RESULTS:

Introduction. Candida auris is a pathogenic yeast that mainly affects immunosuppressed patients and those with implanted medical devices. This pathogen also displays elevated resistance to common antifungals and high survival and spreading capacities. Since no antifungal breakpoints have yet been defined for this pathogen the data obtained here can be useful for further research concerning treatment or implementation of a prevention and disinfection protocol. Our aim was to study the antifungal resistance of C. auris to current antifungals in planktonic and sessile states. Using confocal laser scanning microscopy and viable biomass production we demonstrated the ability of C. auris to develop a mature biofilm. We compared the minimal inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for the C. auris DSM 21092 strain plus two clinical isolates and the results were compared with those obtained for Candida albicans and Candida parapsilosis two species strongly linked to bloodstream infections and infections associated with biomaterials. We found that the clinical isolates of C. auris were resistant to fluconazole and sensitive to echinocandins and polyenes. The C. auris biofilms did not show susceptibility to any antifungal agent showing MBECs that were up to 512-fold higher than the MICs. These findings highlight the importance of biofilm formation as a key factor underlying the resistance of this species to antifungals and suggest that the presence of implantable medical devices is one of the major risk factors in immunocompromised patients.

Purpose. Otomycosis is a mycotic infection of the external auditory canal and can be caused by a wide range of fungal species. In this study we aimed to identify fungal isolates from patients suspected of otomycosis.

Methodology. External ear canal samples were taken from patients referred to the outpatient department of Shahid-Mofatteh Clinic in the city of Yasuj Iran and examined by direct microscopy and culture. DNA of the isolated fungi was tested by internal transcribed spacer PCR restriction fragment length polymorphism analysis for identification of yeasts and β-tubulin sequencing for identification of Aspergillus species.

Results. Among 275 patients suspected of otomycosis 144 cases (83 female and 61 male) were confirmed with otomycosis. For 89% (n=128) of positive cultures microscopy was also positive while there were no cases with a microscopy-positive and culture-negative result. The predominant predisposing factor was self-cleaning of the external ear using unhygienic tools and the main risk occupation was ‘housewife’. The most common isolated fungi were typically Aspergillus (n=120) including 73 isolates of Aspergillus section Nigri 43 of section Flavi 3 of section Terrei and 1 of section Fumigati. After sequencing 44 out of 73 strains primarily identified as Aspergillus niger turned out to be Aspergillus tubingensis. Thirty-five isolates were identified as Candida including Candida parapsilosis (n=22) Candida albicans (n=12) and Candida tropicalis (n=1).

Conclusion. Aspergillus tubingensis was the most common species involved in otomycosis. This work corroborates the difficulty of precise identification of species within the black Aspergilli by morphological characteristics.

Purpose. Candida species have become resistant to commonly used anti-fungal drugs like fluconazole and echinocandins. In our screen a series of quaternary ammonium compounds (QACs) emerged as an alternative treatment choice for drug-resistant Candida infections.

Methodology. Medium alkyl chain cationic lipo-oxazoles comprising six to thirteen twin carbon chains and a quaternary ammonium unit were synthesized and evaluated for their in vitro anti-Candida and biofilm inhibition activity. SEM was performed to visualize membrane distortion.

Results/Key findings. Heptyl and octyl chain analogues (5c 6b and 6c) showed promising anti-fungal activity. Compound 5c was active against both fluconazole-sensitive and resistant clinical isolates of Candida albicans as well as non-albicans Candida strains. 5c also inhibited the adhesion of C. albicans cells to a polystyrene surface and restricted biofilm formation. SEM further confirmed Candida cell membrane distortion by 5c.

Conclusion. A novel class of QACs called cationic lipo-oxazoles was tested and found to exhibit anti-fungal activity against planktonic cells as well as biofilms of Candida.

Purpose. Candida spp. are the most common causes of fungemia. Rapid and accurate diagnostic methods are very important for appropriate management of candidemia. At present blood culture is the essential diagnostic test despite having a long detection time and low sensitivity rate. We aimed to investigate the ways to shorten the turnaround time from blood culture collection to final identification in candidemia.

Methodology. Sixty clinical bloodstream isolates of Candida were included and Plus Aerobic/F Peds Plus/F and Mycosis IC/Fbottles were used with a BACTEC 9240 blood culture instrument. Germ tube production carbohydrate assimilation (API 20C AUX) and peptide nucleic acid fluorescent in situ hybridization yeast traffic light tests were performed directly from positive-signalled bottles.

Results. Time to positivity of blood cultures was affected by species of Candida fungal load and bottle type. Candida tropicalis had the shortest and Candida glabrata had the longest time to positivity. Mycosis IC/F culture bottle had a significant superiority in the isolation of yeasts especially for C. glabrata and if there was a low fungal load in the bottle. Direct germ tube test had 90 % sensitivity and 97.6 % specificity for Candida albicans in two hours after signalling. The compliance between direct and classical assimilation tests was 98.3 %. Sensitivity and specificity of peptide nucleic acid fluorescent in situ hybridization were 100 %.

Conclusion. We think that it is possible to shorten the turnaround time for the identification of Candida in blood culture even with currently available methods.

The role of penetration limitation in Candida biofilm-associated antifungal resistance remains unclear. Most of the previous work has been done on Candida albicans although non-albicans (NAC) species are also implicated in invasive candidiasis and the biofilm matrix has been shown to vary amongst different species. Only a few studies have evaluated clinical isolates. This study aimed to determine the relevance of penetration limitation in the antifungal resistance of biofilms formed by C. albicans and NAC clinical isolates using an agar disk diffusion assay. The penetration of posaconazole and amphotericin B through the biofilms was significantly reduced. Fluconazole voriconazole and caspofungin showed a superior penetration capacity in C. albicans Candida tropicalis and Candida parapsilosis biofilms but exhibited inter-species and strain/isolate variation. Candida krusei biofilms were the most resilient to antifungal permeation. All of the antifungal drugs failed to kill the biofilm cells independent of penetration suggesting that the other factors contribute markedly to the recalcitrance of the biofilms.

Aim. The aim of this work was to characterize the response of Candida albicans to atorvastatin and to assess its in vivo antifungal capability.

Methodology. The effect of atorvastatin on the growth and viability of C. albicans was assessed. The ability of the statin to alter cell permeability was quantified by measuring amino acid and protein leakage. The response of C. albicans to atorvastatin was assessed using label-free quantitative proteomics. The in vivo antifungal activity of atorvastatin was assessed using Galleria mellonella larvae infected with C. albicans.

Results. Atorvastatin inhibited the growth of C. albicans. The atorvastatin-treated cells showed lower ergosterol levels than the controls demonstrated increased calcofluor staining and released elevated quantities of amino acids and protein. Larvae infected with C. albicans showed a survival rate of 18.1±4.2 % at 144 h. In contrast larvae administered atorvastatin (9.09 mg kg−1) displayed a survival rate of 60.2±6.4 % (P<0.05). Label-free quantitative proteomics identified 1575 proteins with 2 or more peptides and 465 proteins were differentially abundant (P<0.05). There was an increase in the abundance of enzymes with oxidoreductase and hydrolase activity in atorvastatin-treated cells and squalene monooxygenase (4.52-fold increase) and lanosterol synthase (2.84-fold increase) were increased in abundance. Proteins such as small heat shock protein 21 (−6.33-fold) and glutathione peroxidase (−2.05-fold) were reduced in abundance.

Conclusion. The results presented here indicate that atorvastatin inhibits the growth of C. albicans and is capable of increasing the survival of G. mellonella larvae infected with C. albicans.

The present study aimed to evaluate the role of biofilm morphology matrix content and surface hydrophobicity in the biofilm-forming capacity of Candida albicans and non-albicans Candida (NAC) spp. Biofilm formation was determined by microtitre plate assay and bright-field and scanning electron microscopy. The matrix carbohydrates proteins and e-DNA were quantified by phenol-sulfuric acid bicinchoninic acid and UV spectroscopy respectively. Specific glycosyl residues were detected by dot blot. The cell-surface hydrophobicity was determined by hydrocarbon adhesion assay. Candida tropicalis was found to exhibit the highest adherence to polystyrene. It formed dense biofilms with extensive pseudohyphae and hyphal elements high hydrophobicity and the greatest amount of matrix carbohydrates proteins and e-DNA. C. albicans displayed higher adherence and a complex biofilm morphology with larger aggregates than Candida parapsilosis and Candida krusei but had lower matrix content and hydrophobicity. Thus the combinatorial effect of increased filamentation maximum matrix content and high hydrophobicity contributes to the enhanced biofilm-forming capacity of C. tropicalis.

Introduction. Polymicrobial infections including yeasts and bacteria are not rare and patients with polymicrobial bloodstream infection have higher early and overall case fatality rates. The diagnosis of invasive fungal and bacterial infections is mainly based on blood culture.

Aim. The aim was to reveal the effect of concomitant bacteraemia on the detection of fungi from blood cultures in the presence of polymicrobial bloodstream infections involving Candida and non-Candida fungi and to show the superiority of blood culture bottles including selective fungal media in such situations.

Methodology. Twenty-four polymicrobial bloodstream infection models – involving one fungus and one bacterium – were constituted by using clinical blood culture isolates ( Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Candida albicans Candida glabrata Fusarium solani and Trichosporon asahii). The Plus Aerobic/F (PAF) and Mycosis IC/F (MICF) culture bottles were used with the BACTEC 9240 device. After a bottle signalled positive direct microscopic examination and subcultures on agar plates were performed.

Results. All of fungi that were inoculated alone and in combination were detected by both direct microscopic examination and subcultures on agar plates from MICF bottles whereas direct microscopic examination only revealed the bacterial agents from PAF bottles including combinations. Furthermore fungal growth was hidden by bacterial growth on blood agar subcultures from PAF bottles including combinations of F. solani C. glabrata or T. asahii with bacteria.

Conclusion. Blood culture bottles including selective fungal media that can allow selective growth of fungi and earlier detection of some species should be preferred in addition to non-selective blood culture bottles especially in specific patient populations. Further the use of selective agar plates such as inhibitory mould agar may contribute to the solution of this problem in clinical laboratories.

Introduction. The presence of Candida biofilms in medical devices is a concerning and important clinical issue for haemodialysis patients who require constant use of prosthetic fistulae and catheters.

Hypothesis/Gap Statement. This prolonged use increases the risk of candidaemia due to biofilm formation. PH151 and clioquinol are 8-hydroxyquinoline derivatives that have been studied by our group and showed interesting anti-Candida activity.

Aim. This study evaluated the biofilm formation capacity of Candida species on polytetrafluoroethylene (PTFE) and polyurethane (PUR) and investigated the synergistic effects between the compounds PH151 and clioquinol and fluconazole amphotericin B and caspofungin against biofilm cells removed from those materials. Further the synergistic combination was evaluated in terms of preventing biofilm formation on PTFE and PUR discs.

Methodology. Susceptibility testing was performed for planktonic and biofilm cells using the broth microdilution method. The checkerboard method and the time–kill assay were used to evaluate the interactions between antifungal agents. Antibiofilm activity on PTFE and PUR materials was assessed to quantify the prevention of biofilm formation.

Results. Candida albicans Candida glabrata and Candida tropicalis showed ability to form biofilms on both materials. By contrast Candida parapsilosis did not demonstrate this ability. Synergistic interaction was observed when PH151 was combined with fluconazole in 77.8 % of isolates and this treatment was shown to be concentration- and time-dependent. On the other hand indifferent interactions were predominantly observed with the other combinations. A reduction in biofilm formation on PUR material of more than 50 % was observed when using PH151 combined with fluconazole.

Conclusion. PH151 demonstrated potential as a local treatment for use in a combination therapy approach against Candida biofilm formation on haemodialysis devices.

Summary: The adherence of Candida albicans to extracellular matrix proteins may be a critical step in the pathogenesis of candidiasis. Yeast cell adherence to type I and IV collagen fibronectin and laminin was blocked by peptide fragments from denatured type I collagen (gelatin). Gelatin fragments were obtained by digestion of the reduced protein with trypsin or CNBr. The fragments did not have antifungal properties presumably inhibiting adherence by blocking receptors (adhesins) on the surface of the fungus. A 10-mer (GQRGVVGLPG) fashioned from the a-1 chain of type I collagen reduced adherence by 68%. However a gelatin peptide possessing 47 amino acids reduced fungal adherence to type I collagen by 100%. Peptides derived from the biocompatible protein gelatin therefore may have a potential role in reducing the adherence of the fungus to host proteins.