Revealing the impact of Rapamycin on the virulence factors of the Candida haemulonii complex.

The incidence of invasive fungal infections caused by Candida species is increasing, particularly in immunocompromised individuals. This increasing incidence poses a dual challenge, comprising escalating antifungal resistance and the necessity for accurate fungal identification. The Candida haemulonii complex further complicates these challenges due to limited identification tools. Like some other Candida species, infections involving this complex show resistance to multiple antifungals, requiring innovative therapeutic approaches. Rapamycin, known for its antifungal properties and immunosuppressive characteristics, was investigated against the C. haemulonii complex species. Results revealed a rapamycin minimal inhibitory concentration (MIC) range of 0.07 to >20 µM, with fungicidal effects in most strains. In vitro analyses using the rapamycin maximum plasma concentration (0.016 µM) showed reduced surface properties and decreased production of extracellular enzymes. Rapamycin also hindered biofilm formation by some strains. Even when treated at the human therapeutic dose, which is lower than the MIC, phenotypic variations in C. haemulonii were detected, hinting at the possible attenuation of some virulence factors when exposed to rapamycin.

Patterns recovered in phylogenomic analysis of Candida auris and close relatives implicate broad environmental flexibility in Candida/Clavispora clade yeasts.

Fungal pathogens commonly originate from benign or non-pathogenic strains living in the natural environment. The recently emerged human pathogen, Candida auris, is one example of a fungus believed to have originated in the environment and recently transitioned into a clinical setting. To date, however, there is limited evidence about the origins of this species in the natural environment and when it began associating with humans. One approach to overcome this gap is to reconstruct phylogenetic relationships between (1) strains isolated from clinical and non-clinical environments and (2) between species known to cause disease in humans and benign environmental saprobes. C. auris belongs to the Candida/Clavispora clade, a diverse group of 45 yeast species including human pathogens and environmental saprobes. We present a phylogenomic analysis of the Candida/Clavispora clade aimed at understanding the ecological breadth and evolutionary relationships between an expanded sample of environmentally and clinically isolated yeasts. To build a robust framework for investigating these relationships, we developed a whole-genome sequence dataset of 108 isolates representing 18 species, including four newly sequenced species and 18 environmentally isolated strains. Our phylogeny, based on 619 orthologous genes, shows environmentally isolated species and strains interspersed with clinically isolated counterparts, suggesting that there have been many transitions between humans and the natural environment in this clade. Our findings highlight the breadth of environments these yeasts inhabit and imply that many clinically isolated yeasts in this clade could just as easily live outside the human body in diverse natural environments and vice versa.

Identification and antifungal susceptibility testing of Candida haemulonii complex isolated from clinical samples.

Candida haemulonii complex (Candida haemulonii [I], Candida duobushaemulonii [II], and Candida haemulonii var. vulnera [III]) has become relevant in recent times, not so much because of a high incidence in human clinical sample cultures but because of its remarkable antifungal resistance. The objective of this study was to evaluate several methods for the identification of this uncommon species of Candida. Ten isolates of C. haemulonii were identified by biochemical and proteomic methods, and their antifungal susceptibility testing was performed by both commercial and reference methods. MALDI-TOF MS (Vitek MS and Vitek MS PRIME) and Vitek2 correctly identified these genera but API method did not. There was a good correlation between the commercial methods and the reference methods for the AST. In conclusion Vitek MS, Vitek MS PRIME, and Vitek2 systems, but not API32C, are reliable for identification of C. haemulonii complex. Furthermore, MALDI-TOF MS systems could identify to the subspecies level. Commercial methods for antifungal susceptibility testing are valid for the study of this species and confirm amphotericin B and to azole resistance.

Cell Aggregation Capability of Clinical Isolates from Candida auris and Candida haemulonii Species Complex.

The opportunistic fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profiles. Candida auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from the host immune responses. In the present study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex (C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera) and C. auris. Subsequently, we evaluated the effects of physicochemical factors on fungal aggregation competence. The results demonstrated that cell-to-cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates of both the C. haemulonii species complex and C. auris exhibited high aggregation after 2 h of incubation at 37 °C. Interestingly, the fungal cells forming the aggregates remained viable. The aggregation of all isolates was not impacted by pH, temperature, ß-mercaptoethanol (a protein-denaturing agent), or EDTA (a chelator agent). Conversely, proteinase K, trypsin, and sodium dodecyl sulfate (SDS) significantly diminished the fungal aggregation. Collectively, our results demonstrated that the aggregation ability of these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation since the presence of proteases and anionic detergents affected the aggregation capability. However, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.

Candida haemulonii Species Complex: A Mini-review.

Candida haemulonii species complex (CHSC) are emerging multidrug-resistant yeast pathogens able to cause life-threatening human infections in at-risk populations for invasive candidiasis worldwide. A recent laboratory survey conducted in 12 medical centers found that prevalence rates of Candida haemulonii complex isolates rose from 0.9 to 1.7% along the period between 2008 and 2019. We present a mini-review addressing recent aspects of the epidemiology, diagnosis and therapy of infections due to CHSC.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst.

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Candida haemulonii Complex and Candida auris: Biology, Virulence Factors, Immune Response, and Multidrug Resistance.

There is worldwide concern about the constant increase in infections caused by Candida species that are multiresistant to antifungal drugs. The most common candidiasis is caused by Candida albicans, however, the species of the Candida haemulonii complex and Candida auris are emerging opportunistic pathogens, which isolation from clinical samples has significantly increased in the past years. The special interest in the study of these species lies in their ability to evade the action of antifungal drugs, such as amphotericin B, azoles, and echinocandins. In addition, the phenotypic changes of these species have given them the ability to easily adapt to environmental changes, including the host milieu and immunity. In this paper, a detailed review of the current literature on the C. haemulonii complex and C. auris is shown, analyzing aspects such as biology, immune response, putative virulence factors, infection, treatment, and the current strategies for diagnosis.

Clonal Dissemination of Antifungal-Resistant Candida haemulonii, China.

Candida haemulonii, a relative of C. auris, frequently shows antifungal resistance and is transmissible. However, molecular tools for genotyping and investigating outbreaks are not yet established. We performed genome-based population analysis on 94 C. haemulonii strains, including 58 isolates from China and 36 other published strains. Phylogenetic analysis revealed that C. haemulonii can be divided into 4 clades. Clade 1 comprised strains from China and other global strains; clades 2-4 contained only isolates from China, were more recently evolved, and showed higher antifungal resistance. Four regional epidemic clusters (A, B, C, and D) were identified in China, each comprising ≥5 cases (largest intracluster pairwise single-nucleotide polymorphism differences <50 bp). Cluster A was identified in 2 hospitals located in the same city, suggesting potential intracity transmissions. Cluster D was resistant to 3 classes of antifungals. The emergence of more resistant phylogenetic clades and regional dissemination of antifungal-resistant C. haemulonii warrants further monitoring.

Candida khanbhai sp. nov., a new clinically relevant yeast within the Candida haemulonii species complex.

Invasive fungal infections caused by non-albicans Candida species are increasingly reported. Recent advances in diagnostic and molecular tools enabled better identification and detection of emerging pathogenic yeasts. The Candida haemulonii species complex accommodates several rare and recently described pathogenic species, C. duobushaemulonii, C. pseudohaemulonii, C. vulturna, and the most notorious example is the outbreak-causing multi-drug resistant member C. auris. Here, we describe a new clinically relevant yeast isolated from geographically distinct regions, representing the proposed novel species C. khanbhai, a member of the C. haemulonii species complex. Moreover, several members of the C. haemulonii species complex were observed to be invalidly described, including the clinically relevant species C. auris and C. vulturna. Hence, the opportunity was taken to correct this here, formally validating the names of C. auris, C. chanthaburiensis, C. konsanensis, C. metrosideri, C. ohialehuae, and C. vulturna.

Although C. albicans remains the major pathogenic yeast, other previously rare or even novel species are on the rise in the clinic. The most notorious example is the rapid global emergence of multidrug-resistant C. auris. Here we describe its novel sibling species C. khanbhai.

First two fungemia cases caused by Candida haemulonii var. vulnera in China with emerged antifungal resistance.

Candida haemulonii var. vulnera is a rare variant of C. haemulonii, which has been previously reported to cause human infections. Owing to the close kinship between C. haemulonii sensu stricto and C. haemulonii var. vulnera, accurate identification of C. haemulonii var. vulnera relied on DNA sequencing assay targeting, for example, rDNA internal transcribed spacer (ITS) region. In this work, two strains of C. haemulonii var. vulnera were collected from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). The identification capacity of three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 YST ID biochemical methods were evaluated against ITS sequencing. In addition, antifungal susceptibility testing was performed using Sensititre YeastOne. Moreover, we comprehensively screened drug-resistant related genes by whole-genome sequencing. The two strains were not correctly identified to species variant level using MALDI-TOF MS and YST ID cards. Both strains were resistant to amphotericin B (minimum inhibitory concentration [MIC] > 2 µg/ml). Moreover, strain F4564 and F4584 exhibited high MIC to fluconazole (>256 µg/ml) and 5-flucytosine (>64 µg/ml), respectively, which were supposed to result from key amino acid substitutions Y132F and G307A in Erg11p and V58fs and G60K substitutions in Fur1p. The rare species C. haemulonii var. vulnera has emerged in China, and such drug-resistant fungal species that can cause invasive diseases require further close attention.

The Threat Called Candida haemulonii Species Complex in Rio de Janeiro State, Brazil: Focus on Antifungal Resistance and Virulence Attributes.

Although considered rare, the emergent Candida haemulonii species complex, formed by C. haemulonii sensu stricto (Ch), C. duobushaemulonii (Cd) and C. haemulonii var. vulnera (Chv), is highlighted due to its profile of increased resistance to the available antifungal drugs. In the present work, 25 clinical isolates, recovered from human infections during 2011-2020 and biochemically identified by automated system as C. haemulonii, were initially assessed by molecular methods (amplification and sequencing of ITS1-5.8S-ITS2 gene) for precise species identification. Subsequently, the antifungal susceptibility of planktonic cells, biofilm formation and susceptibility of biofilms to antifungal drugs and the secretion of key molecules, such as hydrolytic enzymes, hemolysins and siderophores, were evaluated by classical methodologies. Our results revealed that 7 (28%) isolates were molecularly identified as Ch, 7 (28%) as Chv and 11 (44%) as Cd. Sixteen (64%) fungal isolates were recovered from blood. Regarding the antifungal susceptibility test, the planktonic cells were resistant to (i) fluconazole (100% of Ch and Chv, and 72.7% of Cd isolates), itraconazole and voriconazole (85.7% of Ch and Chv, and 72.7% of Cd isolates); (ii) no breakpoints were defined for posaconazole, but high MICs were observed for 85.7% of Ch and Chv, and 72.7% of Cd isolates; (iii) all isolates were resistant to amphotericin B; and (iv) all isolates were susceptible to echinocandins (except for one isolate of Cd) and to flucytosine (except for two isolates of Cd). Biofilm is a well-known virulence and resistant structure in Candida species, including the C. haemulonii complex. Herein, we showed that all isolates were able to form viable biofilms over a polystyrene surface. Moreover, the mature biofilms formed by the C. haemulonii species complex presented a higher antifungal-resistant profile than their planktonic counterparts. Secreted molecules associated with virulence were also detected in our fungal collection: 100% of the isolates yielded aspartic proteases, hemolysins and siderophores as well as phospholipase (92%), esterase (80%), phytase (80%), and caseinase (76%) activities. Our results reinforce the multidrug resistance profile of the C. haemulonii species complex, including Brazilian clinical isolates, as well as their ability to produce important virulence attributes such as biofilms and different classes of hydrolytic enzymes, hemolysins and siderophores, which typically present a strain-dependent profile.

Case report: Catheter related blood stream infection caused by Candida vulturna.

Candida vulturna is a newly emerging candida species belong to Candida haemulonii species complex of Metschnikowiaceae family. Numerous clinical samples have been reported to isolate C. vulturna since discovery. We report a case of catheter related blood stream infection in which C. vulturna was isolated from blood in patient after prolong antibiotic therapy for recurrent infection of retroperitoneal cyst. The blood isolate was identified to species level by molecular assay targeting D1/D2 regions of 26s rDNA gene. The patient improved with administration of intravenous micafungin despite lack of antifungal susceptibility breakpoints.

Phenotypic Switching and Filamentation in Candida haemulonii, an Emerging Opportunistic Pathogen of Humans.

Phenotypic plasticity is a common strategy adopted by fungal pathogens to adapt to diverse host environments. Candida haemulonii is an emerging multidrug-resistant human pathogen that is closely related to Candida auris. Until recently, it was assumed that C. haemulonii is incapable of phenotypic switching or filamentous growth. In this study, we report the identification of three distinct phenotypes in C. haemulonii: white, pink, and filament. The white and pink phenotypes differ in cellular size, colony morphology, and coloration on phloxine B- or CuSO4-containing agar. Switching between the white and pink cell types is heritable and reversible and is referred to as "the primary switching system." The additional switch phenotype, filament, has been identified and exhibits obviously filamentous morphology when grown on glycerol-containing medium. Several unique characteristics of the filamentous phenotype suggest that switching from or to this phenotype poses as a second yeast-filament switching system. The yeast-filament switch is nonheritable and temperature-dependent. Low temperatures favor the filamentous phenotype, whereas high temperatures promote filament-yeast transition. We further demonstrated that numerous aspects of the distinct cell types differ in numerous biological aspects, including their high temperature response, specific gene expression, CuSO4 tolerance, secreted aspartyl protease (SAP) activity, and virulence. Therefore, transition among the three phenotypes could enable C. haemulonii to rapidly adapt to, survive, and thrive in certain host niches, thereby contributing to its virulence. IMPORTANCE The capacity to switch between distinct cell types, known as phenotypic switching, is a common strategy adopted by Candida species to adapt to diverse environments. Despite considerable studies on phenotypic plasticity of various Candida species, Candida haemulonii is considered to be incapable of phenotypic switching or filamentous growth. Here, we report and describe filamentation and three distinct phenotypes (white, pink, and filament) in C. haemulonii. The three cell types differ in cellular and colony appearance, gene expression profiles, CuSO4 tolerance, and virulence. C. haemulonii cells switch heritably and reversibly between white and pink cell types, which is referred to as the "primary switching system." Switching between pink and filamentous phenotypes is nonheritable and temperature-dependent, representing a second switching system. As in other Candida species, switching among distinct morphological types may provide C. haemulonii with phenotypic plasticity for rapid responses to the changing host environment, and may contribute to its virulence.

Functional and Comparative Analysis of Centromeres Reveals Clade-Specific Genome Rearrangements in Candida auris and a Chromosome Number Change in Related Species.

The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 causing systemic infections worldwide and simultaneously evolved in different geographical zones. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional GC-poor centromere landscape lacking pericentromeres or repeats. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Interspecies genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug-susceptible isolates of C. aurisIMPORTANCECandida auris, the killer fungus, emerged as different geographical clades, exhibiting multidrug resistance and high karyotype plasticity. Chromosomal rearrangements are known to play key roles in the emergence of new species, virulence, and drug resistance in pathogenic fungi. Centromeres, the genomic loci where microtubules attach to separate the sister chromatids during cell division, are known to be hot spots of breaks and downstream rearrangements. We identified the centromeres in C. auris and related species to study their involvement in the evolution and karyotype diversity reported in C. auris We report conserved centromere features in 10 related species and trace the events that occurred at the centromeres during evolution. We reveal a centromere inactivation-mediated chromosome number change in these closely related species. We also observe that one of the geographical clades, the East Asian clade, evolved along a unique trajectory, compared to the other clades and related species.

The serine peptidase inhibitor N-ρ-tosyl-l-phenylalanine chloromethyl ketone (TPCK) affects the cell biology of Candida haemulonii species complex.

Candida haemulonii species complex (C. haemulonii, C. haemulonii var. vulnera and Candida duobushaemulonii) is composed by emerging and multidrug-resistant (MDR) yeasts. Candidiasis, the disease caused by these species, is difficult to treat and culminates in clinical failures and patient death. It is well-known that Candida peptidases play important roles in the fungus-host interactions, and hence these enzymes are promising targets for developing new antifungal drugs. Recently, serine-type peptidases were described in clinical isolates of C. haemulonii complex with the ability to cleave relevant key host proteins. Herein, the effects of serine peptidase inhibitors (SPIs) on the cell biology of this fungal complex were evaluated. Initially, eight distinct SPIs (phenylmethylsulfonyl fluoride - PMSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride - AEBSF, N-α-tosyl-l-lysine chloromethyl ketone hydrochloride - TLCK, N-p-tosyl-l-phenylalanine chloromethyl ketone - TPCK, simeprevir, boceprevir, danoprevir and telaprevir) were tested on the fungal growth. TPCK showed the best efficacy in controlling cell proliferation, being selected for the following experiments. This SPI induced changes in the architecture of yeast cells, as observed by scanning electron microscopy, besides injuries at the plasma membrane and reduction in the ergosterol content. TPCK also diminished the ability of yeasts to adhere to abiotic (polystyrene and glass) and biotic (murine macrophages) surfaces in a typically concentration-dependent manner. In addition, the 24 h-treatment of the mature biofilm promoted a decrease in biomass, viability and extracellular matrix. Altogether, our results highlight that SPIs may be promising new therapeutic agents in the treatment of candidiasis caused by emergent, opportunistic and MDR species forming the C. haemulonii complex.

Liver abscess caused by Candida haemulonii var. vulnera. First case report in Peru.

BACKGROUND: Liver abscesses caused by Candida species are mainly found in immunocompromised hosts, associated with conditions (such as neutropenia and mucositis) that facilitate the spreading of microorganisms from the gastrointestinal tract. CASE REPORT: We present the case of a non-immunocompromised 72-year-old woman with a liver abscess caused by Candida haemulonii var. vulnera, in whom potential associated conditions could be polycystic kidney disease and renal replacement therapy. The patient experienced clinical resolution after percutaneous drainage and treatment with caspofungin. CONCLUSIONS: To our knowledge, this is the first case reported in Peru of a liver abscess due to Candida haemulonii var. vulnera, a clinical presentation that has not been described previously. This finding should prompt us to establish active surveillance of causal agents of systemic candidiasis.

Antifungal susceptibility profile of Candida clinical isolates from 22 hospitals of São Paulo State, Brazil

This study aimed to evaluate the frequency of cryptic Candida species from candidemia cases in 22 public hospitals in São Paulo State, Brazil, and their antifungal susceptibility profiles. During 2017 and 2018, 144 isolates were molecularly identified as 14 species; C. parapsilosis (32.6%), C. albicans (27.7%), C. tropicalis (14.6%), C. glabrata (9.7%), C. krusei (2.8%), C. orthopsilosis (2.8%), C. haemulonii var. vulnera (2.1%), C. haemulonii (1.4%), C. metapsilosis (1.4%), C. dubliniensis (1.4%), C. guilliermondii (1.4%), C. duobushaemulonii (0.7%), C. kefyr (0.7%), and C. pelliculosa (0.7%). Poor susceptibility to fluconazole was identified in 6.4% of C. parapsilosis isolates (0.12 to >64 µg/mL), 50% of C. guilliermondii (64 µg/mL), 66.6% of C. haemulonii var. vulnera (16-32 µg/mL), and C. duobushaemulonii strain (MIC 64 µg/mL). Our results corroborated the emergence of C. glabrata in Brazilian cases of candidemia as previously reported. Importantly, we observed a large proportion of non-wild type C. glabrata isolates to voriconazole (28.6%; <0.015 to 4 µg/mL) all of which were also resistant to fluconazole (28.6%). Of note, C. haemulonii, a multidrug resistant species, has emerged in the Southeast region of Brazil. Our findings suggested a possible epidemiologic change in the region with an increase in fluconazole-resistant species causing candidemia. We stress the relevance of routine accurate identification to properly manage therapy and monitor epidemiologic trends.

Candida duobushaemulonii: An Old But Unreported Pathogen.

Candidiasis caused by species of the Candida haemulonii complex (Candida haemulonii and Candida duobushaemulonii) and closely related species, Candida auris and Candida pseudohaemulonii are increasing. These species often show reduced susceptibility to antifungal drugs, such as azoles and amphotericin B or, less frequently, echinocandins. However, conventional phenotypic identification methods are unable to accurately differentiate these species and, therefore, their prevalence may have been underestimated. In this study, 150 isolates that were probably misidentified were reanalyzed using two novel PCR approaches. We found that one isolate previously identified in 1996 as Candida intermedia was C. duobushaemulonii, being one of the oldest isolates of this species described to date. We also found that this isolate had reduced susceptibility to fluconazole, itraconazole, and amphotericin B.

Susceptibility of the Candida haemulonii Complex to Echinocandins: Focus on Both Planktonic and Biofilm Life Styles and a Literature Review.

Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) is well-known for its resistance profile to different available antifungal drugs. Although echinocandins are the most effective class of antifungal compounds against the C. haemulonii species complex, clinical isolates resistant to caspofungin, micafungin and anidulafungin have already been reported. In this work, we present a literature review regarding the effects of echinocandins on this emergent fungal complex. Published data has revealed that micafungin and anidulafungin were more effective than caspofungin against the species forming the C. haemulonii complex. Subsequently, we investigated the susceptibilities of both planktonic and biofilm forms of 12 Brazilian clinical isolates of the C. haemulonii complex towards caspofungin and micafungin (anidulafungin was unavailable). The planktonic cells of all the fungal isolates were susceptible to both of the test echinocandins. Interestingly, echinocandins caused a significant reduction in the biofilm metabolic activity (viability) of almost all fungal isolates (11/12, 91.7%). Generally, the biofilm biomasses were also affected (reduction range 20-60%) upon exposure to caspofungin and micafungin. This is the first report of the anti-biofilm action of echinocandins against the multidrug-resistant opportunistic pathogens comprising the C. haemulonii complex, and unveils the therapeutic potential of these compounds.