In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions

Abstract

An understanding of biofilm behavior of Candida species under different environmental conditions is key to the development of effective preventive measures for candidal infections. Hence in this study we assessed the impact of the environmental milieu on Candida biofilm formation using polystyrene, flat-bottomed 96-well microtiter plates. A total of 20, comprising 10 clinical isolates each of Candida albicans and, non-albicans species of Candida were compared for their biofilm forming ability both under aerobic and anaerobic conditions, and static and dynamic conditions. XTT reduction assay was used to quantify the sessile growth. Biofilm formation of all 10 C. albicans isolates differed significantly between dynamic and static states under both atmospheric conditions (P < 0.05). For non-albicans Candida species, a significant difference in biofilm growth between dynamic and static states was noted only when incubated aerobically (P < 0.05), and no significant difference in biofilm formation was noted between aerobic and anaerobic conditions. Scanning electron microscopy revealed that C. albicans produced a compact multilayered biofilm embedded in noticeably higher quantity of extracellular polymeric matrix in aerobic/dynamic conditions compared with anaerobic/static conditions. Our data indicate that biofilm formation of C. albicans and non-albicans Candida species is modulated by hydrodynamic conditions and ambient oxygen gradients. However, further work is required to fully elucidate how Candida biofilms persist within the oral milieu under such challenging ecological pressures.

Introduction

The yeast pathogen Candida, the most prevalent fungal species in the human oral cavity, has the ability to grow under diverse environmental conditions. Their conversion from commensalism to parasitism and exuberant growth is usually associated with intraoral environmental changes (e.g. unhygienic prostheses, xerostomia) and/or systemic factors such as diabetes and immunodeficiency.¹ A classic example of fungal biofilms in the oral cavity is the denture plaque in Candida-associated denture stomatitis.² Here, the yeast appears to thrive and proliferate in the space between the denture and the mucosa in a milieu low in environmental oxygen.³ Furthermore, recent clinical reports indicate candidal growth and persistence within root canal systems causing polymicrobial infections⁴ and, in periodontal pockets.⁵ These eco-niches are necessarily anaerobic and the growth of Candida species under such conditions has not been well studied.

The phenomenon of biofilm formation by microbes on inert surfaces has been extensively studied in the last decade and there appear to be a direct relationship between the ability of the organisms to form a biofilm and its pathogenicity.⁶ Further, it is thought that the nature of biofilm architecture favors the lowest oxygen levels near the central core area⁷ although how such conditions modify the biofilm growth is yet unclear, particularly for Candida species. A few workers have investigated the growth of Candida under anaerobic conditions with conflicting results.⁸ In early studies, Samaranayake et al.⁹ found faint, yet discernible surface growth of Candida albicans on Sabouraud's dextrose slopes under strict anaerobic incubations. In a later study, Webster and Odds,¹⁰ demonstrated variable anaerobic growth of seven Candida species including C. albicans, except for C. guilliermondii and C. parapsilosis which were dormant, under anaerobiosis. Very recently, Biswas and Chaffin¹¹ attempted to develop C. albicans biofilms anaerobically and found that static or mild shaking conditions did not support such fungal biofilm growth on plastic surfaces or denture acrylic. Apart from the foregoing, there are no detailed studies on the biofilm development of C. albicans and other non-albicans Candida species under anaerobic or static/dynamic conditions. Therefore, the main aim of this study was to evaluate quantitatively the effect of aerobic/anaerobic as well as static/dynamic conditions on biofilm formation of C. albicans and six different non-albicans species of Candida namely, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, and C. kefyr. The opportunity was further taken to evaluate the biofilm ultrastructure of C. albicans and C. glabrata (one strain each) under the foregoing environmental conditions using scanning electron microscopy.