Candida spp. isolated from recreational coastal waters of Rio de Janeiro - Brazil: Focus on antifungal resistance and virulence attributes.

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1-5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87-3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

Candida parapsilosis: heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host immune response. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 FAU, with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

Screening of Candida spp. in wastewater in Brazil during COVID-19 pandemic: workflow for monitoring fungal pathogens.

Fungal diseases are often linked to poverty, which is associated with poor hygiene and sanitation conditions that have been severely worsened by the COVID-19 pandemic. Moreover, COVID-19 patients are treated with Dexamethasone, a corticosteroid that promotes an immunosuppressive profile, making patients more susceptible to opportunistic fungal infections, such as those caused by Candida species. In this study, we analyzed the prevalence of Candida yeasts in wastewater samples collected to track viral genetic material during the COVID-19 pandemic and identified the yeasts using polyphasic taxonomy. Furthermore, we investigated the production of biofilm and hydrolytic enzymes, which are known virulence factors. Our findings revealed that all Candida species could form biofilms and exhibited moderate hydrolytic enzyme activity. We also proposed a workflow for monitoring wastewater using Colony PCR instead of conventional PCR, as this technique is fast, cost-effective, and reliable. This approach enhances the accurate taxonomic identification of yeasts in environmental samples, contributing to environmental monitoring as part of the One Health approach, which preconizes the monitoring of possible emergent pathogenic microorganisms, including fungi.

Manganese(II), copper(II) and silver(I) complexes containing 1,10-phenanthroline/1,10-phenanthroline-5,6-dione against Candida species.

Background: New chemotherapeutics are urgently required to treat Candida infections caused by drug-resistant strains. Methods: The effects of 16 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione/dicarboxylate complexed with Mn(II), Cu(II) and Ag(I) were evaluated against ten different Candida species. Results: Proliferation of Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis and Candida tropicalis was inhibited by three of six Cu(II) (MICs 1.52-21.55 µM), three of three Ag(I) (MICs 0.11-12.74 µM) and seven of seven Mn(II) (MICs 0.40-38.06 µM) complexes. Among these [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O, where oda = octanedioic acid, exhibited effective growth inhibition (MICs 0.4-3.25 µM), favorable activity indexes, low toxicity against Vero cells and good/excellent selectivity indexes (46.88-375). Conclusion: [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O represents a promising chemotherapeutic option for emerging, medically relevant and drug-resistant Candida species.

Candida species are widespread fungi that can cause a variety of infections in humans, and some of them exhibit resistance profile to existing antifungal drugs. Consequently, it is imperative to discover novel treatments for these clinically relevant human infections. Complexes are chemical compounds containing metal ion components that are well-known for their antimicrobial properties, including antifungal activity. In the present study, we investigated the effects of 16 novel complexes against ten medically relevant Candida species, including some strains resistant to commonly used clinical antifungals. Our findings revealed that all complexes containing manganese and silver metals effectively inhibited the growth of all Candida species tested, albeit to varying extents. Some of these complexes exhibited superior antifungal activity and lower toxicity to mammalian cells compared to traditional antifungals, such as fluconazole. In conclusion, these new complexes hold promise as a potential novel approach for treating fungal infections, especially those caused by drug-resistant Candida strains.

Antifungal potential of the new copper(II)-theophylline/1,10-phenanthroline complex against drug-resistant Candida species.

Candida spp. are the commonest fungal pathogens worldwide. Antifungal resistance is a problem that has prompted the discovery of novel anti-Candida drugs. Herein, 25 compounds, some of them containing copper(II), cobalt(II) and manganese(II) ions, were initially evaluated for inhibiting the growth of reference strains of Candida albicans and Candida tropicalis. Eight (32%) of the compounds inhibited the proliferation of these yeasts, displaying minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 µg/mL and minimum fungicidal concentration (MFCs) from 62.5 to 250 µg/mL. Drug-likeness/pharmacokinetic calculated by SwissADME indicated that the 8 selected compounds were suitable for use as topical drugs. The complex CTP, Cu(theo)2phen(H2O).5H2O (theo = theophylline; phen = 1,10-phenanthroline), was chosen for further testing against 10 medically relevant Candida species that were resistant to fluconazole/amphotericin B. CTP demonstrated a broad spectrum of action, inhibiting the growth of all 20 clinical fungal isolates, with MICs from 7.81 to 62.5 µg/mL and MFCs from 15.62 to 62.5 µg/mL. Conversely, CTP did not cause lysis in erythrocytes. The toxicity of CTP was evaluated in vivo using Galleria mellonella and Tenebrio molitor. CTP had no or low levels of toxicity at doses ranging from 31.25 to 250 µg/mL for 5 days. After 24 h of treatment, G. mellonella larvae exhibited high survival rates even when exposed to high doses of CTP (600 µg/mL), with the 50% cytotoxic concentration calculated as 776.2 µg/mL, generating selectivity indexes varying from 12.4 to 99.4 depending on each Candida species. These findings suggest that CTP could serve as a potential drug to treat infections caused by Candida species resistant to clinically available antifungals.

Development of Echinocandin Resistance in Candida haemulonii: An Emergent, Widespread, and Opportunistic Fungal Pathogen.

Echinocandins, used for the prevention and treatment of invasive fungal infections, have led to a rise in breakthrough infections caused by resistant Candida species. Among these species, those belonging to the Candida haemulonii complex are rare multidrug-resistant (MDR) yeasts that are frequently misidentified but have emerged as significant healthcare-associated pathogens causing invasive infections. The objectives of this study were to investigate the evolutionary pathways of echinocandin resistance in C. haemulonii by identifying mutations in the FKS1 gene and evaluating the impact of resistance on fitness. After subjecting a MDR clinical isolate of C. haemulonii (named Ch4) to direct selection using increasing caspofungin concentrations, we successfully obtained an isolate (designated Ch4'r) that exhibited a high level of resistance, with MIC values exceeding 16 mg/L for all tested echinocandin drugs (caspofungin, micafungin, and anidulafungin). Sequence analysis revealed a specific mutation in the resistant Ch4'r strain, leading to an arginine-histidine amino acid substitution (R1354H), occurring at the G4061A position of the HS2 region of the FKS1 gene. Compared to the wild-type strain, Ch4'r exhibited significantly reduced growth proliferation, biofilm formation capability, and phagocytosis ratio, indicating a decrease in fitness. Transmission electron microscopy analysis revealed alterations in cell wall components, with a notable increase in cell wall thickness. The resistant strain also exhibited higher amounts (2.5-fold) of chitin, a cell wall-located molecule, compared to the wild-type strain. Furthermore, the resistant strain demonstrated attenuated virulence in the Galleria mellonella larval model. The evolved strain Ch4'r maintained its resistance profile in vivo since the treatment with either caspofungin or micafungin did not improve larval survival or reduce the fungal load. Taken together, our findings suggest that the acquisition of pan-echinocandin resistance occurred rapidly after drug exposure and was associated with a significant fitness cost in C. haemulonii. This is particularly concerning as echinocandins are often the first-line treatment option for MDR Candida species.

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 palmioleophila: A New Emerging Threat in Brazil?

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal diseases. In addition to climate alterations, the onset of the COVID-19 pandemic has also been associated with the emergence of fungal pathogens. Fungi showed that an inability to grow at high temperatures limits the capacity of fungi to infect mammals. However, fungi can develop thermotolerance, gradually adapting to rising temperatures due to climate change, and generating a greater number of disease-causing organisms. In the present study, we reported the detection and identification of Candida palmioleophila isolates recovered from raw sewage samples in Niteroi city, Rio de Janeiro State, Brazil, during a monitoring program for measuring SARS-CoV-2 presence and concentration. Using polyphasic taxonomy to identify the species and evaluating some virulence aspects of this species, such as biofilm formation and extracellular enzyme production, our data highlight this species as a possible emerging pathogen in Brazil, especially in the pandemic context.

SARS-CoV-2 Post-Infection and Sepsis by Saccharomyces cerevisiae: A Fatal Case Report-Focus on Fungal Susceptibility and Potential Virulence Attributes.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for approximately 6.8 million deaths worldwide, threatening more than 753 million individuals. People with severe coronavirus disease-2019 (COVID-19) infection often exhibit an immunosuppression condition, resulting in greater chances of developing co-infections with bacteria and fungi, including opportunistic yeasts belonging to the Saccharomyces and Candida genera. In the present work, we have reported the case of a 75-year-old woman admitted at a Brazilian university hospital with an arterial ulcer in the left foot, which was being prepared for surgical amputation. The patient presented other underlying diseases and presented positive tests for COVID-19 prior to hospitalization. She received antimicrobial treatment, but her general condition worsened quickly, leading to death by septic shock after 4 days of hospitalization. Blood samples collected on the day she died were positive for yeast-like organisms, which were later identified as Saccharomyces cerevisiae by both biochemical and molecular methods. The fungal strain exhibited low minimal inhibitory concentration values for the antifungal agents tested (amphotericin B, 5-flucytosine, caspofungin, fluconazole and voriconazole), and it was able to produce important virulence factors, such as extracellular bioactive molecules (e.g., aspartic peptidase, phospholipase, esterase, phytase, catalase, hemolysin and siderophore) and biofilm. Despite the activity against planktonic cells, the antifungals were not able to impact the mature biofilm parameters (biomass and viability). Additionally, the S. cerevisiae strain caused the death of Tenebrio molitor larvae, depending on the fungal inoculum, and larvae immunosuppression with corticosteroids increased the larvae mortality rate. In conclusion, the present study highlighted the emergence of S. cerevisiae as an opportunistic fungal pathogen in immunosuppressed patients presenting several severe comorbidities, including COVID-19 infection.

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.

Prospective Medicines against the Widespread, Emergent, and Multidrugresistant Opportunistic Fungal Pathogen Candida auris: A Breath of Hope.

The emergence of the pathogen Candida auris is a real concern worldwide, especially due to its multidrug resistance profile, besides the difficulties in establishing the correct identification by conventional laboratory methods and its capacity of causing outbreaks in healthcare settings. The limited arsenal of available antifungal drugs, coupled with the lack of momentum for the development of new reagents, represent a challenge in the management of such a pathogen. In this perspective, we have focused on discussing new, promising treatment options for C. auris infections. These novel drugs include an antifungal agent already approved for medical use in the United States of America, compounds that are already in clinical trials and those with potential for repurposing use against this important fungal pathogen.

Protease Inhibitors as Promising Weapons against COVID-19: Focus on Repurposing of Drugs used to Treat HIV and HCV Infections.

As a part of the efforts to quickly develop pharmaceutical treatments for COVID-19 through repurposing existing drugs, some researchers around the world have combined the recently released crystal structure of SARS-CoV-2 Mpro in complex with a covalently bonded inhibitor with virtual screening procedures employing molecular docking approaches. In this context, protease inhibitors (PIs) clinically available and currently used to treat infectious diseases, particularly viral ones, are relevant sources of promising drug candidates to inhibit the SARS-CoV-2 Mpro, a key viral enzyme involved in crucial events during its life cycle. In the present perspective, we summarized the published studies showing the promising use of HIV and HCV PIs as potential repurposing drugs against the SARS-CoV-2 Mpro.

Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans-In Silico, In Vitro and In Vivo Approaches.

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

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.

Insights into the Multi-Azole Resistance Profile in Candida haemulonii Species Complex.

The Candida haemulonii complex (C. duobushaemulonii, C. haemulonii, and C. haemulonii var. vulnera) is composed of emerging, opportunistic human fungal pathogens able to cause invasive infections with high rates of clinical treatment failure. This fungal complex typically demonstrates resistance to first-line antifungals, including fluconazole. In the present work, we have investigated the azole resistance mechanisms expressed in Brazilian clinical isolates forming the C. haemulonii complex. Initially, 12 isolates were subjected to an antifungal susceptibility test, and azole cross-resistance was detected in almost all isolates (91.7%). In order to understand the azole resistance mechanistic basis, the efflux pump activity was assessed by rhodamine-6G. The C. haemulonii complex exhibited a significantly higher rhodamine-6G efflux than the other non-albicans Candida species tested (C. tropicalis, C. krusei, and C. lusitaneae). Notably, the efflux pump inhibitors (Phe-Arg and FK506) reversed the fluconazole and voricolazole resistance phenotypes in the C. haemulonii species complex. Expression analysis indicated that the efflux pump (ChCDR1, ChCDR2, and ChMDR1) and ERG11 genes were not modulated by either fluconazole or voriconazole treatments. Further, ERG11 gene sequencing revealed several mutations, some of which culminated in amino acid polymorphisms, as previously reported in azole-resistant Candida spp. Collectively, these data point out the relevance of drug efflux pumps in mediating azole resistance in the C. haemulonii complex, and mutations in ERG11p may contribute to this resistance profile.

Pathogenicity Levels of Colombian Strains of Candida auris and Brazilian Strains of Candida haemulonii Species Complex in Both Murine and Galleria mellonella Experimental Models.

Candida auris and Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) are phylogenetically related species that share some physiological features and habits. In the present study, we compared the virulence of these yeast species using two different experimental models: (i) Galleria mellonella larvae to evaluate the survival rate, fungal burden, histopathology and phagocytosis index and (ii) BALB/c mice to evaluate the survival. In addition, the fungal capacity to form biofilm over an inert surface was analyzed. Our results showed that in both experimental models, the animal survival rate was lower when infected with C. auris strains than the C. haemulonii species complex. The hemocytes of G. mellonella showed a significantly reduced ability to phagocytize the most virulent strains forming the C. haemulonii species complex. Interestingly, for C. auris, it was impossible to measure the phagocytosis index due to a general lysis of the hemocytes. Moreover, it was observed a greater capability of biofilm formation by C. auris compared to C. haemulonii species complex. In conclusion, we observed that C. auris and C. haemulonii complex have different levels of pathogenicity in the experimental models employed in the present study.

Secreted aspartyl peptidases by the emerging, opportunistic and multidrug-resistant fungal pathogens comprising the Candida haemulonii complex.

The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72-96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans, detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of ∼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp.