Identificación de cepas del complejo Candida albicans aisladas de muestras clínicas en la región de Valparaíso, Chile / Identification of strains of the Candida albicans complex isolated clinical samples in the Valparaíso region, Chile

Introducción: C. albicans es reconocida como la especie más virulenta del género y representa la causa más frecuente de candidiasis en humanos. A nivel taxonómico, C.albicans se clasifica como un complejo de especies estrechamente relacionadas que incluye a C. albicans sensu stricto (s.s), C. dubliniensis y C. africana. Objetivo: identificar las especies del complejo C. albicans aisladas desde distintas muestras de pacientes de la quinta región de Valparaíso. Materiales y método: Se identificaron 103 cepas del complejo C. albicans, aisladas desde muestras superficiales y profundas durante el año 2020. La identificación se realizó en base a morfofisiología y la amplificación del gen HWP1. Resultados: Se identificaron 100 cepas como C. albicans s.s, 2 como C. dubliniensis y 1 como C. africana. Dentro de las cepas identificadas como C. albicans s.s se observaron cuatro patrones de tamaños de fragmentos genéticos. Conclusiones: C. albicans s.s fue la especie más frecuente y en base al genotipo de HPW1 se describen cuatro patrones ( H1 a H4). (AU)

Introduction: C. albicans is recognized as the most virulent species of the genus and represents the major cause of candidiasis in humans. At the taxonomic level, C. albicansis classified as a complex of closely related species that includes C. albicans sensu stricto (s.s), C. dubliniensis, and C. africana. Objective: to identify the species of the C. albicans complex isolated from different samples of patients from the fifth region of Valparaíso. Materials and method: 103 strains of the C. albicans complex were identified, isolated from superficial and deep samples during the year 2020. The identification was carried out based on morphophysiology and the amplification of the HWP1 gene. Results: 100 strains were identified as C. albicans s.s, 2 as C. dubliniensis and 1 as C. africana. Within the strains identified as C. albicans s.s, 4 patterns of fragment sizes were observed. Conclusions: C. albicans s.s was the most frequent species and based on the HPW1 genotype, four patterns are described (H1 to H4).(AU)

Rapid identification of microbial contaminants in pharmaceutical products using a PCA/LDA-based FTIR-ATR method

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied

Development of amphotericin b Based organogels against mucocutaneous fungal infections

Amphotericin B is a broad spectrum antifungal agent used to treat fungal infections. Organogel is a semisolid preparation in which the apolar phase gets immobilized within spaces of the three-dimensional structure. The current study aimed at the formulation and comparative evaluation of sorbitan monostearate organogels and pluronic lecithin organogels (PLO). Different compositions of span 60 based organogels were prepared by varying the concentrations of the span 60 and PLO gels were prepared by varying the concentration of Pluronic F 127. The developed organogels were subjected to various characteristics such as pH, viscosity, spreadability, extrudability, and drug release studies. The optimized formulations were evaluated against Candida albicans and carried out ex vivo release study. The optimized formulation was selected from span 60 based organogels, and pluronic lecithin organogels were S1 and P1, respectively. The optimized formulation (S1) showed effective inhibition against Candida albicans. The skin irritation test was carried out on albino mice for optimized formulations and results showed that no irritation to the skin. Based on the results, organogels prepared by sorbitan monostearate showed better antifungal activity, and also all the formulations were found to be stable and safe throughout the study period.

Genotyping of Candidaalbicans isolates from oropharyngeal candidiasis in head and neck cancer patients in Iran: Molecular epidemiology and SAP2 gene expression.

OBJECTIVE: Multilocus sequence typing is a powerful method for genotyping of clinical isolates of Candidaalbicans. Cross-contamination between the patients is an important reason for nosocomial infections. Oropharyngeal candidiasis (OPC) caused by C. albicans is an important problem in patients with head and neck cancer, in Cancer Institute of Tehran. Here we studied the endemic genotypes of C. albicans isolates and the relationship between geographic distributions, potential cross-contaminations and the expression of SAP2 gene, an important gene in oral candidiasis, with MLST groups. MATERIAL AND METHODS: A total of 32 clinical strains of C. albicans isolated from head and neck cancer patients with oropharyngeal candidiasis were subjected to MLST analysis and SAP2 gene expression was analyzed by Real-time PCR. RESULTS: We identified 75 polymorphic sites in 7 loci of C. albicans isolates and 30 diploid sequence types which 27 of them were found as new. After eBURST analysis, our results determined that CC 124 was the most prevalent group among all CCs. SAP2 gene showed high expression in almost all OPC patients' isolates, compared to the control. CONCLUSION: We found few genetically-related as well as identical isolates among the 32 Candida strains which indicated low cross-contaminations among the patients. There was no relationship between C. albicans MLST profiles and their geographic distribution, cancer type and SAP2 gene expression. This lack of correlation was possibly due to the small understudy population; hence, finding more relevance requires studies with a higher number of samples.

Atividade antifúngica de metabólitos produzidos por Streptococcus mutans sobre Candida albicans / Antifungal activity of metabolites produced by Streptococcus mutans on Candida albicans

RESUMO A formação de biofilmes e produção de hifas por Candida albicans são importantes fatores de virulência, principalmente, para a aderência à mucosa e invasão tecidual. A busca por metabólitos secundários produzidos por S. mutans é de suma importância, pois poderá fornecer novas estratégias terapêuticas no combate às infecções por Candida, possibilitando o desenvolvimento de medicamentos capazes de inibir os mecanismos de patogenicidade das espécies desse gênero. Desse modo, o objetivo desse estudo foi obter o extrato bruto e frações a partir do sobrenadante da cultura de 4 h de S. mutans (UA159) e avaliar seus efeitos sobre os mecanismos de patogenicidade de C. albicans (ATCC18804) por meio de estudos in vitro. Após o cultivo de S. mutans, o extrato bruto foi obtido via partição líquido-líquido com acetato de etila (3x) e posteriormente fracionado em coluna de sílica derivatizada C-18 eluída com gradiente MeOH:H2O, fornecendo cinco frações (SM-F1, SM-F2, SM-F3, SM-F4 e SM-F5). A identificação das substâncias contidas no extrato bruto e frações foi realizada utilizando cromatografia gasosa acoplada a espectrometria de massas (CGEM), sendo encontradas as seguintes substâncias: ácido octanóico e uridina no extrato bruto, ácido propanóico, (3R)-3-Methyl-1,4-bis(trimethylsilyl)piperazine-2,5- dione, pirimidina, gulose e oleamida na SM-F1 e ácido nicotínico e triptofano na SM- F2. O extrato bruto e as frações foram submetidos aos ensaios de bioatividade sobre a formação de hifas de C. albicans e analisados por microscopia óptica e microscopia eletrônica de varredura (MEV). O extrato bruto e a fração SM-F2, que resultou em maior inibição das hifas, foram investigados na expressão de genes de C. albicans envolvidos no mecanismo de filamentação (CPH1, EFG1, HWP1, UME6 e YWP1) por PCR quantitativo em tempo real (RT-qPCR). Além disso, o potencial de inibição do extrato bruto, frações SM-F1 e SM-F2 foi avaliado sobre a formação de biofilmes de C. albicans, analisados pela quantificação de células viáveis (UFC/mL) e MEV Os resultados demonstraram inibição significativa na formação de hifas de C. albicans pelo extrato bruto e principalmente, pela SM-F2, com alterações significativas na expressão de todos os genes analisados. O extrato bruto e SM-F2 regularam negativamente a expressão dos genes CPH1, EFG1, HWP1 e UME6, em contrapartida, regularam positivamente a expressão do gene YWP1, envolvido no mecanismo de dispersão das leveduras. Nas análises de UFC/mL, os resultados demonstraram redução nas contagens de C. albicans nos biofilmes formados quando em contato com o extrato bruto, frações SM-F1 e SM-F2 em todas as concentrações testadas, sendo que o extrato bruto reduziu totalmente as células de C. albicans na concentração 15 mg/mL. Os resultados do nosso estudo demonstraram que o extrato bruto e frações de S. mutans apresentaram efeitos inibitórios sobre importantes mecanismos de virulência de C. albicans, fornecendo evidências que S. mutans produz substâncias com ação antifúngica, tornando-o promissor na busca de novos compostos antimicrobianos para prevenção e tratamento das candidoses humanas(AU)

The biofilm formation and hyphae production by Candida albicans are important virulence factors, mainly for mucosal adhesion and tissue invasion. The search for secondary metabolites produced by S. mutans is of great importance, since it may provide new therapeutic strategies against Candida infections, enabling the development of drugs that inhibit the mechanisms of pathogenicity of the species of this genus. Thus, the objective of this study was to obtain the crude extract and fractions from the supernatant of the 4 h culture of S. mutans (UA159) and evaluate their effects on the mechanisms of pathogenicity of C. albicans (ATCC18804) through in vitro studies. After culture of S. mutans, the crude extract was obtained via liquid-liquid partition with ethyl acetate (3x) and then fractionated on a C-18 derivatized silica column eluted with MeOH:H2O gradient, providing five fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The following substances were found: octanoic acid and uridine in crude extract, propanoic acid, (3R) -3-methyl -1,4-bis (trimethylsilyl) piperazine-2,5-dione, pyrimidine, gulose and oleamide in SM-F1 and nicotinic acid and tryptophan in SM-F2. The crude extract and fractions were submitted to bioactivity assays on hyphae formation by C. albicans and analyzed by optical microscopy and scanning electron microscopy (SEM). The crude extract and SM-F2 fraction, which resulted in highest inhibition of hyphae, were investigated in the expression of C. albicans genes involved in the filamentation mechanism (CPH1, EFG1, HWP1, UME6 and YWP1) by quantitative real-time PCR (RT-qPCR). In addition, the potential of inhibition of the crude extract, SM-F1 and SM-F2 fractions in biofilms of C. albicans were evaluated by the quantification of viable cells (CFU/mL) and SEM. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. The results demonstrated a significant inhibition on C. albicans hyphae formation by the crude extract and mainly by SMF2, with significant alterations in the expression of all genes analyzed. The crude extract and SM-F2 negatively regulated the expression of the CPH1, EFG1, HWP1 and UME6 genes, in contrast, positively regulated the expression of the YWP1 gene, involved in the mechanism of yeast dispersion. In the CFU/mL analyzes, the results showed a reduction in the counts of C. albicans in the biofilms formed when in contact with the crude extract, SM-F1 and SM-F2 fractions in all tested concentrations and the crude extract totally reduced C. albicans cells at 15 mg/mL concentration. The results of our study demonstrated that the crude extract and fractions of S. mutans presented inhibitory effects on important mechanisms of virulence of C. albicans, providing evidence that S. mutans produces substances with antifungal action, making it promising in the search for new compounds antimicrobials for the prevention and treatment of human candidoses(AU)

Factores relacionados a candidiasis oral en niños y adolescentes con VIH, caracterización de especies y susceptibilidad antifúngica / Factors related to oral candidiasis in HIV children and adolescents, species characterization and antifungal susceptibility

Resumen Introducción: Se desconocen los factores asociados a la candidiasis oral en población pediátrica con infección por VIH de los países en desarrollo. Objetivo: Identificar los factores asociados a la colonización por Candida, candidiasis oral y la susceptibilidad in vitro a antifúngicos, en niños y adolescentes con infección por VIH institucionalizados en la ciudad de Tijuana, México. Materiales y Métodos: Se examinó la cavidad oral de 30 niños y adolescentes con infección por VIH, se obtuvo una muestra de la mucosa oral para identificar las especies de Candida mediante cultivo y auxonograma. La susceptibilidad a los antifúngicos se determinó de acuerdo al CLSI. Los indicadores del estado inmunológico y falla virológica se clasificaron conforme a la OMS. Resultados: Se identificaron seis especies de Candida, 53% colonizantes y 47% causantes de candidiasis. Los factores asociados a candidiasis fueron alta carga viral (p = 0,001), menor recuento de LTCD4+ (p = 0,002) y esquema TARAA (p ≤ 0,014). La especie prevalente fue C. glabrata (33%); sin embargo, C. albicans (27%) fue más resistente a fluconazol (p = 0,001). Las especies resistentes a itraconazol se identificaron en esquemas que incluyen un INNTR (p = 0,041). Conclusiones: Los niños y adolescentes con infección por VIH institucionalizados mostraron una prevalencia elevada de Candida spp. colonizante y resistencia a los antifúngicos relacionada con los INNTR .

Background: Factors associated with candidiasis and colonization in HIV-positive children and adolescents in developing countries are not well understood. Aim: To identify the factors associated with oral Candida colonization and candidiasis in institutionalized HIV-positive children and adolescents in Tijuana, México, as well as the response of the isolates to antifungals. Materials and Methods: Sample of the oral mucosa of 30 HIV positive children and adolescents were obtained to isolate and identify Candida species by culture and metabolic profile. Antifungal drugs susceptibility was determined according to CLSI. Indicators of immunological and virologic failure were classified in accordance to WHO criteria. Results: Six Candida species were identified from oral mucosa, 53% colonizers and 47% in candidiasis. Factors associated with candidiasis and oral colonization were viral load (p = 0,001), CD4+ counts (p = 0,002) and HAART regimen (p ≤ 0,014). The most prevalent species was C. glabrata (33%), but C. albicans (27%) was more resistant to fluconazole (p = 0,001). Itraconazol resistant species were identified in regimens that include an NNRTI (p = 0,041). Conclusion: HIV-positive children and adolescents living in an orphanage showed high prevalence of colonizing Candida spp. and resistance to antifungals, related to NNRTI.

Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.

Gene duplication facilitates functional diversification and provides greater phenotypic flexibility to an organism. Expanded gene families arise through repeated gene duplication but the extent of functional divergence that accompanies each paralogous gene is generally unexplored because of the difficulty in isolating the effects of single family members. The telomere-associated (TLO) gene family is a remarkable example of gene family expansion, with 14 members in the more pathogenic Candida albicans relative to two TLO genes in the closely-related species C. dubliniensis. TLO genes encode interchangeable Med2 subunits of the major transcriptional regulatory complex Mediator. To identify biological functions associated with each C. albicans TLO, expression of individual family members was regulated using a Tet-ON system and the strains were assessed across a range of phenotypes involved in growth and virulence traits. All TLOs affected multiple phenotypes and a single phenotype was often affected by multiple TLOs, including simple phenotypes such as cell aggregation and complex phenotypes such as virulence in a Galleria mellonella model of infection. No phenotype was regulated by all TLOs, suggesting neofunctionalization or subfunctionalization of ancestral properties among different family members. Importantly, regulation of three phenotypes could be mapped to individual polymorphic sites among the TLO genes, including an indel correlated with two phenotypes, growth in sucrose and macrophage killing. Different selective pressures have operated on the TLO sequence, with the 5' conserved Med2 domain experiencing purifying selection and the gene/clade-specific 3' end undergoing extensive positive selection that may contribute to the impact of individual TLOs on phenotypic variability. Therefore, expansion of the TLO gene family has conferred unique regulatory properties to each paralog such that it influences a range of phenotypes. We posit that the genetic diversity associated with this expansion contributed to C. albicans success as a commensal and opportunistic pathogen.

Coassociation between Group B Streptococcus and Candida albicans Promotes Interactions with Vaginal Epithelium.

Group B Streptococcus (GBS) is a leading cause of neonatal sepsis, pneumonia, and meningitis worldwide. In the majority of cases, GBS is transmitted vertically from mother to neonate, making maternal vaginal colonization a key risk factor for neonatal disease. The fungus Candida albicans is an opportunistic pathogen of the female genitourinary tract and the causative agent of vaginal thrush. Carriage of C. albicans has been shown to be an independent risk factor for vaginal colonization by GBS. However, the nature of interactions between these two microbes is poorly understood. This study provides evidence of a reciprocal, synergistic interplay between GBS and C. albicans that may serve to promote their cocolonization of the vaginal mucosa. GBS strains NEM316 (serotype III) and 515 (serotype Ia) are shown to physically interact with C. albicans, with the bacteria exhibiting tropism for candidal hyphal filaments. This interaction enhances association levels of both microbes with the vaginal epithelial cell line VK2/E6E7. The ability of GBS to coassociate with C. albicans is dependent upon expression of the hypha-specific adhesin Als3. In turn, expression of GBS antigen I/II family adhesins (Bsp polypeptides) facilitates this coassociation and confers upon surrogate Lactococcus lactis the capacity to exhibit enhanced interactions with C. albicans on vaginal epithelium. As genitourinary tract colonization is an essential first step in the pathogenesis of GBS and C. albicans, the coassociation mechanism reported here may have important implications for the risk of disease involving both of these pathogens.

Historia sucinta de Candida albicans, blanca pero no tanto / Brief history of Candida, white but not so much

From the begin of clinical microbiology in the second half of the nineteenth century, the fungi were neglected as contaminants without relevance for health, belonging the major advances of their study to the fields of milk derivatives and beer industries. However, the seek for the etiological agent of thrush, a very common oral pathology affecting the newborn, put the yeasts on the table near 1840 with three capital papers - Berg, Gruby and Bennett - speaking about spores from vegetable as parasites of animal and human beings. The door was open, and very soon, in 1853, came the decisive description by Robin of the Oidium albicans as the causative agent of this painful disease. Seventy years after, in 1923, Christine Marie Berkhout, rejecting this name, defined the genus as Candida, leaving the specie with the iterative Latin name of Candida albicans, that means "White-white". Or, perhaps, with a fine sense of humor, she has made an oxymoron, because "candida" means a brilliant white and "albicans" a matt one, both opposite adjectives. Or, may be, Christine is still saying us: "White…but not so white".

Cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies.

The present study aimed to characterize cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies. It was evaluated 12 clinical isolates of C. albicans from vaginal samples: 4 from asymptomatic women (AS), 4 from women with a single episode of vulvovaginal candidiasis (VVC) and 4 from women with recurrent vulvovaginal candidiasis (RVVC). We evaluated the ability of C. albicans to adhere to human cervical cancer cells (SiHa), the yeast-SiHa cell interactions and cell damage. All of the clinical isolates presented a high adhesion capacity on SiHa cells. However, clinical isolates from symptomatic women (VVC and RVVC) had higher filamentation after contact (24 h) with SiHa cells and a greater capacity to cause cell damage (>80 %). Clinical isolates from symptomatic women had greater potential to invade SiHa cells, suggesting that they are more pathogenic than AS isolates.

Phenotypic and genotypic characteristics of Candida albicans isolates from bloodstream and mucosal infections.

The interaction of Candida albicans with the host is of a complex nature involving fungal factors and host's response. In this study, we concentrated on the phenotypic expression of virulence attributes and genotypic characteristics of C. albicans isolates from two distinct clinical entities of candidiasis-blood stream and vaginal infections, and the possible role of these factors. Hence, we conducted a comparative in vitro assessment of virulence characteristics, including adhesion to epithelial cells and HaCat cell line, biofilm formation, aspartic proteinases and phospholipase activity of 20 C. albicans isolates from patients with C. albicans bloodstream infection and 22 isolates from patients with C. albicans vaginitis. Further, we studied the epigenetic phenotypic switching of the strains and their ploidy, by flow cytometry and CHEF techniques. These studies indicated that although no overall differentiation between the isolates of the two groups (bloodstream infection and vaginitis) could be demonstrated, several characteristics were more specific to one of the groups than the other. While the strains from vaginal infection had higher capacity to adhere, the strains from patients with bloodstream infection had higher activity of phospholipase. Differences were also noted in phenotypic switching, with the strains from bloodstream infection revealing primarily the "white" type colonies, known to be more virulent, and had higher DNA content. This study is unique considering the concurrent comparison of isolates from different clinical entities, at the phenotypic and genotypic level.

Disseminated Candidiasis in a Young, Previously Healthy, Dog and Review of Literature.

BACKGROUND: The reports on disseminated candidiasis in dogs so far describe at least one predisposing factor. This case report, however, highlights candidiasis in a dog without any known predisposition. PATIENT: A 1.5-year-old intact female Hovawart dog was presented with subcutaneous nodules and polyuria/polydipsia. An excisional biopsy revealed a chronic pyogranulomatous and necrotizing inflammation with mycotic structures. The patient became febrile and lethargic, and developed lameness. METHODS: A physical examination, blood tests, urinalysis, thoracic radiographs, abdominal ultrasonography of the abdomen, fine-needle aspiration biopsies, and a culture of a subcutaneous nodule aspirate were obtained. Selected sections of multiple organs were collected for routine histology postmortem. The isolate and a subcutaneous mass were subjected to molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. RESULTS: Clinical, laboratory, and radiological findings were consistent with a granulomatous chronic systemic inflammation. Cytology and histology showed a pyogranulomatous and necrotizing inflammation with myriads of intra- and extra-cellular yeasts and extracellular hyphae. Culture yielded numerous yeast colonies, which appeared Candida albicans-like, but showed a negative serum test and a low identification in API 20 C AUX. Nucleic acid sequences showed homology with the C. albicans-type strain CBS 562. Multilocus sequence typing (MLST) resulted in a new type with designation DST121. The identification of the isolates was confirmed by MALDI-TOF-MS analysis. CONCLUSION AND CLINICAL IMPORTANCE: Future MLST typing and investigation of virulence can provide further evidence whether this MLST-type is associated with clinical cases of disseminated candidiasis without an apparent predisposing condition.

Prevalencia y sensibilidad a los antifúngicos de Candida albicans y sus especies relacionadas, Candida dubliniensis y Candida africana aisladas de muestras vulvovaginales en un hospital de Argentina / Prevalence and antifungal susceptibility of Candida albicans and its related species Candida dubliniensis and Candida africana isolated from vulvovaginal samples in a hospital of Argentina

Candida africana taxonomical status is controversial. It was proposed as a separate species within the Candida albicans species complex; however, phylogenetic analyses suggested that it is an unusual variety of C. albicans. The prevalence of C. albicans-related species (Candida dubliniensis and C. africana) as vulvovaginal pathogens is not known in Argentina. Moreover, data on antifungal susceptibility of isolates causing vulvovaginal candidiasis is scarce. The aims of this study were to establish the prevalence of C. dubliniensis and C. africana in vaginal samples and to evaluate the antifungal susceptibilities of vaginal C. albicans species complex strains. We used a molecular-based method coupled with a new pooled DNA extraction methodology to differentiate C. dubliniensis and C. africana in a collection of 287 strains originally identified as C. albicans isolated from an Argentinian hospital during 2013. Antifungal susceptibilities to fluconazole, clotrimazole, itraconazole, voriconazole, nystatin, amphotericin B and terbinafine were evaluated by using the CLSI M27-A3 and M27-S4 documents. Of the 287 isolates, 4 C. dubliniensis and one C. africana strains (1.39% and 0.35% prevalence, respectively) were identified. This is the first description of C. africana in Argentina and its identification was confirmed by sequencing the ITS2 region and the hwp1 gene. C. dubliniensis and C. africana strains showed very low MIC values for all the tested antifungals. Fluconazole-reduced-susceptibility and azole cross-resistance were observed in 3.55% and 1.41% of the C. albicans isolates, respectively. These results demonstrate that antifungal resistance is still a rare phenomenon in this kind of isolates.

La clasificación taxonómica de Candida africana está en discusión, es considerada una nueva especie dentro del complejo C. albicans o una variedad inusual de C. albicans. La prevalencia de las especies relacionadas a C. albicans (C. dubliniensis y C. africana) como agentes de vulvovaginitis en Argentina se desconoce. Los objetivos de este trabajo fueron determinar la prevalencia de C. dubliniensis y C. africana en muestras vaginales y evaluar la sensibilidad a los antifúngicos de aislamientos vaginales de las especies del complejo C. albicans. Para diferenciar C. dubliniensis y C. africana utilizamos un método molecular asociado a un nuevo método de extracción de ADN. Se utilizó una colección de 287 cepas originalmente identificadas como C. albicans aisladas durante 2013 en un hospital de Argentina. Se evaluó la sensibilidad a fluconazol, clotrimazol, itraconazol, voriconazol, nistatina, anfotericina B y terbinafina utilizando los documentos M27-A3 y M27-S4 del CLSI. De los 287 aislamientos, se identificaron 4 C. dubliniensis y 1 C. africana (1,39 y 0,35% de prevalencia, respectivamente). Esta es la primera descripción de C. africana en Argentina. Su identificación fue confirmada por secuenciación de la región ITS2 y del gen hwp1. Las cepas identificadas como C. dubliniensis y C. africana mostraron valores de CIM muy bajos para todos los antifúngicos probados. En los aislamientos de C. albicans, la sensibilidad reducida al fluconazol y la resistencia cruzada a todos los azoles se observó en el 3,55% y el 1,41%, respectivamente. Estos resultados demuestran que la resistencia a los antifúngicos es todavía un fenómeno raro en este tipo de aislamientos.

Identification of signature volatiles to discriminate Candida albicans, glabrata, krusei and tropicalis using gas chromatography and mass spectrometry.

Oral candidiasis is the most frequent fungal infection of the oral cavity. Clinical diagnoses require mycological confirmation, which is time-consuming in case of culture testing. The aim of the study was to identify signature volatiles to develop a chairside breath test to diagnose oral candidiasis. Headspaces above Candida albicans, glabrata, tropicalis, krusei cultures, and growth media as control were analysed after eight and 24 h using offline gas chromatography and mass spectrometry. The identification of signature volatiles was assisted using various microbial databases. Retrieved volatile patterns enabled Candida species discrimination in vitro. For C. albicans 3-methyl-2-butanone and styrene and for C. krusei a combination of p-xylene, 2-octanone, 2-heptanone and n-butyl acetate were found to be specific. 1-hexanol was found in C. tropicalis, but is emitted by a variety of other microorganisms. C. glabrata was characterised through the absence of these volatiles. The development of a breath test is a promising approach in confirming suspicions of oral candidiasis. To confirm the retrieved results in vivo, breath tests in affected and healthy subjects have to be performed.

PUTATIVE FERROXIDASES IN THE FLAVINOGENIC YEAST PICHIA GUILLIERMONDII ARE REGULATED BY IRON ACQUISITION.

Using similarity search we identified Candida (Pichia) guilliermondii genes involved in iron acquisition. This yeast possesses at least four genes potentially coding for ferri-reductases, four genes encoding iron permeases and two genes codingforferroxidases. Identified C.(P.) guilliermondii genes encoding ferroxidases possess different patterns of expression under iron repletion conditions whereas their expression is activated under iron deficiency conditions or in mutant strains defective in regulation of iron acquisition. C.(P.) guilliermondii has no homologue of Saccharomyces cerevisiae transcriptional regulator of iron metabolism, Aft1p and possess an iron regulatory network similar to that of Candida albicans. Since most of C.(P.) guilliermondii known strains are not pathogenic, in contrast to that of C. albicans, we propose C.(P.) guilliermondii as safe and useful model for studying iron-dependent regulation of metabolism in yeasts belonging to CUG clade.

Molecular surveillance of candidemia due to Candida albicans among cancer patients during 2009 to 2013 by microsatellite typing.

BACKGROUND: Since the high morbidity and mortality of candidemia among cancer patients, the epidemiology has been underlined. In recent years, Candida species genotyping has been established, which could provide detail characteristics of epidemiology and has been underscored for candidemia preventing strategies. METHODS: Data of cancer patients with candidemia and hospitalized in Tianjin Medical University Cancer Institute and Hospital (TMUCIH) during 2009-2013 were reviewed. Species identification was carried out by using VITEK-2 Compact. Microsatellite typing was performed for molecular analysis. SPSS 20.0 and MVSP 3.22 software were used for statistical and clustering analysis, respectively. RESULTS: Total of 36 isolates of Candida albicans were recovered from 36 cancer patients with nosocomial candidemia in TMUCIH during the period of 2009-2013 included in the study. Total of 17 genotypes were identified and 2 of them were endemic genotypes, which caused 21 (58.3%) of 36 episodes of candidemia. Hepatobiliary oncology, ICU and gastrointestinal oncology were the main wards of infections due to endemic strains. Gastrointestinal cancer and insertion of a nasogastric tube were the predictors of infections caused by endemic strains (p = 0.014 and p = 0.041, respectively). For the 36 cases, crude mortality was up to 30.6%, and there was no significant difference between infections due to endemic and non-endemic strains (p = 0.077). CONCLUSIONS: This study proved that endemic stains of C. albicans could exist for a long period and mainly in a few wards. Patients with gastrointestinal cancer or nasogastric tube insertion were more sensitive to endemic C. albicans.

Perfil fitoquímico e determinação da atividade antimicrobiana de Syzygium cumini (L. ) Skeels (Myrtaceae) frente a microrganismos bucais / Phytochemical profile and determination of antimicrobial activity of Syzygiumcumini (L. ) Skeels (Myrtaceae) against oral microorganisms

RESUMO Este estudo teve como objetivo determinar o perfil fitoquímico e avaliar a atividade antimicrobiana in vitro do extrato etanólico da casca do caule de Syzygium cumini(L.) Skeels frente a microrganismos bucais. O perfil fitoquímico do extrato foi traçado através da determinação espectrofotométrica quantitativa para verificar o teor de taninos, flavonóides, saponinas e polifenóis. A atividade antimicrobiana foi determinada através da Concentração Inibitória Mínima (CIM), por meio da técnica de microdiluição em caldo, utilizando-se as seguintes linhagens de microrganismos: Streptococcus mutans (25175), Streptococcus oralis (10557) e Candida albicans (10231). Uma quantidade apreciável de fitocontituintes foi observada, especialmente de taninos (100,58 ± 1,81). Os extratos apresentaram atividade antimicrobiana inibindo o crescimento das linhagens em estudo, destacando-se essa atividade sobre o crescimento de C. albicans (CIM=250 µg/mL). Já as CIMs para Streptococcus foram baixas. Diante dos resultados expostos, pode-se concluir que o perfil fitoquímico foi traçado e que, dentre os microrganismos testados, o extrato etanólico da casca de S. cumini apresentou forte potencial de inibição sobre o crescimento de C. albicans e fraca inibição frente aos Streptococcus testados. Este estudo sugere que mais pesquisas devem ser realizadas dando continuidade à bioprospecção, por meio de análises experimentais com essa espécie vegetal, objetivando, no futuro, que essa planta possa ser utilizada clinicamente para tratar candidose bucal.

ABSTRACT This study aimed to determine the phytochemical profile and to evaluate the in vitro antimicrobial activity of the ethanol extract of stem bark of Syzygium cumini (L.) Skeels against oral microorganisms. The phytochemicalprofile of the extract was traced through a quantitative spectrophotometric determination in order to check the tannin, flavonoids, saponins, and polyphenols content. The antimicrobial activity was determined through minimum inhibitory concentration (MIC) by the broth microdilution technique, using the following strains of microorganisms: Streptococcus mutans (25175), Streptococcus oralis (10557) and Candida albicans (10231). An appreciable amount of fitocontituintes was observed, particularly the tannin (100.58 ± 1.81). The extracts showed antimicrobial activity, inhibiting the growth of the strains under study, with this activity being more intense on the growth of C. albicans ( MIC = 250 mg / mL). On the other hand, the MICs of the Streptococcus were low. In face of the mentioned results, we can conclude that the phytochemical profile was traced and that, among the tested microorganisms, the ethanol extract of S. cumini bark showed strong potential to inhibit the growth of C. albicans and weak inhibition against the Streptococcus tested. This study suggests that more research should be done by proceeding with the bioprospecting, through experimental tests with this plant`s species, aiming that in the future this substance can be used clinically for the treatment of oral candidiasis.

[Antifungal activity of melanin in clinical isolates of Candida spp]. / Actividad antifúngica de melanina en cepas clínicas de Candida spp.

BACKGROUND: Melanocytes are cells located in epidermis and mucous membranes that synthesize melanin and cytokines. It is known that melanin has antimicrobial activity and that melanocytes are melanized in presence of microbial molecules. OBJECTIVE: To study the antifungal activity of melanin on Candida spp. METHODOLOGY: The minimum inhibitory concentration (MIC) to melanin was determined in 4 Candida ATCC strains (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001, C. krusei 6258) and 56 clinical isolates of Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) using a broth microdilution method. In addition, the antifungal activity of melanocytes and mice melanoma cells was tested against C. albicans. RESULTS: Melanin inhibited the tested isolates, including the susceptible dose-dependent and fluconazole-resistant strains; MIC range and MIC50 were 0.09-50 µg/mL and 6.25 µg/mL, respectively. Pigmented cells lysates inhibited C. albicans. CONCLUSIONS: Melanin is able to inhibit clinical isolates of Candida spp. Melanization could be an important protective mechanism of melanocytes.

Actividad antifúngica de melanina en cepas clínicas de Candida spp / Antifungal activity of melanin in clinical isolates of Candida spp

Background: Melanocytes are cells located in epidermis and mucous membranes that synthesize melanin and cytokines. It is known that melanin has antimicrobial activity and that melanocytes are melanized in presence of microbial molecules. Objective: To study the antifungal activity of melanin on Candida spp. Methodology: The minimum inhibitory concentration (MIC) to melanin was determined in 4 Candida ATCC strains (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001, C. krusei 6258) and 56 clinical isolates of Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) using a broth microdilution method. In addition, the antifungal activity of melanocytes and mice melanoma cells was tested against C. albicans. Results: Melanin inhibited the tested isolates, including the susceptible dose-dependent and fluconazole-resistant strains; MIC range and MIC50 were 0.09-50 μg/mL and 6.25 μg/mL, respectively. Pigmented cells lysates inhibited C. albicans. Conclusions: Melanin is able to inhibit clinical isolates of Candida spp. Melanization could be an important protective mechanism of melanocytes.

Introducción: Los melanocitos son células presentes en piel y en mucosas que sintetizan melanina, además de citoquinas. Es sabido que melanina presenta actividad antimicrobiana y que los melanocitos se melanizan al ser expuestos a moléculas microbianas. Objetivo: Estudiar la actividad antifúngica de melanina en cepas clínicas de Candida spp. Metodología: Se midió la concentración inhibitoria mínima (CIM) a melanina, de 4 cepas de Candida ATCC (C. albicans SC5314, C. parapsilosis 22019, C. glabrata 2001 y C. krusei 6258) y 56 aislados clínicos de Candida spp. (33 C. albicans, 12 C. glabrata, 3 C. famata, 3 C. krusei, 3 C. parapsilosis, 2 C. tropicalis) mediante un método de microdilución en caldo. Además se estudió el efecto antifúngico de lisados de melanocitos y células de melanoma de ratón en C. albicans. Resultados: Melanina inhibió las cepas analizadas, incluso cepas susceptibles dosis-dependiente y resistentes a fluconazol, siendo los rangos de CIM y CIM50 de 0,09-50 μg/mL y 6,25 μg/ mL, respectivamente. Los lisados de células pigmentadas inhibieron C. albicans. Conclusiones: Melanina es capaz de inhibir cepas clínicas de Candida spp. La melanización podría ser un importante mecanismo protector de los melanocitos.

A novel renal epithelial cell in vitro assay to assess Candida albicans virulence.

Candida albicans, an opportunistic fungal pathogen, can cause severe systemic infections in susceptible patient groups. Systemic candidiasis is mainly studied in the mouse intravenous challenge model, where progressive infection correlates with increased early renal chemokine levels. To develop a new in vitro assay to assess C. albicans virulence, which reflects the events occurring in the murine infection model, renal M-1 cortical collecting duct epithelial cells were evaluated as the early producers of cytokines in response to C. albicans. We show that renal epithelial cells respond only to live C. albicans cells capable of forming hyphae, producing chemokines KC and MIP-2, with levels correlating with epithelial cell damage. By assaying epithelial cell responses to strains of known virulence in the murine intravenous challenge model we demonstrate that renal epithelial cells can discriminate between virulent and attenuated strains. This simple, novel assay is a useful initial screen for altered virulence of C. albicans mutants or clinical isolates in vitro and provides an alternative to the mouse systemic infection model.