, like headaches, aggravation in rashes, nausea, abdominal pain, diarrhea, and vomiting. In some severe cases, it may also cause very high fever, trouble breathing, jaundice, hepatitis, seizures, abnormalities in blood cells, and abnormal heart rhythm that may even cause death.
But these may sometimes be unavoidable circumstances.
Have you heard of Athlete’s foot, jock itch, ringworm, thrush, or yeast infections? If you have, then you must know these are all fungal infections, caused by certain groups of harmful fungi or overgrowth of relatively non-harmful groups of fungi.
Like any infection, it’s usually our weakened immune system that is to blame for candida invasion. Other reasons may include a high intake of antibiotics, excessive alcohol intake, use of tampons and oral contraceptives, diabetes, and increased stress levels.
Multiple virulence factors of C. albicans are associated with the infection-related host tissue damage processes such as extracellular hydrolytic enzymes and hyphae-specific cell surface proteins. In this study, we tested the expression of virulence-related genes such as lipases, phospholipases, and cell wall proteins related to the RBT family in C. albicans biofilms formed after a 24 h incubation with CBD. The extracellular lipases produced by C. albicans, LIPs, contribute to the delivery of nutrients and support fungal permeation of the host tissues . Phospholipase B (PLBs) are also involved in the virulence of C. albicans by assisting the fungi in crossing host cell membranes with its following destruction . RBTs genes are induced during filamentous growth and contribute to fungal pathogenesis . In this study, we observed noticeable downregulation of all examined virulence genes (LIP2, LIP4, LIP5, PLB1, PLB2, RBT1, RBT4, RBT5) in biofilms formed in the presence of CBD.
C. albicans–GFP biofilms were allowed to form in 24-well polystyrene microtiter plates in the absence or presence of 50 μg/mL CBD in RPMI for 24 h at 37 °C. The washed biofilms were incubated for 45 min in PBS containing 25 μg/mL concanavalin A (ConA)-Alexa Fluor 647 conjugate (excitation wavelength 650 nm and emission at 668 nm) (Invitrogen, Carlsbad, CA, USA). ConA binds to glucose and mannose residues of cell wall exopolysaccharides (EPS) . Stained biofilms (green color for viable fungal cells and blue color for EPS) were observed with a NIKON confocal microscope. Three-dimensional images of the formed biofilms and EPS distribution were constructed using the NIS-Element AR software. At least three random fields were captured and analyzed. The amount of total EPS production and viable cells in each sample was calculated according to the fluorescence intensity using Image J (version 3.91; Java image processing program; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA). The data are presented as total EPS production or total viable cells in each layer of biofilm (5 µm). The percentage of total EPS production or total viable cells in biofilms treated with CBD is presented as area under the curve (AUC) and compared to untreated control. Three independent experiments were performed and one set of representative data is shown.
The resistance rate of C. albicans biofilms to the majority of antifungal drugs is much higher as compared to the planktonic form of fungi, making biofilm-associated infections difficult to treat. In addition, many of the currently used antifungal drugs have the disadvantages of being highly toxic, the possibility of interacting with other drugs, and the need for intravenous administration [10,11]. Therefore, there is an urgent need for alternative compounds capable to treat fungal biofilms.
We initially studied the effect of CBD on biofilm formation. For this purpose, C. albicans was exposed to various concentrations of CBD for 24–72 h, and the metabolic activity of the biofilms was analyzed using the MTT assay. We observed a time- and dose-dependent inhibition of fungal biofilm formation by CBD ( Figure 1 A). After a 24 h of incubation, 12.5 μg/mL CBD inhibited biofilm formation by 37% in comparison to untreated control, while after 72 h, 1.56 μg/mL CBD was sufficient to achieve a similar impact (31% inhibition) ( Figure 1 A). CBD at a dose of 6.25 μg/mL did not reduce the biofilm mass after 24 h, while an inhibition of biofilm mass by 28% and 39% was observed after 48 h and 72 h, respectively ( Figure 1 A). Similarly, increasing the CBD dose to 25 μg/mL caused a pronounced time-dependent inhibitory effect. The amount of metabolically active cells in 24 h-, 48 h- and 72 h-biofilms decreased at this dose by 48%, 64%, and 87%, respectively ( Figure 1 A). CBD exhibited minimal biofilm inhibitory concentration (MBIC) of 90% (MBIC90) at 100 μg/mL based on the finding that C. albicans biofilm was almost totally inhibited at this concentration at all tested time points ( Figure 1 A). No minimal inhibitory concentration (MIC) and no minimal fungicidal concentration (MFC) were detected at the tested doses of CBD towards planktonic C. albicans .
(A) Effect of CBD on C. albicans biofilm formation. C. albicans yeast cells were incubated with various concentrations of CBD for 24, 48, and 72 h at 37 °C; (B) Effect of CBD on preformed biofilms. Biofilms of C. albicans which were 24 h-old were exposed for 24 h to various concentrations of CBD and the metabolic activity of biofilm cells was measured using the MTT assay. The values of the untreated control were set to 100%. * Significantly lower than the untreated control (p < 0.05).
Raw data are available upon request.
Ergosterol is an important virulence factor contributing to normal functioning and structural integrity of the plasma membrane in Candida species . In our study, genes related to ergosterol biosynthesis, ERG11 and ERG20, were downregulated by sub-MBIC90 of CBD which could be attributed to its cell membrane-targeting activity. On the other hand, we recorded notable upregulation of DDP3 gene associated with biosynthesis of farnesol—the molecule able to inhibit C. albicans biofilm formation . Similarly, other natural products have been demonstrated to inhibit C. albicans biofilm formation by upregulating DPP3 gene and by downregulating ergosterol biosynthesis-associated genes .
2.5. Determination of Minimal Inhibitory Concentration (MIC)
C. albicans biofilms were formed in 8-well chamber slide (ibidi GmbH, Gräfelfing, Germany) in the absence or presence of CBD (6.25, 12.5, 25, and 50 μg/mL), washed and stained with 1 μg/mL of Calcofluor White M2R (CFW) (Sigma-Aldrich) for 5 min in the dark. CFW is a fluorophore which emits blue fluorescence after binding to chitin in the fungal cell wall . Total chitin content was measured using the Infinite M20 PRO plate reader (Tecan) (excitation 365 nm, emission 435 nm). Data are presented as percentage of untreated control. In parallel, the CFW-stained biofilms were visualized by NIKON confocal microscope using the DAPI filter set. At least three random fields were observed. Three independent experiments were performed and one set of representative data is shown.
Total RNA was extracted and purified from untreated and CBD (25 µg/mL)-treated fungal biofilms using Tri-Reagent (Sigma-Aldrich) as described . The purified RNA was reverse transcribed to cDNA using the qScript cDNA synthesis kit (Quantabio, Beverly, MA, USA). Real-time qPCR was performed in a CFX96 BioRad Connect Real-Time PCR apparatus using Power Sybr Green Master Mix (Applied Biosystems, Waltham, MA, USA) on 50 ng cDNA and 300 nM of the respective primer sets (Table 3). The relative expression levels of the target genes were analyzed using a Bio-Rad CFX Maestro software (Quantabio, Beverly, MA, USA). Primers for the tested genes were taken from the literature or designed using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/; access on 18 February 2021). For each set of primers, a standard amplification curve (critical threshold cycle vs. exponential of concentration) was plotted, and only those with a slope ≈ −3 were considered reliable. The PCR conditions consisted of a denaturation step at 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 60 s). The expression of ACT1 gene was used as internal standard. Gene expression is given in relative values, setting the expression level of the untreated control to 1 for each gene. The assay was performed in triplicates.