Chilean crab (Aegla cholchol) as a new source of chitin and chitosan with antifungal properties against Candida spp.

In the present work, the chemical composition of the Chilean freshwater crab Aegla cholchol exoskeleton was studied for the first time. α-Chitin was isolated from three main body parts (pincers, legs, carapace), and its content ranged from 9.0-10.4% (w/w). Moreover, chitosan was extracted by alkaline treatment at different temperature and time regimes. Aegla cholchol exoskeleton, chitin and chitosans were characterized by FTIR, TGA, and SEM. Chemical structure of chitin and chitosan was confirmed by NMR spectroscopy. Chitosan molecular weight determinations were carried out by GPC. The obtained chitosan samples had a degree of N-acetylation (DA) between 4 and 15% and molecular weight (Mw) in the range of 65-201 kDa. The antifungal activity of the chitosan samples and the chitooligomer were tested toward twenty isolated clinical strains of Candida yeast. Chitosan with lower DA (4%) and higher molecular weight showed the lowest minimum inhibitory concentration (MIC) values, which was evidenced by the highest antifungal activity toward Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results suggest that Aegla cholchol is an excellent natural source for production of bioactive materials with potential applications in the health system, to prevent infections associated with Candida strains.

Genotyping and Persistence of Candida albicans from Pregnant Women with Vulvovaginal Candidiasis.

OBJECTIVE: To study Candida albicans genotypes using RAPD and their susceptibility to fluconazole in healthy pregnant women and in vulvovaginal candidiasis (VVC) patients after topical treatment with clotrimazole. METHODS: Vaginal swabs were collected at t = 0 and t = 1 (1 month later) in pregnant women (control group, n = 33), and before (t = 0), at 1 month (t = 1) and at 2 months (t = 2) after clotrimazole treatment in pregnant women with VVC. RESULTS: Candida albicans was isolated in 30% of healthy pregnant women and 80% of patients with VVC. A high genetic heterogeneity was observed in C. albicans genotypes between individuals. In patients with VVC, topical antifungal treatment with clotrimazole was clinically effective, but only in a 62% C. albicans was eradicated. In patients in which C. albicans was not eradicated, this microorganism persisted for 1 or 2 months after the antifungal treatment. The persistent colonies were not associated with a specific genotype, but they were associated with higher MICs in comparison with colonies isolated from the control group. CONCLUSIONS: Therapy with topical clotrimazole, despite a good clinical outcome, could not eradicate completely C. albicans allowing the persistence of genotypes, with higher MICs to fluconazole. More studies with higher number of patients are needed to validate this preliminary finding.

[Characterization of azole resistance mechanisms in Chilean clinical isolates of Candida albicans]. / Caracterización de los mecanismos de resistencia a azoles en aislados clínicos chilenos de Candida albicans.

INTRODUCTION: The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. OBJECTIVE: To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. METHODOLOGY: Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). RESULTS: No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). CONCLUSION: The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.

Caracterización de los mecanismos de resistencia a azoles en aislados clínicos chilenos de Candida albicans / Characterization of azole resistance mechanisms in Chilean clinical isolates of Candida albicans

Introduction: The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. Objective: To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. Methodology: Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). Results: No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). Conclusion: The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.

Introducción: Candida albicans es una levadura comensal capaz de causar una infección oportunista en hospederos susceptibles denominada candidiasis, que puede ser superficial o sistémica. En Chile, los antifúngicos más utilizados para el tratamiento de las candidiasis son los azoles. En un estudio previo en nuestro centro, se detectó que 2,1 y 1,6% de cepas clínicas de C. albicans fueron resistentes a fluconazol y voriconazol, respectivamente. Objetivo: Caracterizar los mecanismos de resistencia involucrados en la resistencia a azoles en cepas clínicas chilenas. Metodología: Según los criterios del Clinical Laboratory Standards Institute (CLSI) M27-S3, se seleccionaron ocho cepas resistentes, nueve cepas susceptibles dosis dependiente (SDD) y 10 cepas sensibles (n: 27), aisladas de flujo vaginal y orina. Se evaluó la presencia de mutaciones en la región 408-488 del gen ERG11 por secuenciación y la expresión relativa del gen ERG11 y de los genes de bombas de eflujo CDR1, CDR2 y MDR1 por RPC en tiempo real cuantitativa (q-PCR). Resultados: No se encontraron mutaciones en el gen ERG11 y la sobre-expresión de éste sólo se presentó en 12,5% de las cepas resistentes (1/8). El mecanismo prevalente en la cepas resistentes fue la sobre-expresión de bombas de eflujo encontrándose en 62,5% de las cepas resistentes (5/8). Conclusión: El estudio de la expresión bombas de eflujo por q-PCR podría ser una herramienta diagnóstica útil para la detección temprana de resistencia a azoles en C. albicans.

Melanocytes and melanin represent a first line of innate immunity against Candida albicans.

Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.

[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.

Interacción Candida albicans-Hospedero: un proceso complejo en el que la inmunidad innata juega un importante papel / Candida albicans-host interaction: a complex process in which the innate immunity play an important role

Candida albicans es una levadura comensal, que bajo ciertas circunstancia puede convertirse en patógeno. La capacidad de cambiar se forma constituye un factor de virulencia, permitiéndole a la levadura invadir y diseminarse. El sistema inmune innato es capaz de reconocer las diferentes morfologías de C. albicans activando receptores (PRRs) que reconocen PAMPs o patrones moleculares conservados. Las células inmunes más importantes son los macrófagos y los neutrófilos que desencadenan una respuesta efectora a través de la fagocitosis y la activación de estrés oxidativo contra C. albicans. Las células dendríticas, por su parte expresan la mayoría de los PRRs involucrados en el reconocimiento de C. albicans activando la secreción de citoquinas hacia una respuesta tipo TH1 (inducida por INF tipo1, IL-12, INFγ, ), Treg (inducida por TGFβ, IL-10) y TH17 (inducida por IL-23, IL6). Las células epiteliales no sólo constituyen una barrera física frente a la infección por C. albicans, sino son fundamentales en el reconocimiento primario de este microorganismo. Mediante una respuesta bifásica estas células activan diferencialmente, vías de transducción de señales que determinan que no se active una respuesta de citoquinas frente a la presencia de blastoconidios (forma comensal) y que se active frente a presencia de hifas (forma invasora). Por su parte, C. albicans es capaz de desarrollar mecanismos evasivos de la respuesta inmune. Esta compleja interacción y hongo-hospedero determina si el hospedero será capaz de eliminar a este microorganismo o si éste finalmente invadirá expresando su virulencia.

Candida albicans is a comensal microorganism that under certain circumstances is able to transform into a pathogen. This ability to switch constitute a virulence factor that C. albicans uses to invade and spread. The innate immune system recognize the different forms of C. albicans activating receptors (PRRs) that recognize PAMPs or conserved molecular patterns. The most important immune cells are macrophages and neutrophils that generate an effector response through phagocytosis and oxidative burst against C. albicans. Dendritic cells express the most of PRRs involved in the recognition of C. albicans activating the cytokines synthesis forward to a TH1 (induced by INF tipo1, IL-12, INFγ), Treg (induced by TGFβ, IL-10) y TH17 (induced by IL- 23, IL6) immune response. Epithelial cells not only constitute a physical barrier against C. albicans, but they are crucial in the first recognition of this microorganism. Through a biphasic response these cells differentially activate pathways that determine a no cytokine response when blastoconidia (commensal form) is present and an inflammatory response in presence of hyphae (pathogenic form). C. albicans develops immune evasive mechanism. This complex host-fungi interaction determines if the host will eradicate the infection or if this microorganism will invade the host, expressing it virulence.

[Species distribution and susceptibility pattern of Candida spp.: the importance to survey also strains isolated from the community]. / Distribución de especies y perfil de susceptibilidad de aislados de Candida spp: la importancia de vigilar también cepas de la comunidad.

BACKGROUND: The most of the surveillance studies has been conducted in hospitalized patients with invasive infections. Recently, new clinical breakpoints (CBPs) have been proposed for antifungal susceptibility testing and epidemiological cutoffs (ECVs). AIM: To evaluate species distribution and susceptibility pattern of Candida spp. obtained from in and outpatients in a period of 6 months. MATERIAL AND METHODS: The isolates (n=223) came from vaginal discharge (51.6%), lower respiratory tract (24.7%), urine (20.2%), wounds (1.8%), blood (0.9%), peritoneal fluid (0.4%) and nails (0.4%). RESULTS: The species distribution was C. albicans 84.8% (n: 189), C. glabrata 7.6% (n: 17), C. tropicalis 2.7% (n: 6), C. parapsilosis 2.2% (n: 5), C. kefyr 0.9% (n: 2) and others 1.8% (C. krusei, C. lusitanie, C. guilliermondii, C. intermedia) (n: 4). The susceptibility dose dependence (SDD) and resistance were 3.2% for fluconazole and 2.2% for voriconazole. The most of SDD and resistant strains were isolated from ambulatory patients. Also, a higher percentage of MICs over the new CBPs and ECVs were found in strains from ambulatory patients and especially in C. glabrata isolates to caspofungin. CONCLUSION: Taking into consideration that most of the invasive infections are caused by strains from the endogenous microbiota, and that there is a resistant population of Candida spp. in the community, should be important to include in surveillance studies strains isolated from ambulatory patients.

Distribución de especies y perfil de susceptibilidad de aislados de Candida spp: la importancia de vigilar también cepas de la comunidad / Species distribution and susceptibility pattern of Candida spp.: the importance to survey also strains isolated from the community

Background: The most of the surveillance studies has been conducted in hospitalized patients with invasive infections. Recently, new clinical breakpoints (CBPs) have been proposed for antifungal susceptibility testing and epidemiological cutoffs (ECVs). Aim: To evaluate species distribution and susceptibility pattern of Candida spp. obtained from in and outpatients in a period of 6 months. Material and Methods: The isolates (n=223) came from vaginal discharge (51.6%), lower respiratory tract (24.7%), urine (20.2%), wounds (1.8%), blood (0.9%), peritoneal fluid (0.4%) and nails (0.4%). Results: The species distribution was C. albicans 84.8% (n: 189), C. glabrata 7.6% (n: 17), C. tropicalis 2.7% (n: 6), C. parapsilosis 2.2% (n: 5), C. kefyr 0.9% (n: 2) and others 1.8% (C. krusei, C. lusitanie, C. guilliermondii, C. intermedia) (n: 4). The susceptibility dose dependence (SDD) and resistance were 3.2% for fluconazole and 2.2% for voriconazole. The most of SDD and resistant strains were isolated from ambulatory patients. Also, a higher percentage of MICs over the new CBPs and ECVs were found in strains from ambulatory patients and especially in C. glabrata isolates to caspofungin. Conclusion: Taking into consideration that most of the invasive infections are caused by strains from the endogenous microbiota, and that there is a resistant population of Candida spp. in the community, should be important to include in surveillance studies strains isolated from ambulatory patients.

Introducción: Los estudios de vigilancia de Candida spp. en general, no incluyen cepas de la comunidad. Recientemente, se han propuesto nuevos puntos de corte clínicos (CBPs) para interpretar la susceptibilidad y puntos de corte epidemiológicos (ECVs), para detectar cepas silvestres o con algún tipo de resistencia. Objetivo: Ainalizar la distribución y perfil de susceptibilidad Candida spp. de pacientes hospitalizados y ambulatorios durante seis meses. Material y Métodos: Las cepas (n: 223) provenían desde flujo vaginal (51,6%), tracto respiratorio bajo (24,7%), orina (20,2%), heridas (1,8%), sangre (0,9%), líquido peritoneal (0,4%) y uñas (0,4%). Resultados: La distribución de especies fue C. albicans 84,8% (n: 189), C. glabrata 7,6% (n: 17), C. tropicalis 2,7% (n: 6), C. parapsilosis 2,2% (n: 5), C. kefyr 0,9% (n: 2) y otras 1,8% (C. krusei, C. lusitanie, C. guilliermondii, C. intermedia) (n: 4). La susceptibilidad dosis dependiente (SDD) y resistencia fueron de 3,2% para fluconazol y 2,2% para voriconazol. La mayoría de las cepas SDD resistentes y fueron ambulatorias. Además, en este grupo, se encontró un alto porcentaje de cepas con CIMs sobre los nuevos CPBs y ECVs, especialmente en aislados C. glabrata para caspofungina. Conclusión: Dado que la mayoría de las infecciones invasoras son causadas por cepas endógenas, y que hay cepas con algún grado de resistencia en la comunidad, estas últimas debieran vigilarse.

Antifungal activity of low molecular weight chitosan against clinical isolates of Candida spp.

Chitosan is a natural polymer derived from chitin, a structural component of fungi, insects and shrimp, which exerts antimicrobial effects against bacteria and fungi. The aim of this study was to investigate the in vitro antifungal activity of low molecular weight chitosan (LMWC), and the potential synergy between chitosan and a currently used antifungal drug, fluconazole. The in vitro minimal inhibitory concentrations (MICs) of chitosan and fluconazole against 105 clinical Candida isolates were measured by the broth microdilution method. LMWC exhibited a significant antifungal activity, inhibiting over 89.9% of the clinical isolates examined (68.6% of which was completely inhibited). The species included several fluconazole-resistant strains and less susceptible species such as C. glabrata, which was inhibited at a concentration of 4.8 mg/l LMWC. Although some strains were susceptible at pH 7.0, a greater antifungal activity of LMWC was observed at pH 4.0. There was no evidence of a synergistic effect of the combination of LMWC and fluconazole at pH 7.0. This is the first report in which the antifungal activity of LMWC was investigated with clinical Candida strains. The use of LMWC as an antifungal compound opens new therapeutic perspectives, as the low toxicity of LMWC in humans supports its use in new applications in an environment of pH 4.0-4.5, such as a topical agent for vulvovaginal candidiasis.