Host-pathogen interactions upon Candida auris infection: fungal behaviour and immune response in Galleria mellonella.

Candida auris has globally emerged as a multidrug-resistant fungus linked to healthcare-associated outbreaks. There is still limited evidence on its virulence, pathogenicity determinants, and complex host-pathogen interactions. This study analyzes the in vivo fungal behaviour, immune response, and host-pathogen interactions upon C. auris infection compared to C. albicans and C. parapsilosis in G. mellonella. This was performed by immunolabelling fungal structures and larval plasmatocytes and using a quantitative approach incorporating bioinformatic morphometric techniques into the study of microbial pathogenesis. C. auris presents a remarkably higher immunogenic activity than expected at its moderate degree of tissue invasion. It induces a greater inflammatory response than C. albicans and C. parapsilosis at the expense of plasmatocyte nodule formation, especially in non-aggregative strains. It specifically invades the larval respiratory system, in a pattern not previously observed in other Candida species, and presents inter-phenotypic tissue tropism differences. C. auris filaments in vivo less frequently than C. albicans or C. parapsilosis mostly through pseudohyphal growth. Filamentation might not be a major pathogenic determinant in C. auris, as less virulent aggregative phenotypes form pseudohyphae to a greater extent. C. auris has important both interspecific and intraspecific virulence and phenotype heterogeneity, with aggregative phenotypes of C. auris sharing characteristics with low pathogenic species such as C. parapsilosis. Our work suggests that C. auris owns an important morphogenetic plasticity that distinguishes it from other yeasts of the genus. Routine phenotypic identification of aggregative or non-aggregative phenotypes should be performed in the clinical setting as it may impact patient management.

Haematopoietic cell transplantation outcomes are linked to intestinal mycobiota dynamics and an expansion of Candida parapsilosis complex species.

Allogeneic haematopoietic cell transplantation (allo-HCT) induces profound shifts in the intestinal bacterial microbiota. The dynamics of intestinal fungi and their impact on clinical outcomes during allo-HCT are not fully understood. Here we combined parallel high-throughput fungal ITS1 amplicon sequencing, bacterial 16S amplicon sequencing and fungal cultures of 1,279 faecal samples from a cohort of 156 patients undergoing allo-HCT to reveal potential trans-kingdom dynamics and their association with patient outcomes. We saw that the overall density and the biodiversity of intestinal fungi were stable during allo-HCT but the species composition changed drastically from day to day. We identified a subset of patients with fungal dysbiosis defined by culture positivity (n = 53) and stable expansion of Candida parapsilosis complex species (n = 19). They presented with distinct trans-kingdom microbiota profiles, characterized by a decreased intestinal bacterial biomass. These patients had worse overall survival and higher transplant-related mortality independent of candidaemia. This expands our understanding of the clinical significance of the mycobiota and suggests that targeting fungal dysbiosis may help to improve long-term patient survival.

The gut commensal fungus, Candida parapsilosis, promotes high fat-diet induced obesity in mice.

Gut fungi is known to play many important roles in human health regulations. Herein, we investigate the anti-obesity efficacy of the antifungal antibiotics (amphotericin B, fluconazole and 5-fluorocytosine) in the high fat diet-fed (HFD) mice. Supplementation of amphotericin B or fluconazole in water can effectively inhibit obesity and its related disorders, whereas 5-fluorocytosine exhibit little effects. The gut fungus Candida parapsilosis is identified as a key commensal fungus related to the diet-induced obesity by the culture-dependent method and the inoculation assay with C. parapsilosis in the fungi-free mice. In addition, the increase of free fatty acids in the gut due to the production of fungal lipases from C. parapsilosis is confirmed as one mechanism by which C. parapsilosis promotes obesity. The current study demonstrates the gut C. parapsilosis as a causal fungus for the development of diet-induced obesity in mice and highlights the therapeutic strategy targeting the gut fungi.

Enhancing the chemical transformation of Candida parapsilosis.

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20-55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at -80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

Broad Diversity of Fungi in Hospital Water.

INTRODUCTION: Some studies have reported the occurrence of microorganisms isolated from water. Considering these microorganisms, fungi are known to occur ubiquitously in the environment, including water, and some are pathogenic and may cause health problems, especially in immunocompromised individuals. The aim of this study was to identify fungi in hospital water samples and to correlate their presence with the concentration of free residual chlorine. METHODS: Water samples (100 mL) were collected from taps (n = 74) and water purifiers (n = 14) in different locations in a university hospital. Samples were filtered through a nitrocellulose membrane and placed on Sabouraud dextrose agar and incubated for 24 hours at 30°C. Fungi were identified according to established methods based on macroscopic and microscopic characteristics (filamentous) and physiological tests (yeasts). Free chlorine residual content was measured at the time of sample collection. RESULTS: Seventy species of fungi were identified in the water samples and about 56% of the water samples contained culturable fungi. Cladosporium oxysporum, Penicillium spinulosum, and Aspergillus fumigatus were the most common filamentous fungi. Aureobasidium pullulans and Candida parapsilosis were the most common yeasts. Chemical analyses revealed that free residual chlorine was present in 81.8% of the samples within recommended concentrations. Among samples from water purifiers, 92.9% showed low levels of free residual chlorine (<0.2 mg/L). There was no significant association between chlorine concentrations (either within or outside the recommended range) and the presence of filamentous fungi and yeasts. CONCLUSIONS: This study showed that hospital water can be a reservoir for fungi, some of which are potentially harmful to immunocompromised patients. Free residual chlorine was ineffective in some samples.

Anti-biofilm effect by the combined action of fluconazole and acetylsalicylic acid against species of Candida parapsilosis complex.

The Candida parapsilosis complex has been associated with highly refractory infections mainly due to the presence of biofilms. High glucose levels enable the development of this virulence factor which can aggravate the clinical condition of patients with diabetes mellitus, those using parenteral nutrition, with invasive medical device, including others. Combined antifungal therapy, such as azole and cyclooxygenase inhibitors, may be an alternative in such infections since they modulate prostaglandin production favoring the adhesion and development of biofilms. Thus, the present study aimed to evaluate the influence of glucose supplementation in the formation and detection of Candida parapsilosis complex biofilms and to treat them using fluconazole and a cyclooxygenase inhibitor in combination. Protein spectra evaluation allowed the differentiation between species from the complex (score > 2) in our studies. All isolates were able to form active biofilms at different glucose concentrations. In addition, a significant reduction in biofilm formation was observed when fluconazole and acetylsalicylic acid were combined. The ultrastructural analysis presented typical biofilm characteristics by species from the complex. These data support new combined therapies for the treatment of fungal infections, especially with those which are resistant and therapeutic failure is associated with virulence factors.

Lack of efficacy of echinocandins against high metabolic activity biofilms of Candida parapsilosis clinical isolates.

Candida parapsilosis produces biofilm, which colonizes catheters and other invasive medical devices that are manipulated by health care workers. In previous studies, C. parapsilosis in vitro biofilms have exhibited high resistance rates against conventional antifungals, but susceptibility to both echinocandins and lipid formulations of amphotericin B (lipid complex and liposomal). However, a recent study showed good activity of amphotericin B deoxycholate on the biomass of C. parapsilosis biofilms. Although moderate activity of echinocandins has been demonstrated against low metabolic activity biofilms of C. parapsilosis, few studies have analyzed the action of these drugs on high metabolic activity biofilms. Moreover, high biofilm-forming isolates have been associated with central venous catheter-related fungemia outbreaks and higher mortality rates. Therefore, it is relevant to verify the activity of the main antifungal drugs against high metabolic activity biofilms of C. parapsilosis. Our study aimed to evaluate the in vitro activity of amphotericin B deoxycholate, anidulafungin, caspofungin, and micafungin against high biofilm-forming and high metabolic activity clinical isolates of C. parapsilosis. Our results showed good activity of amphotericin B against C. parapsilosis biofilms, but none of the echinocandin drugs was effective. This suggests that amphotericin B deoxycholate may be a better choice than echinocandins for the treatment of biofilm-associated infections by C. parapsilosis, mainly in countries with insufficient health care resources to purchase lipid formulations of amphotericin B. These results warn of the possibility of persistent catheter-related candidemia caused by high biofilm-forming C. parapsilosis strains when treated with echinocandin drugs.

Physiological and Transcriptional Responses of Candida parapsilosis to Exogenous Tyrosol.

Tyrosol plays a key role in fungal morphogenesis and biofilm development. Also, it has a remarkable antifungal effect at supraphysiological concentrations. However, the background of the antifungal effect remains unknown, especially in the case of non-albicans Candida species such as Candida parapsilosis We examined the effect of tyrosol on growth, adhesion, redox homeostasis, virulence, as well as fluconazole susceptibility. To gain further insights into the physiological consequences of tyrosol treatment, we also determined genome-wide gene expression changes using transcriptome sequencing (RNA-Seq). A concentration of 15 mM tyrosol caused significant growth inhibition within 2 h of the addition of tyrosol, while the adhesion of yeast cells was not affected. Tyrosol increased the production of reactive oxygen species remarkably, as revealed by a dichlorofluorescein test, and it was associated with elevated superoxide dismutase, glutathione peroxidase, and catalase activities. The interaction between fluconazole and tyrosol was antagonistic. Tyrosol exposure resulted in 261 and 181 differentially expressed genes with at least a 1.5-fold increase or decrease in expression, respectively, which were selected for further study. Genes involved in ribosome biogenesis showed downregulation, while genes related to the oxidative stress response and ethanol fermentation were upregulated. In addition, tyrosol treatment upregulated the expression of efflux pump genes, including MDR1 and CDR1, and downregulated the expression of the FAD2 and FAD3 virulence genes involved in desaturated fatty acid formation. Our data demonstrate that exogenous tyrosol significantly affects the physiology and gene expression of C. parapsilosis, which could contribute to the development of treatments targeting quorum sensing in the future.IMPORTANCECandida-secreted quorum-sensing molecules (i.e., farnesol and tyrosol) are key regulators in fungal physiology, which induce phenotypic adaptations, including morphological changes, altered biofilm formation, and synchronized expression of virulence factors. Moreover, they have a remarkable antifungal activity at supraphysiological concentrations. Limited data are available concerning the tyrosol-induced molecular and physiological effects on non-albicans Candida species such as C. parapsilosis In addition, the background of the previously observed antifungal effect caused by tyrosol remains unknown. This study reveals that tyrosol exposure enhanced the oxidative stress response and the expression of efflux pump genes, while it inhibited growth and ribosome biogenesis as well as several virulence-related genes. Metabolism was changed toward glycolysis and ethanol fermentation. Furthermore, the initial adherence was not influenced significantly in the presence of tyrosol. Our results provide several potential explanations for the previously observed antifungal effect.

Correlating Genotype and Phenotype in the Asexual Yeast Candida orthopsilosis Implicates ZCF29 in Sensitivity to Caffeine.

Candida orthopsilosis is diploid asexual yeast that causes human disease. Most C. orthopsilosis isolates arose from at least four separate hybridizations between related, but not identical, parents. Here, we used population genomics data to correlate genotypic and phenotypic variation in 28 C. orthopsilosis isolates. We used cosine similarity scores to identify 65 variants with potential high-impact (deleterious effects) that correlated with specific phenotypes. Of these, 19 were Single Nucleotide Polymorphisms (SNPs) that changed stop or start codons, or splice sites. One variant resulted in a premature stop codon in both alleles of the gene ZCF29 in C. orthopsilosis isolate 185, which correlated with sensitivity to nystatin and caffeine. We used CRISPR-Cas9 editing to introduce this polymorphism into two resistant C. orthopsilosis isolates. Introducing the stop codon resulted in sensitivity to caffeine and to ketoconazole, but not to nystatin. Our analysis shows that it is possible to associate genomic variants with phenotype in asexual Candida species, but that only a small amount of genomic variation can be easily explored.

Comparison of biofilm-producing ability of clinical isolates of Candida parapsilosis species complex.

OBJECTIVE: Candida parapsilosis is one of the main emerging non-Candida albicans species leading to superficial and systemic fungal infections in humans. Candida has the ability to produce biofilms associated with pathogenesis. The aim of the study was to estimate biofilm-producing ability of clinical isolates of C. parapsilosis sp. complex. METHODS: Clinical samples of C. parapsilosis complex have been analyzed. Crystal violet (CV) staining and tetrazolium reduction assay (MTT) have been used to analyze the clinical isolates ability to produce biofilms. The biofilm's structural characteristics have been assessed by using scanning electron microscopy. RESULTS: All 65 isolates were able to form biofilm. In addition, no significant difference was found between biofilm quantification based on two assays at different time intervals (24h, 48h, 72h, 96h) (P>0.05), with the exception of Candida orthopsilosis, which exhibited higher metabolic activity at 24h time point (P<0.05). Moreover, metabolic activity and production of biofilm biomass demonstrated statistically significant correlation (r=0.685, P<0.01). According to microscopic observations, the investigated clinical strains formed the similar surface topography with the slight differences in morphology; in addition, there was no statistically significant difference between efficiency of two assays to quantify biofilm. CONCLUSION: It was shown that, similar to C. parapsilosissensu stricto, two cryptic identified species (C. orthopsilosis and Candida metapsilosis) obtained from different clinical samples, were biofilm producers, while C. parapsilosissensu stricto exhibited the highest biofilm production.

Biofilm formation by blood isolates of Candida parapsilosis sensu stricto in the presence of a hyperglycidic solution at comparable concentrations of total parenteral nutrition.

INTRODUCTION: Administration of total parenteral nutrition (TPN) via catheters increases the risk for candidemia from Candida parapsilosis. METHODS: C. parapsilosis sensu stricto blood isolates were evaluated for ability total biomass biofilm formation and morphogenesis in presence of glucose at TPN equivalent concentrations. RESULTS: Biofilms were increased at high glucose concentrations (25-30%) compared to the control medium. Significant increase in filamentous forms was observed in cultures with 30% glucose. CONCLUSIONS: Biofilm formation by C. parapsilosis sensu stricto in hyperglycidic medium may contribute to its pathogenic potential for fungemia related to TPN catheters.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Nicotine enhances the thickness of biofilm and adherence of Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019.

Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019 hyphal-wall protein 1 (HWP1) are involved in hyphae formation and pathogenesis. The transcriptional agglutinin-like sequence 3 (ALS3) genes in both species are responsible for the development of biofilm and colonization on tooth surfaces. Therefore, we investigated the expression of HWP1 and ALS3 quantitatively in C. albicans and C. parapsilosis and examined the biofilm structure upon exposure to various nicotine concentrations. In vitro, biofilms of Candida species were developed directly on slides using the Lab-Tek Chamber Slide System and visualized by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to measure HWP1 and ALS3 expression in C. albicans ATCC 14053 and C. parapsilosis ATCC 22019. The results indicated that nicotine multiplied the number of yeast cells and increased the extracellular polysaccharides of Candida species. We also found that 1-2 mg/mL nicotine could enhance the formation of biofilm. The findings also revealed that the expression of HWP1 and ALS3 in Candida species were increased as the nicotine concentration increased. Therefore, nicotine influences the biofilm development of oral-associated C. albicans ATCC 14053 and C. parapsilosis ATCC 22019.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

Biofilm formation by blood isolates of candida parapsilosis sensu stricto in the presence of a hyperglycidic solution at comparable concentrations of total parenteral nutrition

Abstract INTRODUCTION: Administration of total parenteral nutrition (TPN) via catheters increases the risk for candidemia from Candida parapsilosis. METHODS: C. parapsilosis sensu stricto blood isolates were evaluated for ability total biomass biofilm formation and morphogenesis in presence of glucose at TPN equivalent concentrations. RESULTS: Biofilms were increased at high glucose concentrations (25-30%) compared to the control medium. Significant increase in filamentous forms was observed in cultures with 30% glucose. CONCLUSIONS: Biofilm formation by C. parapsilosis sensu stricto in hyperglycidic medium may contribute to its pathogenic potential for fungemia related to TPN catheters.

Adhesion and biofilm formation of Candida parapsilosis isolated from vaginal secretions to copper intrauterine devices.

INTRODUCTION: Candida parapsilosis is one of the main species that is able to adhere to forming biofilms on inert materials. Adhesion is the first step towards the colonization and invasion of host cells during the infectious process. Among the infections, vulvovaginal candidiasis is increasingly common. The objective was to evaluate the profile of adherence and biofilm formation of eight isolates of C. parapsilosis on the metal used in intrauterine devices (IUDs). METHODS: Eight strains of C. parapsilosis presenting strong adhesion and biofilm formation properties were isolated from vaginal secretions in a previous study. To assay the adhesion and biofilm formation, copper fragments were made and cultivated in tubes containing 3 mL of phosphate-buffered saline and incubated for 6 and 24 h at 37 °C to evaluate biofilm formation. After incubation, the intensity of adherence and of biofilm formation on copper fragments were determined by performing a colony count. RESULTS: All isolates were able to form biofilms and the isolate Cp62 showed many cells joined in a planktonic mode forming biofilms. The use of an IUD is one of the main factors that favors vulvovaginal candidiasis, and the presence of copper in this device increases the chance of recurrent vulvovaginal candidiasis (CVVR) due to the ease with which species of the genus Candida can adhere to inert surfaces. CONCLUSION: This research showed that the clinical isolates studied adhered to IUD copper fragments and formed biofilms, further increasing their virulence.

Efficacy of liposomal amphotericin B against four species of Candida biofilms in an experimental mouse model of intravascular catheter infection.

The formation of Candida biofilms on implanted medical devices is crucial to the development of infections and an important clinical problem because of elevated resistance to antifungals. The aim of this study was to compare the in vitro activity of liposomal amphotericin B (L-AMB) and micafungin (MCFG) against four species of Candida biofilms, and the efficacy of systemic plus lock therapy with L-AMB and MCFG in a Candida biofilm-associated catheter infection model. An XTT-reduction assay was used to measure the metabolic activity of the biofilms to evaluation of in vitro antibiofilm activity. MCFG had better in vitro activity than L-AMB against Candida glabrata biofilms, whereas L-AMB had better activity than MCFG against Candida albicans and Candida tropicalis biofilms. L-AMB and MCFG had comparable efficacy against Candida parapsilosis biofilms. In an in vitro lock therapy model, 2 mg/ml L-AMB, unlike 2 mg/ml MCFG, significantly reduced the metabolic activity of all the strains of biofilms by >96%. Systemic and intraluminal lock treatment with L-AMB for 3-days resulted in more than about 2 log10 reduction of Candida compared with that of systemic treatment and the control group in the C. albicans SP-20012, C. glabrata SP-20040, C. glabrata SP-20131, C. parapsilosis SP-20137, and C. tropicalis SP-20047 infection models. L-AMB was more effective at eradicating Candida biofilms in 3-day course of systemic and lock therapy than MCFG. L-AMB may be useful for the treatment of catheter-related Candida biofilm infections, but this finding will need to be confirmed by further studies including a long treatment duration.

Do raspberry extracts and fractions have antifungal or anti-adherent potential against Candida spp.?

Candida spp., especially Candida albicans, is one of the main colonisers of the oral cavity. Due to its ability to form biofilms, it can be implicated in dental caries, periodontal disease and denture stomatitis. Microbial cells in biofilms are minimally impacted by conventional drugs. The aim of this study was to find new substances able to inhibit the adhesion of Candida spp. in order to prevent biofilm formation in the oral cavity. This study focused on the red raspberry (Rubus idaeus) fruit, known for its richness in potentially antimicrobial tannins. Extraction with a polarity gradient was performed on acetone extracts from frozen ripe and unripe fruits, resulting in eight extracts. The antifungal and anti-adhesion effects of the extracts were determined using broth microdilution and XTT methods, respectively, against C. albicans, Candida glabrata and Candida parapsilosis strains. Interestingly, four extracts (hexane and ethyl acetate) displayed anti-adhesion activity against C. albicans at low concentrations [50% inhibitory concentration (IC50) 15.6-62.5 µg/mL]. Bioassay-guided fractionation by chromatographic methods of the most active extract obtained from ripe fruit (ethyl acetate extract) led to two subfractions enriched in anti-adhesion compounds, identified by mass spectrometry analysis as hydrolysable and condensed tannins. Their activities were dose-dependent with maximum inhibition at 80% (IC50 = 25 µg/mL and 12.5 µg/mL). Regarding antifungal activity, no extract was active against planktonic cells of the tested strains. This work highlights for the first time the potential of raspberries to prevent oral C. albicans biofilms.

Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential.

PURPOSE: Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex. METHODOLOGY: Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were ß-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure. CONCLUSION: These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.