Molecular Evaluation of the mRNA Expression of the ERG11, ERG3, CgCDR1, and CgSNQ2 Genes Linked to Fluconazole Resistance in Candida glabrata in a Colombian Population.

INTRODUCTION: The study of Candida glabrata genes associated with fluconazole resistance, from a molecular perspective, increases the understanding of the phenomenon with a view to its clinical applicability. OBJECTIVE: We sought to establish the predictive molecular profile of fluconazole resistance in Candida glabrata by analyzing the ERG11, ERG3, CgCDR1, and CgSNQ2 genes. METHOD: Expression was quantified using RT-qPCR. Metrics were obtained through molecular docking and Fisher discriminant functions. Additionally, a predictive classification was made against the susceptibility of C. glabrata to fluconazole. RESULTS: The relative expression of the ERG3, CgCDR1, and CgSNQ2 genes was higher in the fluconazole-resistant strains than in the fluconazole-susceptible, dose-dependent strains. The gene with the highest relative expression in the fluconazole-exposed strains was CgCDR1, and in both the resistant and susceptible, dose-dependent strains exposed to fluconazole, this was also the case. The molecular docking model generated a median number of contacts between fluconazole and ERG11 that was lower than the median number of contacts between fluconazole and ERG3, -CgCDR1, and -CgSNQ2. The predicted classification through the multivariate model for fluconazole susceptibility achieved an accuracy of 73.5%. CONCLUSION: The resistant strains had significant expression levels of genes encoding efflux pumps and the ERG3 gene. Molecular analysis makes the identification of a low affinity between fluconazole and its pharmacological target possible, which may explain the lower intrinsic susceptibility of the fungus to fluconazole.

Microevolution of Candida glabrata (Nakaseomyces glabrata) during an infection.

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

Production of new ent-hardwickiic acid derivatives by microbial transformation and their antifungal activity.

Ent-hardwickiic acid is the major compound of Copaifera pubiflora Benth oleoresin traditionally used in Brazilian folk medicine as an antimicrobial agent. Microbial transformation of ent-hardwickiic by Cunninghamella elegans ATCC 10028b resulted in two and five antifungal derivatives (four new ones) produced in the Czapek modified and Koch's K1 media, respectively. The derivatives were isolated and their structures were determined by spectral analysis, namely 1D/2D NMR and HR-ESIMS. All compounds were tested for cytotoxic and antifungal activities and they were not cytotoxic to the tested cell lines, but all derivatives showed fungicidal activity against Candida glabrata and Candida krusei, which have emerged as resistant to fluconazole. One of the yet unreported biotransformation products displayed the strongest activity with minimum fungicidal concentration values smaller than the other compounds, including fluconazole.

[Acute cholangitis due to Candida glabrata and Klebsiella pneumoniae]. / Colangitis aguda por Candida glabrata y Klebsiella pneumoniae.

Acute cholangitis is a bile duct infection associated with bile duct obstruction. Bile culture is positive in most cases, and the most frequent etiological agent is Escherichia coli. Candida sp acute cholangitis is a rare finding, which is more common in patients with immunosuppression, use of corticosteroids, prolonged antibiotic treatment or surgical procedures of the bile duct. We present the case of a 67-year-old woman with none of the above-mentioned history who consulted for fever, abdominal pain and jaundice. MRI of the abdomen revealed a lithiasic image in the common bile duct with dilation. It required endoscopic drainage of the biliary tract. Direct microscopic examination of the bile fluid revealed gram-negative bacilli and yeast, and in the culture of bile fluid Klebsiella pneumoniae producing extended spectrum beta-lactamase (ESBL) and Candida glabrata were isolated. The patient completed the antibiotic treatment with piperacillin tazobactam and anidulafungin with good evolution. Bile duct infection by association of Gram-negative bacilli and Candida sp is a rare entity, more in patients without underlying diseases.

La colangitis aguda es una infección de la vía biliar, asociada a la obstrucción de esta. El cultivo de la bilis es positivo en la mayoría de los casos y el agente etiológico más frecuente es Escherichia coli. La colangitis aguda por Candida sp es un hallazgo poco común, que es más frecuente en pacientes con inmunocompromiso, uso de corticoides, tratamiento antibiótico prolongado o procedimientos quirúrgicos de la vía biliar. Presentamos el caso de una mujer de 67 años, que no presentaba ninguno de los antecedentes mencionados, y que consultó por fiebre, dolor abdominal e ictericia. En la resonancia magnética nuclear de abdomen se constató imagen litiásica en el colédoco con dilatación de la vía biliar. Requirió drenaje endoscópico del tracto biliar. En el examen microscópico directo del líquido biliar se evidenciaron levaduras y bacilos Gram negativos, y en el cultivo se aisló Klebsiella pneumoniae productora de betalactamasa de espectro extendido (BLEE) y Candida glabrata. La paciente completó el tratamiento antibiótico con piperacilina tazobactam y anidulafungina con buena evolución. La infección de la vía biliar por la asociación de bacilos Gram negativos y Candida sp es una entidad poco frecuente, más en pacientes sin enfermedades subyacentes.

Colangitis aguda por Candida glabrata y Klebsiella pneumoniae / Acute cholangitis due to Candida glabrata and Kleb siella pneumoniae

Resumen La colangitis aguda es una infección de la vía biliar, asociada a la obstrucción de esta. El cultivo de la bilis es positivo en la mayoría de los casos y el agente etio lógico más frecuente es Escherichia coli. La colangitis aguda por Candida sp es un hallazgo poco común, que es más frecuente en pacientes con inmunocompromiso, uso de corticoides, tratamiento antibiótico prolongado o procedimientos quirúrgicos de la vía biliar. Presenta mos el caso de una mujer de 67 años, que no presen taba ninguno de los antecedentes mencionados, y que consultó por fiebre, dolor abdominal e ictericia. En la resonancia magnética nuclear de abdomen se constató imagen litiásica en el colédoco con dilatación de la vía biliar. Requirió drenaje endoscópico del tracto biliar. En el examen microscópico directo del líquido biliar se evidenciaron levaduras y bacilos Gram negativos, y en el cultivo se aisló Klebsiella pneumoniae productora de betalactamasa de espectro extendido (BLEE) y Candida glabrata. La paciente completó el tratamiento antibiótico con piperacilina tazobactam y anidulafungina con buena evolución. La infección de la vía biliar por la asociación de bacilos Gram negativos y Candida sp es una entidad poco frecuente, más en pacientes sin enfermedades subyacentes.

Abstract Acute cholangitis is a bile duct infection associated with bile duct obstruction. Bile culture is positive in most cases, and the most frequent etiological agent is Escherichia coli. Candida sp acute cholangitis is a rare finding, which is more common in patients with im munosuppression, use of corticosteroids, prolonged antibiotic treatment or surgical procedures of the bile duct. We present the case of a 67-year-old woman with none of the above-mentioned history who consulted for fever, abdominal pain and jaundice. MRI of the ab domen revealed a lithiasic image in the common bile duct with dilation. It required endoscopic drainage of the biliary tract. Direct microscopic examination of the bile fluid revealed gram-negative bacilli and yeast, and in the culture of bile fluid Klebsiella pneumoniae produc ing extended spectrum beta-lactamase (ESBL) and Can dida glabrata were isolated. The patient completed the antibiotic treatment with piperacillin tazobactam and anidulafungin with good evolution. Bile duct infection by association of Gram-negative bacilli and Candida sp is a rare entity, more in patients without underlying diseases.

Therapeutic Applications of Essential Oils from Native and Cultivated Ecuadorian Plants: Cutaneous Candidiasis and Dermal Anti-Inflammatory Activity.

Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, α-phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF-α, IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation.

The Anti-Virulence Effect of Vismia guianensis against Candida albicans and Candida glabrata.

In folk medicine, Vismia guianensis is used to treat skin diseases and mycoses in the Amazon region. We evaluated the anti-Candida activity of the hydroalcoholic extract from the leaves of Vismia guianensis (EHVG). HPLC-PDA and FIA-ESI-IT-MSn were used to chemically characterize EHVG. The anti-Candida activity was determined in vitro by the minimum inhibitory concentrations (MIC) against Candida glabrata (ATCC-2001); Candida albicans (ATCC-90028, ATCC-14053, and ATCC-SC5314), and C. albicans clinical isolates. EHVG effects on adhesion, growth, and biofilm formation were also determined. Molecular docking was used to predict targets for EHVG compounds. The main compounds identified included anthraquinone, vismione D, kaempferol, quercetin, and vitexin. EHVG was fungicidal against all tested strains. C. albicans ATCC 14053 and C. glabrata ATCC 2001 were the most sensitive strains, as the extract inhibited their virulence factors. In silico analysis indicated that vismione D presented the best antifungal activity, since it was the most effective in inhibiting CaCYP51, and may act as anti-inflammatory and antioxidant agent, according to the online PASS prediction. Overall, the data demonstrate that EHVG has an anti-Candida effect by inhibiting virulence factors of the fungi. This activity may be related to its vismione D content, indicating this compound may represent a new perspective for treating diseases caused by Candida sp.

Extracts from Argentinian native plants reverse fluconazole resistance in Candida species by inhibiting the efflux transporters Mdr1 and Cdr1.

BACKGROUND: The development of multidrug resistance (MDR) associated with the overexpression of the efflux transporters Mdr1 and Cdr1 in Candida species impedes antifungal therapies. The urgent need for novel agents able to inhibit the function of both pumps, led us to evaluate this property in 137 extracts obtained from Argentinian plants. METHODS: The ability of the extracts to reverse efflux pump-mediated MDR was determined with an agar chemosensitization assay using fluconazole (FCZ) resistant Mdr1- and Cdr1-overexpressing clinical isolates of Candida albicans and Candida glabrata as well as Saccharomyces cerevisiae strains selectively expressing Mdr1 (AD/CaMDR1) or Cdr1 (AD/CaCDR1). The resistance-reversing activity of the most potent extracts was further confirmed using a Nile Red accumulation assay. RESULTS: Fifteen plant extracts overcame the FCZ resistance of Candida albicans 1114, which overexpresses CaMdr1 and CaCdr1, and AD/CaMDR1, with those from Acalypha communis and Solanum atriplicifolium being the most effective showing 4- to 16-fold reversal of resistance at concentrations ≥ 25 µg/mL. Both extracts, and to a lesser extent that from Pterocaulon alopecuroides, also restored FCZ sensitivity in CgCdr1-overexpressing C. glabrata 109 and in AD/CaCDR1 with fold reversal values ranging from 4 to 32 and therefore demonstrating a dual effect against Mdr1 and Cdr1. Both, A. communis and S. atriplicifolium extracts at concentrations ≥ 12.5 and ≥ 25 µg/mL, respectively, increased the intracellular Nile Red accumulation in all yeast strains overexpressing efflux pumps. CONCLUSIONS: The non-toxic and highly active extracts from A. communis and S. atripicifolium, provide promising sources of compounds for potentiating the antifungal effect of FCZ by blocking the efflux function of Mdr1 and Cdr1 transporters.

Emphysematous pyelonephritis caused by C. glabrata / Pielonefrite enfisematosa por C. glabrata

Abstract Emphysematous pyelonephritis (EPN) is a rare acute necrotizing infection of the kidney and surrounding tissues, with gas in the renal parenchyma, collecting system or perirenal tissue. The bacterial etiology predominates; mainly Gram-negative bacilli; Candida spp. and C. albicans are rarely described. We describe a case of EPN caused by C. glabrata, sensitive to fluconazole in a young, hypertensive woman with undiagnosed diabetes mellitus (DM), with renal dysfunction upon admission; her abdominal CT scan found a volumetric increase in the left kidney, signs of gas collections and perirenal blurring. Despite the antimicrobial therapy instituted, due to clinical refractoriness, a double J catheter and subsequent total nephrectomy were indicated, with good postoperative evolution. Her uroculture showed C. glabrata sensitive to fluconazole, and the pathology study showed tubular atrophy and intense interstitial inflammatory infiltrate. Despite the serious, potentially fatal condition, we could control the infection and the patient recovered fully. Poor DM management is an important triggering factor, and it is of great relevance to identify the EPN through imaging exams due to the peculiarities of its clinical and potentially surgical management

Resumo A pielonefrite enfisematosa (PNE) é uma infecção aguda rara necrotizante do rim e dos tecidos adjacentes, com presença de gás no parênquima renal, sistema coletor ou tecido perirrenal. Predomina a etiologia bacteriana, principalmente bacilos Gram-negativos; Candida spp. e na maioria das vezes C. albicans são raramente descritas. Descreve-se um caso de PNE causada por C. glabrata sensível a fluconazol em mulher jovem, hipertensa e com diabetes mellitus (DM) não diagnosticada, com disfunção renal à admissão; tomografia computadorizada de abdome constatou aumento volumétrico do rim esquerdo, sinais de coleções gasosas e borramento perirrenal. Apesar da terapia antimicrobiana instituída, devido à refratariedade clínica, foi indicado cateter duplo J e posterior nefrectomia total, com boa evolução pós-operatória. A urocultura evidenciou C. glabrata sensível a fluconazol, e o anatomopatológico demonstrou atrofia tubular e intenso infiltrado inflamatório intersticial. Apesar da condição grave, potencialmente fatal, houve controle do foco infeccioso e plena recuperação da paciente. O mau manejo do DM é um importante fator desencadeante, e é de grande relevância identificar a PNE por meio de exames de imagem devido às peculiaridades de seu manejo clínico e potencialmente cirúrgico.

Bioactive compounds with antifungal activity against pathogens isolated from pregnant woman: Gallesia integrifolia (garlic wood) is a promising treatment for vulvovaginal candidiasis.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves, bark, and roots of Gallesia integrifolia are consumed in folk medicine through infusion, decoction, and topical preparation by crushing because of its pharmacological properties in several peripheral system disorders, including microbial infections. The presence of various molecules in different parts of the plant likely confers this species' fungicidal action, but scientific evidence is lacking. Vulvovaginal candidiasis mainly affects women of reproductive age. When left untreated, it can cause pregnancy complications. Currently available antifungals often cause undesirable side effects. New alternative therapeutic strategies based on medicinal plants have been proposed. AIM: To investigate the antifungal activity of G. integrifolia against vulvovaginal candidiasis secretion in pregnant women. MATERIALS AND METHODS: Antifungal activity was determined by the minimum inhibitory concentration (MIC), determined by broth microdilution method using Candida spp (NEWP1210), C. albicans (CCCD-CC001), C. tropicalis (CCCD-CC002) standard and clinical isolates from pregnant women with vulvovaginal candidiasis. Nystatin and fluconazole were used as positive controls. The chemical composition of essential oils that were extracted from leaves, flowers, and fruits of G. integrifolia was determined by gas chromatography coupled to mass spectrometry. Reverse docking was used to suggest a possible target in Candida. Conventional docking was used to identify the most probable compound that inhibits fungal growth. RESULTS: A total of 24 compounds were identified, accounting for ∼99% of volatile constituents in the essential oils. Leaves of G. integrifolia contained 3,5-dithiahexanol-5,5-dioxide (40.93%), flowers contained methionine ethyl ester (46.78%), and fruits contained 2,8-dithianonane (54.01%) as the most abundant compounds. The MICs of essential oils of leaves, flowers, and fruits of G. integrifolia against standard strains of Candida spp, C. albicans, and C. tropicalis ranged from 13.01 to 625.00 µg/mL. The essential oil of flowers more effectively inhibited Candida spp. Essential oils of leaves and flowers were similar to fluconazole against C. albicans. Essential oils of flowers and fruits were similar to fluconazole against C. tropocalis. In Candida yeast species that were isolated from vaginal secretion samples from pregnant patients, the MICs of leaves and flowers ranged from 52.08 to 5000.00 µg/mL. The essential oil of leaves (277.77 µg/mL) was the most active against C. albicans. No significant differences were found between the essential oils of leaves and flowers against C. glabrata. Docking simulations suggested that phytol in leaves and flowers was responsible for the antimicrobial effect. CONCLUSION: The present results suggest the potential therapeutic use of G. integrifolia, especially its leaves and flowers, against Candida and vulvovaginal candidiasis.

A Denture Use Model Associated with Candida spp. in Immunocompetent Male and Female Rats.

Denture stomatitis (DS) is a common infection in denture wearers, especially women. This study evaluated the induction of DS using acrylic devices attached to the palate of rats combined with inoculation of Candida spp. Immunocompetent male and female rats received a carbohydrate-rich diet. Impressions were taken from the rats' palate to individually fabricate acrylic devices. Mono- and multispecies biofilms of C. albicans, C. glabrata, and C. tropicalis were grown on the devices, which were then cemented on posterior teeth and kept in the rats' palate for four weeks. Microbial samples from the palate and the device were quantified. Oral microbiome of rats inoculated with C. albicans was analyzed by 16S rRNA gene sequencing. Log10(CFU/mL) were analyzed by mixed or two-way MANOVA (α = 0.05). Candida spp. and acrylic device did not induce palatal inflammation macroscopically nor microscopically. Although there was an increase (p < 0.001) of the total microbiota and female rats demonstrated higher (p = 0.007) recovery of Candida spp. from the palate, the gender differences were not biologically relevant. The microbiome results indicate an increase in inflammatory microbiota and reduction in health-associated micro-organisms. Although Candida spp. and acrylic device did not induce DS in immunocompetent rats, the shift in microbiota may precede manifestation of inflammation.

Effects of ß-lapachone and ß-nor-lapachone on multidrug efflux transporters and biofilms of Candida glabrata.

Infections caused particularly by Candida glabrata are hard to treat due to the development of antifungal resistance that occurs mainly through the production of efflux pumps and biofilm. Thus, a promising strategy to overcome infections caused by C. glabrata could be to use a substance able to inhibit efflux pumps and eradicate biofilms. Lapachones are natural naphthoquinones that possess a variety of pharmacological properties. Previous studies show that these substances inhibit the growth, virulence factors and efflux pumps of C. albicans. The aim of the present study was to evaluate whether lapachones are able to inhibit efflux pumps related to antifungal resistance in C. glabrata and either prevent biofilm formation or affect mature biofilms. Assays were performed with Saccharomyces cerevisiae strains that overexpress C. glabrata transporters (CgCdr1p and CgCdr2p). One C. glabrata clinical isolate that overexpresses CgCdr1p was also used. Both ß-lapachone and ß-nor-lapachone affected the growth of S. cerevisiae and C. glabrata when combined to fluconazole, and this action was inhibited by ascorbic acid. Both lapachones stimulated ROS production, inhibited efflux activity, adhesion, biofilm formation and the metabolism of mature biofilms of C. glabrata. Data obtained on the present study point to the potential use of ß-lapachone and ß-nor-lapachone as antibiofilm agents and adjuvants on the antifungal therapy related to resistant infections caused by C. glabrata.

Candida glabrata is a successful pathogen: An artist manipulating the immune response.

In recent years, Candida glabrata has emerged as a clinically important pathogen causing invasive infections and death. This review highlights the main virulence factors of this fungus and its strategies to subvert the cell response after invasion. Additionally, we emphasize the intracellular behavior and relationship of C. glabrata with several cells. We contrast these features with some fascinating strategies exhibited by M. tuberculosis to highlight that C. glabrata is a successful pathogen and an artist manipulating the immune response like M. tuberculosis.

Perfil de resistencia antifúngica en el tratamiento de candidiasis vaginal: Un diagnóstico de agentes etiológicos / Antifungal resistance profile in the treatment of vaginal candidiasis: A diagnosis of etiologic agents

Introducción: En las micosis, la resistencia a los antifúngicos aumenta debido a los diagnósticos y tratamientos incorrectos. Por tanto, se ha sugerido la vigilancia y el seguimiento de las cepas resistentes para garantizar una terapia adecuada. Objetivo: Determinar las especies de Candida y el perfil de resistencia a fluconazol y voriconazol, que presentan los aislados obtenidos de muestras almacenadas durante los meses diciembre 2017 y marzo 2018 de pacientes provenientes del Laboratorio del Hospital Regional "Miguel Ángel Mariscal Llerena" del departamento de Ayacucho, Perú. Material y Métodos: Estudio descriptivo transversal, en el cual se aislaron 110 cepas de Candida sp almacenadas por un período de cuatro meses, y procesadas por métodos estandarizados y aprobados por el Ministerio de Salud (MINSA) y el Instituto Nacional de Salud (INS) del Perú relacionados con el diagnóstico de agentes etiológicos de micosis humanas y la sensibilidad antifúngica con los métodos estandarizados de Clinical Laboratory Standard Institute (CLSI). Resultados: Se observó C. albicans en 86,4 % de los aislados seguida por C. glabrata con 9,1 %, C. parapsilosis 2,7 % y 0,9 % de C. tropicalis y C. krusei. El 10,5 % de C. albicans fue resistente al fluconazol y voriconazol con CMI ≥ 128 μg/mL y ≥ 16 μg/mL respectivamente, mientras que 20 % de C. glabrata mostró sensibilidad dosis dependiente y 10 % de resistencia al fluconazol. Conclusiones: Existe una gran variedad de especies de Candida, siendo la C. albicans la más común, seguida por C. glabrata, con un mayor porcentaje de aislamiento en comparación con otras especies. Se puede observar que estas especies poseen grados de vulnerabilidad considerables a la aplicación de fármacos como el fluconazol y voriconazo(AU)

Introduction: In mycoses, resistance to antifungals increases due to incorrect diagnosis and treatment. Therefore, surveillance and monitoring of resistant strains has been suggested to ensure adequate therapy. Objective: To determine the Candida species and the resistance profile to fluconazole and voriconazole presented by the isolates obtained from samples stored during the months of December 2017 and March 2018 from patients coming from the Laboratory of the Regional Hospital "Miguel Ángel Mariscal Llerena" in the department of Ayacucho, Peru. Material and Methods: Cross-sectional descriptive study in which 110 strains of Candida sp were isolated and stored for a period of four (04) months, and processed by standardized methods approved by the Ministry of Health (MINSA) and the National Institute of Health (INS) of Peru related to the diagnosis of etiological agents of human mycosis and antifungal sensitivity with the standardized methods of the Clinical Laboratory Standard Institute (CLSI). Results: C. albicans was observed in 86,4 % of isolates, followed by C. glabrata (9,1 %), C. parapsilosis (2,7 %), and 0,9 % of C. tropicalis and C. krusei; 10,5 % of C. albicans was resistant to fluconazole and voriconazole with MIC ≥ 128 μg/mL and ≥ 16 μg/mL respectively, while 20 % of C. glabrata showed dose dependent sensitivity and 10 % resistance to fluconazole. Conclusions: There is a wide variety of Candida species, with C. albicans being the most common, followed by C. glabrata, with a higher percentage of isolation compared to other species. It can be seen that these species have considerable degrees of vulnerability to the application of drugs such as fluconazole and voriconazole(AU)

Synergistic activity of dobutamine combined with azoles and its mechanism of action against strains of Candida glabrata.

Aims: To evaluate the antifungal effect of dobutamine against Candida glabrata as well as its synergism with azoles and its action on biofilm. Methods: The M27-A3 protocol and flow cytometry were used for elucidation of the possible mechanism of action. Results: The tested isolates presented MICs ranging from 2 to 32 µg/ml for dobutamine, with fungistatic effect. A total of 82% of the strains showed synergism with fluconazole, with 90% showing synergism with itraconazole. The effect on biofilm formation was nonsignificant. Cytometry tests showed that dobutamine induced mitochondrial depolarization. Conclusion: Dobutamine has an antifungal effect on strains of C. glabrata and synergistic activity with azoles. This effect is probably mediated by increased oxidative damage to the membrane.

FLO8 deletion leads to decreased adhesion and virulence with downregulated expression of EPA1, EPA6, and EPA7 in Candida glabrata.

BACKGROUND: The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. OBJECTIVES: The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. METHODS: We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. RESULTS: Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. CONCLUSIONS: These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.

Ent-hardwickiic acid from C. pubiflora and its microbial metabolites are more potent than fluconazole in vitro against Candida glabrata.

The incidence of Candida glabrata infections has rapidly grown and this species is among those responsible for causing invasive candidiasis with a high mortality rate. The diterpene ent-hardwickiic acid is a major constituent in Copaifera pubiflora oleoresin and the ethnopharmacological uses of this oleoresin by people from Brazilian Amazonian region point to a potential use of this major constituent as an antimicrobial. Therefore, the goal of this study was to evaluate the antifungal activity of ent-hardwickiic acid against Candida species and to produce derivatives of this diterpene by using microbial models for simulating the mammalian metabolism. The microbial transformations of ent-hardwickiic acid were carried out by Aspergillus brasiliensis and Cunninghamella elegans and hydroxylated metabolites were isolated and their chemical structures were determined. The antifungal activity of ent-hardwickiic acid and its metabolites was assessed by using the microdilution broth method in 96-well microplates and compared with that of fluconazole. All the diterpenes showed fungistatic effects (ranging from 19·7 to 75·2 µmol l-1 ) against C. glabrata at lower concentrations than fluconazole (163·2 µmol l-1 ) and were more potent fungicides (ranging from 39·5 to 150·4 µmol l-1 ) than fluconazole, which showed fungicidal effect at the concentration of 326·5 µmol l-1 .

Candida glabrata Hst1-Rfm1-Sum1 complex evolved to control virulence-related genes.

C. glabrata is an opportunistic fungal pathogen and the second most common cause of opportunistic fungal infections in humans, that has evolved virulence factors to become a successful pathogen: strong resistance to oxidative stress, capable to adhere and form biofilms in human epithelial cells as well as to abiotic surfaces and high resistance to xenobiotics. Hst1 (a NAD+-dependent histone deacetylase), Sum1 (putative DNA binding protein) and Rfm1 (connector protein) form a complex (HRS-C) and control the resistance to oxidative stress, to xenobiotics (the antifungal fluconazole), and adherence to epithelial cells. Hst1 is functionally conserved within the Saccharomycetaceae family, Rfm1 shows a close phylogenetic relation within the Saccharomycetaceae family while Sum1 displays a distant phylogenetic relation with members of the family and is not conserved functionally. CDR1 encodes for an ABC transporter (resistance to fluconazole) negatively controlled by HRS-C, for which its binding site is located within 223 bp upstream from the ATG of CDR1. The absence of Hst1 and Sum1 renders the cells hyper-adherent, possibly due to the overexpression of AED1, EPA1, EPA22 and EPA6, all encoding for adhesins. Finally, in a neutrophil survival assay, HST1 and SUM1, are not required for survival. We propose that Sum1 in the HRS-C diverged functionally to control a set of genes implicated in virulence: adherence, resistance to xenobiotics and oxidative stress.

Synergistic interaction of fluconazole/sodium bicarbonate on the inhibition of Candida glabrata phospholipase gene

Abstract Candida glabrata infections are responsible for deaths of people globally. Fluconazole is known to be less effective against C. glabrata, which developed many strategies to evade being destroyed by fluconazole. To achieve enhanced efficacy of fluconazole against C. glabrata, the interaction of fluconazole with sodium bicarbonate was investigated using the CLSI guidelines. The efficacy of fluconazole alone and in combination with sodium bicarbonate was evaluated using the time-kill and phospholipase production assays. Eventually, the expression of PLB was assessed using semi-quantitative RT-PCR to investigate the inhibitory properties of fluconazole alone and in combination with sodium bicarbonate against C. glabrata. The fluconazole/sodium bicarbonate combination displayed synergistic and antagonistic effects (FICI= 0.375-4.25). In C. glabrata ATCC, SN 152, and SN 164, the fluconazole/sodium bicarbonate combination exhibited a significant fungicidal activity (p< 0.05) but antagonistic effect in the case of SN 283. With exception of SN 283, a significant reduction was noted in phospholipase production in clinical isolates of C. glabrata treated with fluconazole/sodium bicarbonate combination. The PLB was down-regulated significantly by 0.168-0.515 fold in C. glabrata treated with fluconazole/sodium bicarbonate. The results suggested fluconazole/sodium bicarbonate to have a potential synergistic interaction in C. glabrata, and the underlying mechanism may be associated with phospholipase gene

Abf1 Is an Essential Protein That Participates in Cell Cycle Progression and Subtelomeric Silencing in Candida glabrata.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.