Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Pichia kudriavzevii.

The pathogenic yeast Pichia kudriavzevii, previously known as Candida krusei, is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in P. kudriavzevii. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in P. kudriavzevii is similar to Saccharomyces cerevisiae and C. albicans and is comprised of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, P. kudriavzevii cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of P. kudriavzevii yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 33 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in P. kudriavzevii including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of P. kudriavzevii.

Antimicrobial Peptides with Anti-Candida Activity.

Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality. Candida albicans remains the prevalent etiologic agent, but the incidence of other species such as Candida parapsilosis, Candida glabrata and Candida auris keeps increasing. These pathogens frequently show a reduced susceptibility to commonly used antifungal drugs, including polyenes, triazoles and echinocandins, and the incidence of emerging multi-drug-resistant strains of these species continues to increase. Therefore, the need to search for new molecules that target these pathogenic species in a different manner is now more urgent than ever. Nature is an almost endless source of interesting new molecules that could meet this need. Among these molecules, antimicrobial peptides, present in different sources in nature, possess some advantages over conventional antifungal agents, even with their own drawbacks, and are considered as a promising pharmacological option against a wide range of microbial infections. In this review, we describe 20 antimicrobial peptides from different origins that possess an activity against Candida.

Development and Characterization of Monoolein-Based Liposomes of Carvacrol, Cinnamaldehyde, Citral, or Thymol with Anti-Candida Activities.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti-Candida activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. In vitro antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of Candida albicans, Candida auris, Candida dubliniensis, and Candida tropicalis Finally, the ability of macrophage cells to kill Candida isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 µg/ml of cinnamaldehyde, 256 µg/ml of citral, and 128 µg/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (∼41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages.

Identification of Candida auris and related species by multiplex PCR based on unique GPI protein-encoding genes.

BACKGROUND: The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, and however, the fungus has often been misidentified by commercial systems. OBJECTIVES: To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol (GPI)-modified protein-encoding genes. METHODS: Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up. RESULTS: All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different-sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum. CONCLUSIONS: PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up.

Caenorhabditis elegans as a Model System To Assess Candida glabrata, Candida nivariensis, and Candida bracarensis Virulence and Antifungal Efficacy.

Although Candida albicans remains the major etiological agent of invasive candidiasis, Candida glabrata and other emerging species of Candida are increasingly isolated. This species is the second most prevalent cause of candidiasis in many regions of the world. However, clinical isolates of Candida nivariensis and Candida bracarensis can be misidentified and are underdiagnosed due to phenotypic traits shared with C. glabrata Little is known about the two cryptic species. Therefore, pathogenesis studies are needed to understand their virulence traits and their susceptibility to antifungal drugs. The susceptibility of Caenorhabditis elegans to different Candida species makes this nematode an excellent model for assessing host-fungus interactions. We evaluated the usefulness of C. elegans as a nonconventional host model to analyze the virulence of C. glabrata, C. nivariensis, and C. bracarensis The three species caused candidiasis, and the highest virulence of C. glabrata was confirmed. Furthermore, we determined the efficacy of current antifungal drugs against the infection caused by these species in the C. elegans model. Amphotericin B and azoles showed the highest activity against C. glabrata and C. bracarensis infections, while echinocandins were more active for treating those caused by C. nivariensisC. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

Utility of two PCR-RFLP-based techniques for identification of Candida parapsilosis complex blood isolates.

BACKGROUND: Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei. OBJECTIVE: To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. METHODS: Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods. RESULTS: In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. CONCLUSION: The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected.

Effect of biomaterials hydrophobicity and roughness on biofilm development.

Most hospitalized patients are carriers of biomedical devices. Infections associated with these devices cause great morbidity and mortality, especially in patients in intensive care units. Numerous strategies have been designed to prevent biofilm development on biodevices. However, biofilm formation is a complex process not fully clarified. In the current study, roughness and hydrophobicity of different biomaterials was analyzed to assess their influences on the biofilm formation of four leading etiological causes of healthcare-associated infections, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans, using a CDC biofilm reactor. Hydrophobic materials allowed the formation of more abundant and profuse biofilms. Roughness had effect on biofilm formation, but its influence was not significant when material hydrophobicity was considered.

Design and validation of a multiplex PCR protocol for microsatellite typing of Candida parapsilosis sensu stricto isolates.

BACKGROUND: Analysis of polymorphic microsatellite markers (STR) is a helpful genotyping technique to differentiate Candida parapsilosis sensu stricto isolates. The aim of this study is to develop and perform an initial validation of an alternative protocol for the reliable and accurate microsatellite genotyping of C. parapsilosis sensu stricto isolates using high-throughput multiplex PCR. To achieve this, the results obtained using the new protocol were compared to the ones obtained using a previously described reference method. To that end, diagnostic accuracy, informativeness and discrimination parameters were estimated. RESULTS: Our results showed good concordance between both methods (Kappa index: 0.920), leading to a high sensitivity (1; CI(95%) (0.991-1)) and specificity (1; CI(95%) (0.772-1)) after the validation of the new protocol. Moreover, the electropherograms profiles obtained with the new PCR scheme showed a high signal to noise ratio (SNR). CONCLUSIONS: The new multiplex protocol is valuable for the differentiation of C. parapsilosis sensu stricto, with direct clinical applications. Besides, the new protocol represents a shortening the hands-on time, reducing the sample manipulation (dismissing the possibility of cross-contamination), maintaining the quality of the results (when compared to the ones obtained with the reference method), and helping to the standardization and simplification of the genotyping scheme.

Molecular identification of Candida auris by PCR amplification of species-specific GPI protein-encoding genes.

The emerging multidrug-resistant pathogenic yeast Candida auris causes life-threatening invasive infections and shows a capacity for hospital transmission that is uncommon in other Candida species. Rapid and accurate diagnosis of C. auris infections is crucial; however, the fungus is frequently misidentified. Here, we present a rapid and easily applicable PCR assay for reliable identification of C. auris by designing primers from unique GPI protein-encoding genes. Specificity of the used primers for C. auris was verified with a panel of 19 different Candida species including the clinically most relevant and phylogenetically closely related species. Efficacy of the PCR approach was validated by correctly identifying 112 C. auris isolates from an outbreak in a Spanish hospital, 20% of which were not reliably identified by MALDI-TOF MS, and 27 genotypically diverse C. auris isolates originating from hospitals in various countries, in a test that included (blind) negative controls. By employing two GPI protein primer pairs in a single PCR, a double screening can be performed, which enhances the robustness of the PCR assay and avoids potential false negatives due to recent evolutionary events, as was observed for two isolates. Our PCR method, which is based on the uniqueness of selected GPI protein-encoding genes, is useful for easy, low-cost, and accurate identification of C. auris infections in a clinical setting.

Vancomycin heteroresistant community associated methicillin-resistant Staphylococcus aureus ST72-SCCmecIVa strain colonizing the nostrils of a five-year-old Spanish girl.

BACKGROUND AND OBJECTIVES: During a community methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization study, an MRSA strain with vancomycin hetero-resistance (h-VISA) was isolated from a five year-old girl with tetralogy of Fallot without previous exposure to vancomycin. An extended nasal colonization study was performed on all her close relatives. RESULTS: Only the patient and her sister were colonized by an h-VISA MRSA strain (clone USA 700, ST72, t148, agr 1 and SCCmec IVa). Mupirocin decolonisation was effective in the elder sister. A new nasal decolonisation in the younger girl using fusidic acid was also successful. However, after decolonisation both sisters were colonized by a methicillin-susceptible S. aureus (ST30, t012 and agr 3) previously isolated from their mother's nostrils. CONCLUSION: As S. aureus have a great capacity to spread among people in close contact, knowledge of a patients' colonization status, tracing contacts, and a correct management are critical issues for the successful containment of multiresistant staphylococci.

Usefulness of the Non-conventional Caenorhabditis elegans Model to Assess Candida Virulence.

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.

In Vitro Antifungal Susceptibility of Oral Candida Isolates from Patients Suffering from Caries and Chronic Periodontitis.

Caries and chronic periodontitis are common oral diseases where a higher Candida colonization is reported. Antifungal agents could be adjuvant drugs for the therapy of both clinical conditions. The aim of the current study has been to evaluate the in vitro activities of conventional and new antifungal drugs against oral Candida isolates from patients suffering from caries and/or chronic periodontitis. In vitro activities of amphotericin B, fluconazole, itraconazole, miconazole, nystatin, posaconazole and voriconazole against 126 oral Candida isolates (75 Candida albicans, 18 Candida parapsilosis, 11 Candida dubliniensis, six Candida guilliermondii, five Candida lipolytica, five Candida glabrata, four Candida tropicalis and two Candida krusei) from 61 patients were tested by the CLSI M27-A3 method. Most antifungal drugs were highly active, and resistance was observed in less than 5% of tested isolates. Miconazole was the most active antifungal drug, being more than 98% of isolates susceptible. Fluconazole, itraconazole, and the new triazoles, posaconazole and voriconazole, were also very active. Miconazole, fluconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent suitable treatment for a hypothetically adjunctive therapy of caries and chronic periodontitis.

Caries and Candida colonisation in adult patients in Basque Country (Spain).

Candida albicans is one of the most frequent pathogens of the oral cavity, as a major cause of opportunistic disease. Moreover, Candida could be a cofactor of common oral diseases, such as dental caries. The aim of this study was to analyse the oral yeast colonisation in adults with dental caries and to evaluate its relationship with this clinical entity. We studied 190 patients distributed into controls (58 patients) and patients with caries (132 patients). Oral samples were collected by oral rinse and cultured in a chromogenic agar. C. albicans was the most prevalent species isolated from oral specimens in both groups. Patients with caries had a greater Candida colonisation (74 patients, 56.1%), than persons without caries (18 patients, 31%, P < 0.01). Patients with caries were significantly more colonised by non-C. albicans species than individuals without caries (P = 0.006). Moreover, the diversity of Candida species was richer in patients suffering from caries. The odds ratio of the colonisation of patients with caries was 3.144 (95% CI 1.525-5.478). There is a significant clinical correlation between dental caries and oral Candida colonisation in adults.

In vitro fungicidal activities of anidulafungin, caspofungin, and micafungin against Candida glabrata, Candida bracarensis, and Candida nivariensis evaluated by time-kill studies.

Anidulafungin, caspofungin, and micafungin killing activities against Candida glabrata, Candida bracarensis, and Candida nivariensis were evaluated by the time-kill methodology. The concentrations assayed were 0.06, 0.125, and 0.5 µg/ml, which are achieved in serum. Anidulafungin and micafungin required between 13 and 26 h to reach the fungicidal endpoint (99.9% killing) against C. glabrata and C. bracarensis. All echinocandins were less active against C. nivariensis.

Efficacy of the combination of amphotericin B and echinocandins against Candida auris in vitro and in the Caenorhabditis elegans host model.

IMPORTANCE: Multidrug resistance is a rising problem among non-Candida albicans species, such as Candida auris. This therapeutic problem has been very important during the COVID-19 pandemic. The World Health Organization has included C. auris in its global priority list of health-threatening fungi, to study this emerging multidrug-resistant species and to develop effective alternative therapies. In the present study, the synergistic effect of the combination of amphotericin B and echinocandins has been demonstrated against blood isolates of C. auris. Different susceptibility responses were also observed between aggregative and non-aggregative phenotypes. The antifungal activity of these drug combinations against C. auris was also demonstrated in the Caenorhabditis elegans host model of candidiasis, confirming the suitability and usefulness of this model in the search for solutions to antimicrobial resistance.

PK/PD modeling and simulation of the in vitro activity of the combinations of isavuconazole with echinocandins against Candida auris.

In vitro combination of echinocandins and isavuconazole against the emerging species Candida auris is mainly synergistic. However, this combination has not been evaluated in clinical settings. A pharmacokinetic/pharmacodynamic modeling and simulation approach based on in vitro data may be helpful to further study the therapeutic potential of these combinations. Therefore, the aims of this study were to characterize the time course of growth and killing of C. auris in response to the combination of the three approved echinocandins and isavuconazole using a semimechanistic model and to perform model-based simulations in order to predict the in vivo response to combination therapy. In vitro static time-kill curve data for isavuconazole and echinocandins combinations against six blood isolates of C. auris were best modeled considering the total killing of the fungal population as dependent on the additive effects of both drugs. Once assessed, the predictive performance of the model using simulations of different dosing and fungal susceptibility scenarios were conducted. Model-based simulations revealed that none of the combinations at standard or higher dosages would be effective against the studied isolates of C. auris and it was predicted that the combinations of isavuconazole with anidulafungin or caspofungin would be effective for minimum inhibitory concentrations up to 0.03 and 0.06 mg/L respectively, whereas the combination with micafungin would lead to treatment failure. The current approach highlights the importance of bridging the in vitro results to the clinic.

Assessing pH-dependent activities of virulence factors secreted by Candida albicans.

Candida albicans is an opportunistic pathogen that can thrive under adverse conditions including suboptimal pH, nutrient scarcity, and low levels of oxygen. Its pathogenicity is associated with the production of virulence factors such as extracellular hydrolytic enzymes and toxins. This study was aimed at determining the effect of external pH, substrate nature, and strain origin on protease, lipase, and hemolysin production. To achieve this objective, agar plate assays were performed at pH 5.0, 6.5, and 7.5 with substrates suitable for the detection of each family of enzymes. Moreover, the study was conducted with 20 clinical C. albicans isolates from blood, oral cavity, skin, urine, and vagina. The hydrolytic zones formed around the colonies were further measured to calculate the Ez (enzymatic zone) indexes. We found that detection of proteases in skim milk agar plates was possible for most isolates only at pH 5 (80%) and pH 6.5 (75%), whereas BSA plates could confer protease detection exclusively at pH 5 (80%). Similarly, the percentage of isolates possessing lipolytic activities was higher at pH 5 (90%) than at pH 6.5 (70%) and pH 7.5 (35%). In contrast, hemolytic activities were detected in all isolates at pH 6.5 and 7.5 but not at pH 5. Further analysis revealed that some differences in the detected activities could potentially be attributed to the anatomical origin of these isolates. Collectively, these findings suggest that the pH of the site of infection might be critical for mimicking the microenvironment employed to experimentally discover the key virulence factors.

In Vitro and In Vivo Activity of Citral in Combination with Amphotericin B, Anidulafungin and Fluconazole against Candida auris Isolates.

Candida auris is an emerging fungal pathogen responsible for hospital outbreaks of invasive candidiasis associated with high mortality. The treatment of these mycoses is a clinical challenge due to the high resistance levels of this species to current antifungal drugs, and alternative therapeutic strategies are needed. In this study, we evaluated the in vitro and in vivo activities of combinations of citral with anidulafungin, amphotericin B or fluconazole against 19 C. auris isolates. The antifungal effect of citral was in most cases similar to the effect of the antifungal drugs in monotherapy. The best combination results were obtained with anidulafungin, with synergistic and additive interactions against 7 and 11 of the 19 isolates, respectively. The combination of 0.06 µg/mL anidulafungin and 64 µg/mL citral showed the best results, with a survival rate of 63.2% in Caenorhabditis elegans infected with C. auris UPV 17-279. The combination of fluconazole with citral reduced the MIC of fluconazole from > 64 to 1-4 µg/mL against 12 isolates, and a combination of 2 µg/mL fluconazole and 64 µg/mL citral was also effective in reducing mortality in C. elegans. Amphotericin B combined with citral, although effective in vitro, did not improve the activity of each compound in vivo.

Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR.

Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the "gold standard" for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.