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.3–5C. 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.6–8 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.1,9–13C. 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.7,14–16

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.

All tested isolates of C. albicans and C. dubliniensis produced biofilm on polystyrene (Tables 1 and 2 and Fig. 1). However, a great variability in biofilm production was observed in both species. The biofilm metabolic activities (A492nm) of C. albicans isolates at 24h 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 24h biofilms than in oral isolates (0.955±0.379 vs. 0.805±0.328, respectively, P=0.28). A similar pattern was observed for C. dubliniensis (0.763±0.195 vs. 0.634±0.192, respectively, P=0.17).

Fig. 1.

Distribution of Candida albicans and Candida dubliniensis isolates on the basis of their capability for producing biofilms at 37°C for 24h (A) and 48h (B).

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Biofilm metabolic activities of C. albicans isolates at 48h were higher than at 24h (0.945±0.378 vs. 0.869±0.352, respectively, P=0.44) with independence of the origin of these isolates (blood vs. oral isolates: 1.007±0.401 vs. 0.900±0.366, respectively, P=0.48). However, at 48h the biofilms of oral isolates of C. dubliniensis showed more metabolic activity (0.640±0.163) than the biofilms of blood isolates (0.522±0.198) (P=0.19) (Table 2 and Fig. 1B). Most C. dubliniensis isolates belonged to genotype I, except isolates 00-133 and 00-135, which were from the genotype II (Fig. 1). There were no statistically significant differences in biofilm production between both genotypes.

C. albicans included a non-statistically significant higher number of isolates classified as high producers of biofilm than C. dubliniensis (P=0.86) at 48h. Ten out of 28 C. albicans isolates (35.7%) were classified as high producers of biofilm at 24h. Of these, 5 were from blood (41.7% of the blood isolates) and 5 from the oral cavity (31.3% of the oral isolates) (P=0.86). This category of high producers of biofilm only grouped 2 out of 19 C. dubliniensis isolates (10.5%) at 24h, but this difference between isolates of both species was not statistically significant (P=0.109). However, at 48h, 10 out of 28 C. albicans isolates (35.7%) and 0 out of 19 C. dubliniensis isolates were classified as high producers of biofilm (P=0.01). At this incubation time, 5 out of 12 (41.7%) blood isolates of C. albicans were high producers of biofilm in comparison to 5 out of 16 oral isolates (31.3%) (P=0.86).

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. dubliniensis oral and blood isolates develop biofilms on polystyrene plates. We used the colorimetric method based on the XTT reduction in the current study because it correlates well with other quantitative methods, such as ATP or CFU assays used in cross-comparison of species variation in biofilm production.29 The XTT method is more versatile and less-time consuming, and is also particularly suited for assays using microtitre plates that allow the screening a large number of isolates.

Shin et al.28 observed that a very low percentage of isolates of C. albicans produced biofilm, regardless of the invasive (bloodstream) or non-invasive origin of the isolates (8% vs. 7%). Conversely, the biofilm production was high when isolates from other species were tested, and those from blood and deep tissues were statistically significant higher producers than those isolates from other clinical specimens. Kumar and Menon,30 using the same medium and method for biofilm production, reported that 11 out of 18 C. albicans isolates (61%) were biofilm producers. Three out of 7 bloodstream isolates (43%) produced biofilm. Non-C. albicans isolates were higher producers of biofilm than those of C. albicans. These authors included 5 reference strains of C. dubliniensis, observing that all of them produced biofilm. In contrast, other authors have reported that isolates of C. albicans produced statistically significantly more biofilm than other Candida species.27,29,31–34 All isolates in our study showed a variable capability to produce biofilm, being distributed into 3 categories (from high to low producers of biofilm) considering their metabolic activities, and those isolated from the bloodstream were non-statistically significantly higher producers of biofilm than oral ones.

These discrepancies could be associated with differences in the methodology used, to the potential variability associated to the geographic origin of the isolates, or to other isolate-associated factors. Shin et al.28 and Kumar and Menon30 used a Sabouraud dextrose broth, and a RPMI-based broth was used in our comparison. Sabouraud dextrose broth with a high content of glucose (8%) has been claimed to simulate the hyperglycaemic milieu found in those patients receiving total parenteral nutrition.28,30 However, RPMI-based broth is a better promoter of filamentation than Sabouraud dextrose broth,35 a fact that is essential for the first step (adhesion) of biofilm production and can be influenced by different cultural and host factors.36–38 This influence of the RPMI in germ tube and hypha development could explain the production of biofilm by all the C. albicans and C. dubliniensis isolates tested in the current study.

The geographical origin of isolates in the different studies can influence the biofilm production capability, as has been observed for other phenotypic traits of Candida isolates, such as virulence factors or in vitro antifungal susceptibilities.29,39 This can be another potential explanation of the differences encountered, as Shin et al.28 studied isolates from Korea, and Kumar and Menon30 from India, while in the current study all C. albicans isolates were from Spain. Li et al.,40 studying Canadian isolates of C. albicans, reported that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variations in biofilm formation. Something similar was described by Borecka-Melkusova and Bujdakova41 in C. albicans and C. dubliniensis isolates from Slovakia. Thein et al.29 also reported that growth and virulence of the different species of Candida were highly dependent on the isolates chosen for each study, hence it is important to use multiple clinical isolates and strains for the same species to draw firm conclusions in this regard.

There was a high variability in the biofilm production among C. albicans isolates when the interspecies variation of C. albicans and C. dubliniensis isolates was studied. This variability was lower among C. dubliniensis isolates. Kuhn et al.42 observed that XTT results can vary in accordance with the different sensitivity of C. albicans and Candida parapsilosis to tetrazolium salts. C. dubliniensis is more closely related to C. albicans than Candida parapsilosis and probably has a similar sensitivity to XTT than C. albicans. Nevertheless, we found that both Candida species, C. albicans and C. dubliniensis, exhibited good biofilm forming capability on polystyrene surfaces under aerobic conditions. These findings are in agreement with those of Ramage et al.24 who also reported that C. dubliniensis exhibited good biofilm growth on the surface of polystyrene plates.

One of the key morphogenesis factors for the phase transition in these dimorphic fungi is the nature of the environment, as the hypha growth is promoted by low oxygen and, nutrient starvation, as occurs in this aerobic and static conditions assay.29 Such a phase transition is dependent upon strain as well as species characteristics, as shown here by the CA-2 hypha-deficient strain of C. albicans, which always exists in the yeast phase, irrespective of its environmental milieu. Both species formed heterogeneous biofilms with strain variations in their morphology and XTT metabolic activity, as has been described previously by Henriques et al.32

In conclusion, there are important differences in biofilm production by C. albicans and C. dubliniensis isolates. These differences should be taken into account when the anti-biofilm activity of antifungal agents or other virulence factors are tested in vitro. This capability for biofilm development may enable C. albicans and C. dubliniensis to maintain their oral ecological niches as commensal microorganisms and can be a major virulence factor during invasive candidiasis with important clinical repercussions.

The authors have no conflict of interest to declare.