Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene.

This work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.

Galleria mellonella-A Model for the Study of aPDT-Prospects and Drawbacks.

Galleria mellonella is a promising in vivo model insect used for microbiological, medical, and pharmacological research. It provides a platform for testing the biocompatibility of various compounds and the kinetics of survival after an infection followed by subsequent treatment, and for the evaluation of various parameters during treatment, including the host-pathogen interaction. There are some similarities in the development of pathologies with mammals. However, a limitation is the lack of adaptive immune response. Antimicrobial photodynamic therapy (aPDT) is an alternative approach for combating microbial infections, including biofilm-associated ones. aPDT is effective against Gram-positive and Gram-negative bacteria, viruses, fungi, and parasites, regardless of whether they are resistant to conventional treatment. The main idea of this comprehensive review was to collect information on the use of G. mellonella in aPDT. It provides a collection of references published in the last 10 years from this area of research, complemented by some practical experiences of the authors of this review. Additionally, the review summarizes in brief information on the G. mellonella model, its advantages and methods used in the processing of material from these larvae, as well as basic knowledge of the principles of aPDT.

Effect of Quorum Sensing Molecule Farnesol on Mixed Biofilms of Candida albicans and Staphylococcus aureus.

The natural bioactive molecule farnesol (FAR) is widely studied mainly for its antibiofilm and antimicrobial properties. In addition, it increases the effectiveness of some antimicrobial substances, which makes it interesting for the development of combined therapy. In the present work, the effect of FAR either alone or in combination with oxacillin (OXA) on mixed biofilms formed by clinically relevant pathogens, Candida albicans and Staphylococcus aureus, was studied. S. aureus isolates used for biofilm formation originated from blood cultures and central venous catheters (CVC) were characterized in terms of antimicrobial resistance. The minimal biofilm inhibitory concentration (MBIC50) for FAR of 48 h mixed biofilms formed by the C. albicans and methicillin-sensitive S. aureus (MSSA) was determined to be 125 µM, and for the mixed biofilms with methicillin-resistant S. aureus (MRSA) was determined to be 250 µM. Treatment of mixed biofilms with OXA (2 mg/mL) showed ≤4% inhibition; however, the combination of OXA (2 mg/mL) and FAR (300 µM) resulted in 80% inhibition of biofilms. In addition, planktonic cells of S. aureus exhibited an increased susceptibility to OXA, cefoxitin and kanamycin in the presence of FAR (150 and 300 µM). Scanning electron microscopy (SEM) micrographs confirmed patchy biofilm and lack of candidal hyphae in the samples treated with FAR and FAR/OXA in comparison to control and mixed biofilms treated only with OXA. Intriguingly, in a pilot experiment using fluorescence in situ hybridization (FISH), considerable differences in activity (as indicated by ribosome content) of staphylococcal cells were detected. While the activity rate of the staphylococci in mixed biofilms treated with FAR was high, no FISH-positive signal for staphylococcal cells was found in the biofilm treated with FAR/OXA.

Photodynamic Inactivation Effectively Eradicates Candida auris Biofilm despite Its Interference with the Upregulation of CDR1 and MDR1 Efflux Genes.

Candida auris, in recent years, has emerged as a dangerous nosocomial pathogen. It represents a challenge for effective treatment because of its multiresistance. Photodynamic inactivation (PDI) is a promising way to solve problems with a wide range of resistant microorganisms. This study aimed to use PDI for the eradication of C. auris biofilms. Moreover, the regulation of the CDR1, CDR2, and MDR1 resistance genes was studied. Experiments were performed on 24 h biofilms formed by three clinical isolates of C. auris in vitro. PDI was performed in the presence of the photosensitizer methylene blue (0.25 mM) and samples were irradiated with a red laser (λ = 660 nm, 190 mW/cm2) for 79, 120, and 300 s. To confirm the PDI effect, confocal laser scanning microscopy was performed after treatment. Effective PDI was achieved in all strains. The highest inhibition was observed after 300 s irradiation, with over 90% inhibition compared with the non-irradiated control sample. PDI was observed to upregulate the expression of the CDR1 gene, but mainly the MDR1 gene. Despite this observation, PDI significantly decreased the survival of C. auris biofilm cells and proved to have great potential for the eradication of problematic resistant yeasts.

Synthesis and structure-activity relationship of berkeleylactone A-derived antibiotics.

Berkeleylactone A is a potent 16-membered macrolactone antibiotic, recently isolated from a coculture of Berkeley Pit Lake fungi. Although its antimicrobial activity has already been investigated, little is known about the structure-activity relationship. Based on our previous synthetic studies, a series of berkeleylactone A derivatives were synthesized and evaluated for their in vitro antimicrobial activities against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) strains. Our data confirmed the essential role of the embedded conjugated system and suggest a reversible sulfa-protection of the Michael acceptor as a viable option. Structurally simplified achiral macrolactam 8 showed the best inhibitory activity against S. aureus L12 (MRSA) with MIC50 values of 0.39 µg mL-1, 8-fold lower than those of berkeleylactone A. These studies may be of value in the development of more advanced candidates for antibiotic applications.

Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis.

Biofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism's distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.

Farnesol Boosts the Antifungal Effect of Fluconazole and Modulates Resistance in Candida auris through Regulation of the CDR1 and ERG11 Genes.

Candida auris is considered a serious fungal pathogen frequently exhibiting a high resistance to a wide range of antifungals. In this study, a combination of the quorum-sensing molecule farnesol (FAR) and fluconazole (FLU) was tested on FLU-resistant C. auris isolates (C. auris S and C. auris R) compared to the susceptible C. auris H261. The aim was to assess the possible synergy between FAR and FLU, by reducing the FLU minimal inhibitory concentration, and to determine the mechanism underlying the conjunct effect. The results confirmed a synergic effect between FAR and FLU with a calculated FIC index of 0.75 and 0.4 for C. auris S and C. auris R, respectively. FAR modulates genes involved in azole resistance. When FAR was added to the cells in combination with FLU, a significant decrease in the expression of the CDR1 gene was observed in the resistant C. auris isolates. FAR seems to block the Cdr1 efflux pump triggering a restoration of the intracellular content of FLU. These results were supported by observed increasing accumulation of rhodamine 6G by C. auris cells. Moreover, C. auris treated with FAR showed an ERG11 gene down-regulation. Overall, these results suggest that FAR is an effective modulator of the Cdr1 efflux pump in C. auris and, in combination with FLU, enhances the activity of this azole, which might be a promising strategy to control infections caused by azole-resistant C. auris.

Persistence and multi-ward dissemination of vancomycin-resistant Enterococcus faecium ST17 clone in hospital settings in Slovakia 2017-2020.

OBJECTIVES: Hospital vancomycin-resistant Enterococcus faecium (VREfm) were evaluated in term of resistance and phylogenetic relatedness to estimate the location and possible route of transmission of resistance determinants. METHODS: Hospital VREfm (n = 49) were collected in the northern part of Slovakia during 2017-2020. The collection was analysed for the presence of the van operon and 10 representatives were subjected to whole-genome sequencing using Illumina MiSeq platform. Obtained sequences were de novo assembled and the draft genome assemblies were analysed with respect to sequence type (ST), plasmid content, resistance and virulence genes, and the phylogenetic relatedness in single nucleotide polymorphisms (SNP). RESULTS: All strains possessed the vanA operon. Ten selected evaluated isolates belonged to the clinically relevant clonal complex (CC) 17 and carried the vanHAX gene cluster conferring vancomycin resistance on mobile genetic elements, except for the isolate M17773 carrying the vanHAX gene cluster chromosomally. All isolates encoded resistance to quinolones (gyrA and parC mutations) and aminoglycosides [aac(6')-aph(2'')]. Four isolates from different wards and patients belonging to ST17 were closely related (6-50 SNP), suggesting long-term persistence of VREfm ST17 in hospital settings. CONCLUSION: VREfm proved to harbour many genetic determinants of antimicrobial resistance. The plasmids carrying the vanA genes belonged to the conjugative broad-host families Inc18 and RepA_N, posing a threat to human health, especially in hospital settings. Moreover, four clinical isolates were phylogenetically related, pointing towards stable circulation of the ST17 VREfm clone in the hospital setting and underlining the necessity for continuous surveillance of glycopeptide-resistant pathogens.

Surface Characterization and Anti-Biofilm Effectiveness of Hybrid Films of Polyurethane Functionalized with Saponite and Phloxine B.

The main objective of this work was to synthesize composites of polyurethane (PU) with organoclays (OC) exhibiting antimicrobial properties. Layered silicate (saponite) was modified with octadecyltrimethylammonium cations (ODTMA) and functionalized with phloxine B (PhB) and used as a filler in the composites. A unique property of composite materials is the increased concentration of modifier particles on the surface of the composite membranes. Materials of different compositions were tested and investigated using physico-chemical methods, such as infrared spectroscopy, X-ray diffraction, contact angle measurements, absorption, and fluorescence spectroscopy in the visible region. The composition of an optimal material was as follows: nODTMA/mSap = 0.8 mmol g-1 and nPhB/mSap = 0.1 mmol g-1. Only about 1.5% of present PhB was released in a cultivation medium for bacteria within 24 h, which proved good stability of the composite. Anti-biofilm properties of the composite membranes were proven in experiments with resistant Staphylococcus aureus. The composites without PhB reduced the biofilm growth 100-fold compared to the control sample (non-modified PU). The composite containing PhB in combination with the photodynamic inactivation (PDI) reduced cell growth by about 10,000-fold, thus proving the significant photosensitizing effect of the membranes. Cell damage was confirmed by scanning electron microscopy. A new method of the synthesis of composite materials presented in this work opens up new possibilities for targeted modification of polymers by focusing on their surfaces. Such composite materials retain the properties of the unmodified polymer inside the matrix and only the surface of the material is changed. Although these unique materials presented in this work are based on PU, the method of surface modification can also be applied to other polymers. Such modified polymers could be useful for various applications in which special surface properties are required, for example, for materials used in medical practice.

Impact of Healthcare-Associated Infections Connected to Medical Devices-An Update.

Healthcare-associated infections (HAIs) are caused by nosocomial pathogens. HAIs have an immense impact not only on developing countries but also on highly developed parts of world. They are predominantly device-associated infections that are caused by the planktonic form of microorganisms as well as those organized in biofilms. This review elucidates the impact of HAIs, focusing on device-associated infections such as central line-associated bloodstream infection including catheter infection, catheter-associated urinary tract infection, ventilator-associated pneumonia, and surgical site infections. The most relevant microorganisms are mentioned in terms of their frequency of infection on medical devices. Standard care bundles, conventional therapy, and novel approaches against device-associated infections are briefly mentioned as well. This review concisely summarizes relevant and up-to-date information on HAIs and HAI-associated microorganisms and also provides a description of several useful approaches for tackling HAIs.

Physico-Chemical Characterization and Antimicrobial Properties of Hybrid Film Based on Saponite and Phloxine B.

This research was aimed at the preparation of a hybrid film based on a layered silicate saponite (Sap) with the immobilized photosensitizer phloxine B (PhB). Sap was selected because of its high cation exchange capacity, ability to exfoliate into nanolayers, and to modify different surfaces. The X-ray diffraction of the films confirmed the intercalation of both the surfactant and PhB molecules in the Sap film. The photosensitizer retained its photoactivity in the hybrid films, as shown by fluorescence spectra measurements. The water contact angles and the measurement of surface free energy demonstrated the hydrophilic nature of the hybrid films. Antimicrobial effectiveness, assessed by the photodynamic inactivation on hybrid films, was tested against a standard strain and against methicillin-resistant bacteria of Staphylococcus aureus (MRSA). One group of samples was irradiated (green LED light; 2.5 h) and compared to nonirradiated ones. S. aureus strains manifested a reduction in growth from 1-log10 to over 3-log10 compared to the control samples with Sap only, and defects in S. aureus cells were proven by scanning electron microscopy. The results proved the optimal photo-physical properties and anti-MRSA potential of this newly designed hybrid system that reflects recent progress in the modification of surfaces for various medical applications.

Opportunist Coinfections by Nontuberculous Mycobacteria and Fungi in Immunocompromised Patients.

Nontuberculous mycobacteria (NTM) and many fungal species (spp.) are commonly associated with opportunistic infections (OPIs) in immunocompromised individuals. Moreover, occurrence of concomitant infection by NTM (mainly spp. of Mycobacterium avium complex and Mycobacterium abscessus complex) and fungal spp. (mainly, Aspergillus fumigatus, Histoplasma capsulatum and Cryptococcus neoformans) is very challenging and is associated with poor patient prognosis. The most frequent clinical symptoms for coinfection and infection by single agents (fungi or NTM) are similar. For this reason, the accurate identification of the aetiological agent(s) is crucial to select the best treatment approach. Despite the significance of this topic it has not been sufficiently addressed in the literature. This review aims at summarizing case reports and studies on NTM and fungi coinfection during the last 20 years. In addition, it briefly characterizes OPIs and coinfection, describes key features of opportunistic pathogens (e.g., NTM and fungi) and human host predisposing conditions to OPIs onset and outcome. The review could interest a wide spectrum of audiences, including medical doctors and scientists, to improve awareness of these infections, leading to early identification in clinical settings and increasing research in the field. Improved diagnosis and availability of therapeutic options might contribute to improve the prognosis of patients' survival.

The Contribution of Photodynamic Inactivation vs. Corsodyl Mouthwash to the Control of Streptococcus mutans Biofilms.

This work compared the inhibition effect of the commercially available mouthwash Corsodyl, containing 0.1% chlorhexidine digluconate, and photodynamic inactivation (PDI) employing methylene blue (MB) with irradiation from a red laser on 24-h biofilms formed by Streptococcus mutans strains on hydroxyapatite surfaces. The cytotoxicity of Corsodyl and MB was evaluated by Galleria mellonella surviving assay. The viability of biofilm cells after exposure to mouthwash and PDI was determined by counting colony-forming units. The inhibitory effect of antimicrobial agents was confirmed by confocal scanning laser microscopy. MB did not exhibit a cytotoxic effect on larval survival. Non-diluted Corsodyl slightly decreased the survival of larvae. Using our PDI parameters achieved better inhibition than with non-PDI, proving a significant effect on the eradication of S. mutans biofilms and therefore could be an appropriate supplement for the eradication of dental caries.

Synergy Over Monotherapy.

Pathogenic fungi, as an increasing global threat to human health, represent a sizable risk. However, significant attention should also be paid to the yeast biofilms. One promising strategy for combating resistant microbes, as well as fungal biofilms, is to extend the lifespan and efficacy of our currently employed drugs by using combination therapy. Since the application of combined therapy of fungal infections is currently accepted, we have decided to verify the efficacy of derivative H in combination with fluconazole on C. albicans biofilm. The main advantage of synergy over monotherapy lies in reducing or even completely preventing the induction of resistance of fungal cells. We have decided to verify the derivative H (1,4-dihydropyridine-2,3,5-tricarboxylate), an intermediate of nilvadipine synthesis, in the resistance of C. albicans to fluconazole. Therefore, we have focused on the influence of derivative H on the gene expression of the main C. albicans adhesin (ALS3), which is important for the tissue colonization during the infection process. Our results show that the newly synthesized derivative H had an impact on biofilm eradication. The effect of biofilm diminution could, therefore, be explained as derivative H preventing the adherence of C. albicans cells. This study supports even more the attractiveness of this substance as a potential agent that could be used in synergy with commonly used azoles to treat various fungal infections.

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Impact of Farnesol as a Modulator of Efflux Pumps in a Fluconazole-Resistant Strain of Candida albicans.

Aim: This work studied the impact of the quorum-sensing molecule, farnesol (FAR), on fluconazole (FLC)-resistant Candida albicans isolate CY 1123 compared with the susceptible standard strain C. albicans SC5314. The genes encoding efflux pumps belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies, together with overexpression or point mutation of the ERG11 gene, are the main resistance mechanisms to azole antifungal drugs. Results: The upregulation of genes coding for CDR1, CDR2, and MDR1 were confirmed by qPCR with respect to the housekeeping gene ACT1 in the resistant strain. The contribution of the ERG11 gene was also observed. Markedly, increased pump activity (Cdr1 and/or Cdr2) in the CY 1123 strain was confirmed using diS-C3(3) assay. However, the addition of FAR to the yeasts diminished the difference in staining levels between the SC5314 and CY 1123 strains, demonstrating the concentration-dependent character that could be caused by an effective modulation of Cdr pumps. FAR (60 and 100 µM) was also able to decrease the minimal inhibitory concentrations (MIC50), denoting the inhibition of planktonic cells by 50%, from 8 to 4 µg/mL of FLC when the resistant strain CY 1123 was not cultivated with FLC. However, when it was exposed to 64 µg/mL of FLC, the MIC50 shifted from 64 to 8 µg/mL. Conclusion: Besides the many other effects of FAR on eukaryotic and prokaryotic cells, it also affects ABC efflux transporters, resulting in changes in resistance to azoles in C. albicans isolates. However, this effect is dependent on FAR concentrations.

Activity of anti-CR3-RP polyclonal antibody against biofilms formed by Candida auris, a multidrug-resistant emerging fungal pathogen.

Fungal biofilm has remained a serious medical problem that complicates treatment of mycoses. In particular, once biofilms are formed, they display high levels of resistance against most common antifungals. Candida auris is currently considered as a serious emerging fungal pathogen frequently exhibiting high levels of resistance to antifungals. Recent studies have confirmed that C. auris shares similarity with Candida albicans in regards to virulence-associated proteins involved in adherence and biofilm development. Complement receptor 3-related protein (CR3-RP) is one of the key surface antigens expressed by Candida species during biofilm formation. Here, we have investigated the presence of this cell surface moiety on the surface of C. auris, as well as the potential of anti-CR3-RP polyclonal antibody (Ab) to inhibit biofilm formation by this emerging fungal pathogen. Using indirect immunofluorescence and ELISA, we were able to confirm the presence of CR3-RP in C. auris cells within biofilms. Further, not only anti-CR3-RP Ab was able to inhibit biofilm formation by multiple C. auris strains when added during the adherence phase, but it also demonstrated activity against C. auris 24-h pre-formed biofilms, which compared favorably to levels of inhibition achieved by treatment with current conventional antifungals fluconazole, amphotericin B, and caspofungin. Overall, our data demonstrate the presence of this antigen on the surface of C. auris and points to the potential of anti-CR3-RP Ab in eradication of biofilms formed by this novel fungal pathogen.

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in Galleria mellonella Infected with Candida Yeasts Displaying Different Virulence Traits.

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p = 0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.

Anti-biofilm activity of antibody directed against surface antigen complement receptor 3-related protein-comparison of Candida albicans and Candida dubliniensis.

Candida albicans and C. dubliniensis are related yeasts that differ in the expression of virulence-associated proteins involved in adherence and biofilm development. CR3-RP (complement receptor 3-related protein) is one of the surface antigens expressed by Candida species. The main objective of this research was to elucidate the effect of the polyclonal anti-CR3-RP antibody (Ab) on adherence and the biofilm formed by C. albicans SC5314 and C. dubliniensis CBS 7987 and two clinical isolates in vitro, ex vivo and in vivo. A comparison of species, and of treated vs. non-treated with the anti-CR3-RP Ab showed a reduction in adherence (22%-41%) that was dependent on the time point of evaluation (60, 90 or 120 min), but did not prove to be species-dependent. Confocal microscopy revealed a decreased thickness in biofilms formed by both species after pre-treatment with the anti-CR3-RP Ab. This observation was confirmed ex vivo by immunohistochemistry analysis of biofilms formed on mouse tongues. Moreover, anti-CR3-RP Ab administration, 1 h post-infection, has been shown to promote larval survival compared to the control group in a Galleria mellonella infection model. Our data suggest a potential activity of the anti-CR3-RP Ab relevant to immunotherapy or vaccine development against biofilm-associated Candida infections.

The impact of farnesol in combination with fluconazole on Candida albicans biofilm: regulation of ERG20, ERG9, and ERG11 genes.

Farnesol (FAR) has already demonstrated an inhibitory effect on Candida albicans biofilm. The aim of this work was to determine the effectiveness of externally added FAR in combination with fluconazole (FLC) on Candida albicans biofilm and on regulation of the ergosterol genes ERG20, ERG9, and ERG11. The effectiveness of compounds was determined by MTT assay and evaluated by the minimal inhibitory concentrations reducing a sessile biofilm to 50% activity (0.5 µg/mL and 200 µmol/L for FLC and FAR, respectively). These concentrations as well as 30 and 100 µmol/L FAR were selected for a study of the effectiveness of the FAR/FLC combination. The reduction in biofilm robustness mainly caused by the presence of 200 µmol/L FAR-alone or in combination with FLC-was accompanied by a significant inhibition of the yeast-to-hyphae transition that was observed by light microscopy and CLSM. Results from qRT-PCR indicated that while 30 µmol/L FAR only slightly regulated the expression of all 3 genes in the 48-h biofilm, the presence of 200 µmol/L FAR downregulated all the tested genes. However, the addition of 0.5 µg/mL of FLC to the samples with 200 µmol/L FAR restored the downregulation of the ERG20 and ERG11 genes to the control level. Moreover, the gene ERG9 was slightly upregulated. In summary, FAR acted via multiple effects on the C. albicans biofilm, but only a higher concentration of FAR proved to be effective.