Comparative analysis of biofilm formation by Candida albicans and Candida krusei in different types of contact lenses / Análise comparativa da formação de biofilmes, por Candida albicans e Candida krusei, em diferentes tipos de lentes de contato

ABSTRACT Objective: To evaluate the Candida krusei and Candida albicans biofilm formation abilities on 5 different types of contact lenses and compare their metabolic activities and biomass. Methods: After biofilm formation by both the test species, their metabolic activity was assessed by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay with menadione, while the biomass was determined by staining with 0.4% crystal violet dye for further statistical analysis. Results: Both the Candida species could form biofilms on different types of contact lenses, with greater metabolic activities and lower biomass formation in rigid gas permeable lenses. Conclusion: Biofilm formation with greater metabolic activity and greater biomass were expected on soft contact lenses considering their surface hydrophobicity. However, the results demonstrated a greater metabolic activity on rigid contact lenses. This result has a great significance with regards to the increasing risk of microbial keratitis, although further studies are warranted to better elucidate the formation of biofilms on different types of contact lens materials in the future.

RESUMO Objetivo: Avaliar a capacidade de formação de bio­filmes de Candida krusei e Candida albicans em cinco tipos de lentes de contato, comparando atividade metabólica e biomassa dos mesmos. Métodos: Após a formação de biofilme de ambas as espécies, a atividade metabólica foi avaliada por ensaio de redução 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-te­tra­zolium-5-carboxanilide com Menadiona, e a biomassa foi avaliada por coloração com Cristal Violeta 0,4% para posterior análise estatística. Resultados: Ambas as espécies de Candida foram capazes de formar biofilmes nos diferentes tipos de lentes de contato, havendo em lentes rígidas gás permeável maior atividade metabólica e menor biomassa formada. Conclusão: Esperava-se a obtenção de biofilmes de maior atividade metabólica e maior biomassa em lentes de contato gelatinosas com base no fundamento da Hidrofobicidade Superficial. Porém, o resultado apontou para maior atividade metabólica em lentes de contato rígidas. Apesar de observados resultados significativos, trata-se de um assunto de grande importância frente ao aumento do número de ceratites microbianas, mostrando-se necessários outros estudos para melhor elucidar a formação de biofilmes em diferentes tipos de materiais de lentes de contato.

First reported case series of Candida krusei peritonitis secondary to a perforated viscus

Candida peritonitis is associated with high mortality and multiple organ failure. With an evolving epidemiology of candidaemia indicating an increasing prevalence of rare Candida species worldwide, consideration of multidrug-resistant fungal pathogens as a cause of abdominal sepsis is paramount. We report three cases of Candida krusei as a cause of secondary and tertiary peritonitis. These cases highlight that the early use of an echinocandin class antifungal in patients not responding to standard regimens warrants consideration.

Study on the anti-Candida krusei in vitro combined with tacrolimus and itraconazole / 安徽医科大学学报

Objective The use of the antifungal agent itraconazole and the calcineurin inhibitor tacrolimus alone, together with the above two drugs, was evaluated in vitro for growth of Candida krusei. Whether or not the combina-tion is more effective than the antifungal alone can inhibit the growth of Candida krusei. Methods According to the guidelines of the American Institute of Clinical and Laboratory Standards, 12 Candida krusei strains were divided into blank control group, itraconazole group, tacrolimus group, and combination group by microdilution method, and then 12 the Candida krusei strain was incubated for 24 h and its growth was observed. Results In comparison with the alone use of isotriconazole in Candida krusei, the combined use of itraconazole,its minimal inhibitory con-centration was much less than that of alone, with a maximum multiple of 32 fold, a minimum multiple up to 4 times; the same, In comparison with the alone use of tacrolimus in Candida krusei, the combined use of tacroli-mus, its maximum multiple of 32 times, the lowest multiple of 2 times. Conclusion The combined use of itracon-azole and tacrolimus in 12 strains of Candida krusei displayed a powerful synergistic effect in vitro.

Study of Microbial Interaction Formed by "Candida krusei" and "Candida glabrata": "In Vitro" and "In Vivo" Studies

Abstract Recently, the non-albicans Candida species have become recognized as an important source of infection and oral colonization by association of different species in a large number of immunosuppressed patients. The objective of this study was to evaluate the interactions between C. krusei and C. glabrata in biofilms formed in vitro and their ability to colonize the oral cavity of mouse model. Monospecies and mixed biofilms were developed of each strain, on 96-well microtiter plates for 48 h. These biofilms were analyzed by counting colony-forming units (CFU/mL) and by determining cell viability, using the XTT hydroxide colorimetric assay. For the in vivo study, twenty-four mice received topical applications of monospecie or mixed suspensions of each strain. After 48 h, yeasts were recovered from the mice and quantified by CFU/mL count. In the biofilm assays, the results for the CFU/mL count and the XTT assay showed that the two species studied were capable of forming high levels of in vitro monospecie biofilm. In mixed biofilm, the CFU of C. krusei increased (p=0.0001) and C. glabrata decreased (p=0.0001). The metabolic activity observed in XTT assay of mixed biofilm was significantly reduced compared with a single C. glabrata biofilm (p=0.0001). Agreeing with CFU in vitro count, C. glabrata CFU/mL values recovered from oral cavity of mice were statistically higher in the group with single infection (p=0.0001) than the group with mixed infection. We concluded that C. krusei inhibits C. glabrata and takes advantage to colonize the oral cavity and to form biofilms.

Resumo Recentemente, as espécies não albicans tem se tornado uma importante fonte de infecção e de colonização oral pela associação de espécies em um grande número de pacientes imunossuprimidos. O objetivo desse estudo foi avaliar a interação entre C. krusei e C. glabrata em biofilmes formados in vitro e sua capacidade em colonizar a cavidade oral em modelo de camundongo. Biofilmes monoespécies e mistos foram formados em placas de 96 poços por 48 h. Esses biofilmes foram analisados pela contagem de UFC/mL e pela determinação da viabilidade celular, usando ensaio de XTT. Para o estudo in vivo, vinte e quatro camundongos receberam aplicações tópicas de suspensões monoespécies e mistas de cada espécie. Após 48 h, as leveduras foram recuperadas dos camundongos e quantificadas por UFC/mL. Nos ensaios de biofilme, os resultados da contagem de UFC/mL e do ensaio de XTT mostraram que as duas espécies estudadas foram capazes de formar grande quantidade de biofilme monoespécie in vitro. Nos biofilmes mistos, a UFC/mL de C. krusei aumentou (p=0,0001) e de C. glabrata diminuiu (p=0,0001). A atividade metabólica observada no ensaio de XTT nos biofilmes mistos foi significantemente reduzida comparada com o biofilme formado apenas de C. glabrata (p=0,0001). Concordado com as contagens in vitro, os valores de UFC/mL de C. glabrata recuperados da cavidade oral dos camundongos foram estatisticamente maior no grupo com infecção simples (p=0,0001) do que do grupo com infecção mista. Nós concluímos que C. krusei inibe C. glabrata e possui vantagem em colonizar a cavidade oral e formar biofilmes.

Meningoencefalitis por Candida krusei / Meningoencephalitis by Candida krusei

Los procesos infecciosos del sistema nervioso presentan una elevada morbilidad y mortalidad, debido a esto es necesario un diagnóstico certero y rápido por parte del equipo médico de urgencia. En este trabajo se reporta el caso de un paciente tratado en la Unidad de Cuidados Intensivos (UCI) del Hospital Andino de la ciudad de Riobamba de Ecuador. Se trata de un paciente de 46 años de edad que presenta un síndrome convulsivo de etiología infecciosa que se interpreta como una meningoencefalitis. El resultado del cultivo de líquido cefalorraquídeo señala como agente etiológico una especie de Candida poco frecuente: Candida krusei. Desde su ingreso el paciente necesitó ventilación mecánica invasiva y se le administró tratamiento antibiótico y antiviral hasta ir descantando etiologías bacterianas, virales o parasitarias. Al quinto día se aislaron más de 35 000 Unidades Formadoras de Colonias de Candida krusei, por lo que se indicó tratamiento antimicótico con Voriconazol. Se realizaron estudios de imagen, lo que incluyó tomografía axial computarizada (TAC) y resonancia magnética (RM) que mostraron edema cerebral en corteza temporal excluyéndose procesos expansivos. Paciente se retira del ventilador mecánico y es dado de alta de la UCI con afasia sensitiva y hemiparesia izquierda. Durante su estancia en la sala de medicina interna el paciente se somete a rehabilitación con evolución favorable.

Infectious processes of the nervous system produce high morbidity and mortality and because of this, it is necesary an accurate and opportune diagnosis. In this paper we report the case of a patient treated in the Intensive Care Unit (ICU) of the Hospital Andino of the city of Riobamba of Ecuador. It consist of a 46 years old patient that presents a seizure syndrome of infectious etiology that is is interpreted as meningoencephalis. Results of cerebrospinal fluid culture shows the presence of a rare specie of Candida: Candida krusei. From the first day of hospitalization the patient needed invasive mechanical ventilation, antimicrobial and antiviral treatment while a bacterial, viral or parasitic etiology was identified. On the 5th day more than 35 000 Colony Forming Units of Candida krusei were isolated, therefore an antifungal treatment with Voriconazole was indicated. Imaging studies were carried out including, Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), which showed cerebral edema in the temporal cortex and that expansive processes were excluded. The patient is removed from the mechanical ventilator and was discharged form the ICU presenting sensory aphasia and left hemiparesis. During his stay in the internal medicine unit of the hospital, the patient underwent rehabilitation and showed a favorable evolution.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Novel synthetic anti-fungal tripeptide effective against Candida krusei.

Introduction: Candida species are the major fungal pathogens of humans. Among them, Candida krusei have emerged as a notable pathogen with a spectrum of clinical manifestations and is known to develop resistance against azoles mainly fl uconazole. Anti-microbial peptides play important roles in the early mucosal defence against infection and are potent anti-fungal agents since they fi ght against fungal infection as well as have ability to regulate host immune defence system. The aim of the study was to synthesize a small anti fungal peptide. Materials and Methods: The series of tripeptides were synthesized and screened for antifungal activity against Candida strains according to CLSI guidelines. Toxicity effect of peptide was tested with human erythrocytes. The mode of action of peptide on fungus was resolved by scanning electron microscopy (SEM) studies Results: The tripeptide FAR showed a prominent anti fungal activity among the series. The minimum inhibitory concentration and minimum fungicidal concentration of tripeptide FAR was found to be 171.25 μg/ml and 685 μg/ml, respectively against Candida krusei. The therapeutic index was 2.9. The haemolytic experiment revealed that this peptide is non - toxic to human cells. The SEM studies showed disruption of cell wall and bleb-like surface changes and irregular cell surface. Conclusion: The peptide showed a signifi cant antifungal activity against C. krusei. Thus, it can set a platform for the design of new effective therapeutic agents against C. krusei.

Antifungal Effect of Flavonoid 5, 7, 4’- Trimethoxyflavone against Candida krusei Strains.

Most of the flavonoids are considered as constitutive antimicrobial ingredients, especially those belonging to prenylated flavonoids, flavones and isoflavones. In the study, the flavonoid 5,7,4’- trimethoxyflavone was evaluated for its antifungal effects. Four fungal strains were used in the study for activities, Candida krusei – LM 9700, Candida krusei – LM 656, Candida krusei– LM 13 and Candida krusei – LM08. All the microorganism strains were obtained from the Laboratory of Mycology collection. Microdilution method was used for antifungal assay of the flavonoid. The results were also compared with the standard drug, Nistatin (100 UI/mL). The obtained results showed activity fungistatic against Candida krusei strains.

Inhibitory effects of curcumin on hyphal development and biofilm formation of five kinds of non-Candida albicans / 中草药

Objective: To observe the inhibitory effects of curcumin on hyphal development and biofilm formation of five kinds of non-Candida albicans. Methods: Serial 2-fold dilution assay was used to determine the MICs of curcumin to C. tropicalis, C. glabrata, C. parapsilokis, C. krusei, C. guilliermondii; XTT assay was used to determine the SMIC50 of curcumin to the five non-C. albicans. Inverted microscope and scanning electron microscope (SEM) were applied to inspectting the morphological change of non-C. albicans treated by curcumin, The dilution method was applied to inspecting the hyphae around the colonies. Results: The MICs of curcumin to C. tropicalis, C. glabrata, C. parapsilokis, C. Krusei, C. guilliermondii were 64, 128, 256, 256, and 128 μg/mL, and the SMIC50 were 512, 512, >512, >512, and 512 μg/mL, respectively. We found that curcumin could inhibit the hyphal and biofilm formation of the five kinds of non-C. albicans by observation with inverted microscope and SEM, and curcumin could inhibit the hyphal development around the colonies by observation with optical microscope. Conclusion: Curcumin could inhibit the hyphal development and biofilm formation of the five kinds of non-C. albicans.

Composición química y evaluación de la actividad antifúngica del aceite esencial de corteza de Vismia baccifera var. dealbata / Chemical composition and evaluation of anti-fungal activity of essential oil from Vismia baccifera var. dealbata bark

El objetivo de este trabajo fue evaluar la susceptibilidad de levaduras del género Candida y el Complejo Cryptococcus neoformans al aceite esencial de Vismia baccifera var. dealbata. El aceite, procedente de Chiguará, estado Mérida-Venezuela, fue analizado por CG/ EM logrando la identificación de trece componentes, que constituyeron el 97,7% de la mezcla; tres de ellos se apreciaron como productos mayoritarios, representando el 70,4% de la totalidad (Óxido de cariofileno 31,4%, β-cariofileno 26,4% y α-zingibireno 12,6%). El ensayo de actividad antifúngica mostró que dicho aceite inhibió el crecimiento de varias cepas de los géneros Candida y el Complejo C. neoformans, evaluadas cualitativamente por difusión en agar con disco a una concentración de 1.000 μg/mL; se observaron halos de inhibición entre 8 y 12 mm, exhibiendo valores de CMI entre 1,6 y 1.000 μg/mL en el ensayo cuantitativo por el método “Spot on a lawn”. Los datos obtenidos fueron sometidos a análisis de varianza y la prueba de Tukey, obteniéndose una actividad inhibitoria relevante contra Candida krusei. Este estudio constituye el primer reporte, tanto de la composición química como de evaluación antifúngica del aceite esencial extraído a partir de especies del género Vismia.

The purpose of this study was to evaluate the susceptibility of Candida genus yeasts and the Cryptococcus neoformans Complex to essential oil from Vismia baccifera var. dealbata. This oil, obtained at Chiguará, Mérida State, was analyzed by GC/MS, which identified thirteen components that constituted 97.7% of the mixture; three of them were established as majority products, representing 70.4% of the total: (cariophyllene oxide 31.4%, β-cariophyllene 26.4%, and α-zingibirene 12.6%). The anti-fungal assay showed that this oil inhibited the growth of several Candida genus strains and of the C. neoformans Complex, qualitatively evaluated by agar disk diffusion at a 1,000 μg/ mL concentration; inhibition halos of a between 8 to 12 mm diameter were observed, showing MIC values between 1.6 and 1,000 μg/mL in the “Spot on a lawn” quantitative assay. The data obtained were submitted to ANOVA and Tukey’s test, demonstrating a relevant inhibitory activity against Candida krusei. This study constitutes the first report, both of the chemical composition and of the anti-fungal evaluation, of the essential oil extracted from Vismia genus species.

The Evaluation of the Combination of Caspofungin with Voriconazole and Amphotericin B against Clinical Candida krusei Isolates by Etest and Disk Diffusion Methods.

Background: C. krusei is an opportunistic fungal pathogen that known of its intrinsic fluconazole resistance and its frequency is increasing especially among hematology patients. The increase in the frequency of high mortality fungal infections have accelerated the efforts of new drug development with broad spectrum, low toxicity and the studies including their combination. However, there is no standardized method to evaluate the activity of drug combinations. Aims: To evaluate the activity of caspofungin (CAS) with voriconazole (VOR) and amphotericin B (AMB) alone and in combination and the utility of Etest and disk diffusion methods for antifungal combinations. Methodology: The minimum inhibitory concentrations of VOR, CAS and AMB against 30 clinical C. krusei isolates were determined by using Etest, disk diffusion and reference broth microdilution methods. Combinations of CAS with VOR and CAS with AMB were evaluated using disk diffusion (three different ways) and Etest (two different ways) methods. Results: All isolates tested were susceptible to VOR and CAS in vitro by all three methods. Categorical agreements of Etest and disk diffusion methods with reference microdilutiontest were 100% for CAS and VOR (for each method), 86.7% and 50% for AMB, respectively. In the all ways of both combination methods, we did not observe distinctly antagonistic or synergic interaction. Conclusion: Etest and disk diffusion could be easy, convenient, and nontime-consuming alternative methods to evaluate the antifungal combinations. The combinations of CAS with VOR and AMB exhibited promising results because of an apparent antagonistic interaction was not detected in this study.

In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei and Aspergillus fumigatus / Atividade antifúngica in vitro e os efeitos citotóxicos de óleos essenciais e extratos de plantas medicinais e aromáticas contra Candida krusei e Aspergillus fumigatus

The plants are usually used in traditional medicine as antimicrobial agents and their essential oils and extracts have been known to possess antifungal activity. The aim of this study was to evaluate in vitro the activity of 32 essential oils and 29 extracts against C. krusei and A. fumigatus as well as the cytotoxic effect on Vero cells. Time-kill curve and interaction between antifungals and the most active sample against C. krusei, was also evaluated. The oils from C. ambrosioides and the extract of M. cucullata showed antifungal activity against C. krusei (GM-MIC 7.82 and 31.25 µg/mL, respectively). L. citriodora was actives against C. krusei and A. fumigates (GM-MIC = 99.21 µg/mL and 62.5 µg/mL respectively). Time-kill assays done with C. ambrosioides oil showed fungicidal activity at 4x MIC. The interaction of C. ambrosioides oil with itraconazole and amphotericin B was tested following the chequerboard technique. No interaction was detected for the combination of C. ambrosioides oil with amphotericin B and itraconazole (FICI range = 1.03-1.06 and 1.03-1.00, respectively). Cytotoxicity assays for all samples were carried out with MTT. Only the oil from Hedyosmun sp. and L. dulcis were cytotoxic.

As plantas são geralmente utilizadas na medicina tradicional como agentes antimicrobianos e seus óleos essenciais e extratos foram conhecidos por possuir atividade antifúngica. O objetivo deste estudo foi avaliar in vitro a atividade de 32 óleos essenciais e 29 extratos contra Candida krusei e Aspergillus fumigatus, bem como o efeito citotóxico em células Vero. A curva do tempo-morte e a interação entre antifúngicos e Chenopodium ambrosioidese do extrato de Myrcia cucullata mostraram atividade antifúngica contra C. krusei (geometric means of the minimal inhibitory concentration [GM-MIC] 7,82 e 31,25 µg/mL, respectivamente). Lippia citriodora foi ativa contra C. krusei e A. fumigatus (GM-CIM = 99,21 µg/mL e 62,5 µg/mL, respectivamente). Os testes de tempo-morte feitos com óleo de C. ambrosioides mostraram atividade fungicida em 4x MIC. A interação do óleo C. ambrosioides com itraconazol e anfotericina B foi testada pela técnica de xadrez. Nenhuma interação foi detectada pela combinação do óleo C. ambrosioides com anfotericina B e itraconazol (intervalo fractional inhibitory index [FICI] = 1,03-1,06 e 1,03-1,00, respectivamente). Os ensaios de citotoxicidade para todas as amostras foram realizadas com MTT. Apenas os óleos Hedyosmun sp. e L. dulcis foram citotóxicos.

Effect of Different Osmotica on the Metabolism of Candida krusei / 微生物学通报

NaCl.Glycerol and trehalose were the most prominent compatible solutes in C.krusei.The glycerol production was enhanced with the osmotica concentration increasing and NaCl,KCl were much effective than manntiol.The glycerol concentration was increased 74%,63%,57% and the intracellular glycerol was 3.1,2.4,1.8 fold to the control respectively when 0.6mol/L NaCl,KCl and mannitol existed.During the early stage of fermentation,the trehalose all decreased compared with the control.However,the trehalose content was higher than the control in the late stage of fermentation when 0.6mol/L KCl and mannitol existed.The trehalose content was 1.6 and 1.4 fold to that of control respectively.