Original article
Variation in biofilm formation among blood and oral isolates of Candida albicans and Candida dubliniensisEvaluación de la capacidad de formación de biopelícula de aislamientos clínicos de Candida albicans y Candida dubliniensis

https://doi.org/10.1016/j.eimc.2011.06.009Get rights and content

Abstract

Introduction

Biofilm production is considered a potential virulence factor of some Candida species. For this reason, an understanding of biofilm behavior of Candida albicans and its closely related species Candida dubliniensis is key to the development of effective preventive measures for invasive and oral candidiasis. The aim of this study was to compare the capacity of biofilm production by blood and oral isolates of C. albicans and C. dubliniensis using polystyrene, flat-bottomed 100-well microtiter plates.

Methods

A total of 47 isolates, consisting of 28 C. albicans (16 oral and 12 blood isolates) and 19 C. dubliniensis (11 oral and 8 blood isolates) were compared for their biofilm forming ability under aerobic and static conditions. XTT reduction assay was used to quantify the sessile growth.

Results

All tested isolates produced biofilm, measured as XTT metabolic activity. Biofilm formation by C. albicans isolates was statistically significantly higher than biofilm formation by C. dubliniensis isolates at 24 h (P = 0.03) and 48 h (P = 0.0001). There was a higher percentage (41.7%) of high producers of biofilms among C. albicans blood isolates than among oral isolates (31.3%), without statistically significant differences.

Conclusions

This capability may allow C. albicans and C. dubliniensis to maintain their oral ecological niches as commensal or pathogenic microorganisms and can be a major virulence factor during invasive candidiasis. However, the differences in biofilm production among isolates should be taken into account when the anti-biofilm activity of antifungal agents or other virulence factors are tested in vitro.

Resumen

Introducción

El desarrollo de biopelículas o biocapas se considera un factor de virulencia potencial de algunas especies de Candida. Una comprensión mejor del comportamiento de las biopelículas de Candida albicans y de la especie cercana Candida dubliniensis es clave para desarrollar medida preventyivas eficaces de las candidiasis superficiales (orales) e invasoras. El objetivo de este trabajo ha sido comparar la capacidad de producir biopelícula por parte de los aislamientos orales y de sangre de C. albicans y C. dubliniensis en placas de microtitulación de poliestireno de 100 pocillos con fondo plano.

Métodos

Se estudiaron 47 aislamientos: 28 C. albicans (16 orales y 12 hemáticos) y 19 C. dubliniensis (11 orales y 8 hemáticos). Se empleó una prueba de cuantificación de la actividad metabólica de las biopelículas (reducción de la sal de tetrazolio denominada XTT).

Resultados

Todos los aislamientos mostraron actividad metabólica pero la formación de biopelícula por los aislamientos de C. albicans era significativamente mayor que por los de C. dubliniensis a 24 h (P = 0,03) y 48 h (P = 0,0001). Eran más numerosos, los aislamientos de C. albicans muy productores de biopelícula procedentes de sangre (41.7%) que de boca (31.3%) pero las diferencias no eran significativas.

Conclusiones

La capacidad de desarrollar biopelículas, podría permitir a C. albicans y C. dubliniensis mantenerse en el nicho oral como comensales o patógenos y ser un importante factor de virulencia en las candidiasis invasoras. Sin embargo, las diferencias encontradas entre los aislamientos productores de biopelícula deben tenerse en cuenta, sobre todo en los estudios in vitro de virulencia o de la acción anti-biopelícula de los fármacos antifúngicos.

Introduction

Invasive candidiasis represents about 10% of nosocomial invasive infections. Although other species, such as Candida parapsilosis, Candida glabrata or Candida tropicalis, are being isolated with increasing frequency; Candida albicans is the most frequent aetiological agent of candidiasis.1, 2 Many candidiasis are associated with prostheses, catheters and other indwelling medical devices, where these microorganisms develop biofilms. The presence of extracellular polymers and a different cellular phenotype, called sessile is an important feature in the structure of these microbial comunities. Biofilms impede the actions of the immune system and other defence mechanisms and become recalcitrant to current antifungal treatment.3, 4, 5 C. albicans is also an important pathogen of the oral cavity and other mucosae. This yeast has the ability to grow under diverse oral environmental conditions (e.g. unhygienic dentures, xerostomia) and/or systemic factors, such as diabetes and immunodeficiency. The presence of dentures that are overlaid with proteins and other oral components encourages the development of Candida biofilms and denture stomatitis.4

Candida dubliniensis is an emerging species associated to oral candidiasis, mainly in HIV-infected patients.6, 7, 8 The frequence of deep-seated infections caused by C. dubliniensis is low but probably understimated because C. dubliniensis is closely related to C. albicans and many laboratories are not capable of differentiating between both species.1, 9, 10, 11, 12, 13 C. dubliniensis shares many properties with C. albicans and, in addition, shows an important capacity to develop resistance to fluconazole and other antifungal agents under repeated exposure.7, 14, 15, 16

The aim of the current study has been to compare the capacity of biofilm production by blood and oral isolates of C. albicans and C. dubliniensis from infected patients.

Section snippets

Microorganisms

A total of 28 C. albicans (16 oral isolates and 12 blood isolates) and 19 C. dubliniensis (11 oral isolates and 8 blood isolates) from the Laboratorio de Micología Médica at the University of the Basque Country were studied. The clinical origin of the isolates and the infections caused have been described previously.8, 9, 10, 17 Two strains from the National Collection of Pathogenic Fungi (NCPF), C. albicans NCPF 3153 and C. dubliniensis NCPF 3949, as well as C. albicans NCPF 3153

Results

All tested isolates of C. albicans and C. dubliniensis produced biofilm on polystyrene (Table 1, Table 2 and Fig. 1). However, a great variability in biofilm production was observed in both species. The biofilm metabolic activities (A492 nm) of C. albicans isolates at 24 h ranged between 0.291 and 1.506 (mean 0.869 ± 0.352) and those of C. dubliniensis isolates ranged between 0.438 and 1.145 (mean 0.688 ± 0.199) (P = 0.03) (Fig. 1A). C. albicans blood isolates showed a higher metabolic activity in 24 h

Discussion

The phenomenon of biofilm formation by microbes on inert surfaces has been extensively studied in bacteria and to a lesser extent in fungi, and there appears to be a direct relationship between the capability of the organisms to form a biofilm and their pathogenicity. However, few studies have compared biofilm production among C. albicans bloodstream and oral isolates28 and much less, between oral and blood isolates of C. dubliniensis. The current results confirm that most C. albicans and C.

Conflict of interests

The authors have no conflict of interest to declare.

Acknowledgements

María Villar-Vidal and Cristina Marcos-Arias had scholarships from the Universidad del País Vasco-Euskal Herriko Unibertsitatea. We thank José Manuel Aguirre-Urizar, Antonio Cassone, Carmen Rubio, Ricardo Salesa, and Derek Sullivan for kindly providing some of the C. albicans and C. dubliniensis isolates and strains used in this study. This work has been funded in part by projects GIC07 123-IT-222–07 (Departamento de Educación, Universidades e Investigación, Gobierno Vasco), S-PE08UN35 and

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