A new approach by optical coherence tomography for elucidating biofilm formation by emergent Candida species

Abstract

The majority of microorganisms present a community lifestyle, establishing biofilm ecosystems. However, little is known about its formation in emergent Candida species involved in catheter-related infections. Thus, various techniques may be used in the biofilm detection to elucidate structure and clinical impact. In this context, we report the ability of emergent Candida species (Candida haemulonii, C. lusitaniae, C. pelliculosa, C.guilliermondii, C. famata and C. ciferrii) on developing well structured biofilms with cell viability and architecture, using optical coherence tomography (OCT). This new approach was compared with XTT analyses and Scanning Electron Microscopy (SEM). A positive correlation between oxidative activity (XTT) and OCT results (r = 0.8752, p < 0.0001) was observed. SEM images demonstrated cells attachment, multilayer and morphologic characteristics of the biofilm structure. C. lusitaniae was the emergent species which revealed the highest scattering extension length and oxidative metabolism when evaluated by OCT and XTT methods, respectively. Herein, information on C. ciferri biofilm structure were presented for the first time. The OCT results are independently among Candida strains and no species-specific pattern was observed. Our findings strongly contribute for clinical management based on the knowledge of pathogenicity mechanisms involving emergent yeasts.

Fig 1. Oxidative activity biofilm for emergent Candida strains developed at 24 and 48 hours.

Data represent the mean and standard deviation (SD) of the XTT absorbance during biofilm production in two independent experiments with at least three replicates (n≥ 6). For the analysis, Tukey's multiple comparisons test was performed for all averages obtained at the 5% level of significance. Different capital letters indicate significant difference in biofilm production in relation to time (24 and 48 hours) for a single Candida isolate. Different lowercase letters indicate significant difference in biofilm production among Candida isolates. The "#" symbol represents the isolates that have excelled in biofilm production in relation to the others, but they do not differ each other.

Fig 2

Optical coherence tomography indicating the the extension of changes in the sample structure due to the presence of emergent yeast in catheter discs: (A) Candida guilliermondii, (B) C. pelliculosa, (C) C. haemulonii, (D) C. lusitaniae, (E) C. famata, (F) C. ciferri and (G) C. albicans ATCC 90028. The control (H) is disc free of biofilm.

Fig 3. Ample structure changes and metabolism of emergent Candida biofilms observed by optical coherence tomography (OCT) and oxidative activity (XTT).

The results correlation shows positive distribution (Pearson correlation test, r = 0.8752, p < 0.0001).

Fig 4

Scanning electron microscopy of 24h (A1-G1) and 48h (A2-G2) biofilms formed on catheter discs for emergent Candida strains. Candida guilliermondii (A1, A2), C. pelliculosa (B1, B2), C. haemulonii (C1, C2), C. lusitaniae (D1, D2), C. famata, (E1, E2) and C. ciferri (F1, F2). The control (G1, G2) is C. albicans ATCC90028. Arrow indicates the presence of the extracellular matrix within the biofilm. Magnification x4.000.