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.

Full text

RESEARCH ARTICLE
A new approach by optical coherence
tomography for elucidating biofilm formation
by emergent Candida species
Melyna Chaves Leite de Andrade
1
, Marcos Andre Soares de Oliveira
2
, Franz de Assis
Graciano dos Santos
1
, Pamella de Brito Ximenes Vilela
1
, Michellangelo Nunes da Silva
1
,
Danielle Patrı
´cia Cerqueira Macêdo
3
, Reginaldo Gonc¸alves de Lima Neto
4
, Henrique Jonh
Pereira Neves
1
, Ildnay de Souza Lima Brandão
1
, Guilherme Maranhão Chaves
5
, Renato
Evangelista de Araujo
2
, Rejane Pereira Neves
1
*
1Department of Mycology, Federal University of Pernambuco, Recife, Brazil, 2Department of Electronics
and Systems, Federal University of Pernambuco, Recife, Brazil, 3Department of Pharmaceutical Sciences,
Federal University of Pernambuco, Recife, Brazil, 4Department of Tropical Medicine, Federal University of
Pernambuco, Recife, Brazil, 5Department of Clinical and Toxicological Analysis, Federal University of Rio
Grande do Norte, Natal, Brazil
*rejadel@yahoo.com.br
Abstract
The majority of microorganisms present a community lifestyle, establishing biofilm ecosys-
tems. 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 Can-
dida 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 oxida-
tive 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.
Introduction
Several biological mechanisms involved in fungal infections are not yet fully understood.
Therefore, a major concern involves the understanding and characterization of emergent Can-
dida species [1]. The sudden emergence of previously uncommon and apparently harmless
yeast species as agents of invasive candidiasis may be attributed to several factors such as the
PLOS ONE | https://doi.org/10.1371/journal.pone.0188020 November 16, 2017 1 / 12
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OPEN ACCESS
Citation: Leite de Andrade MC, Soares de Oliveira
MA, Santos FdAGd, Ximenes Vilela PdB, da Silva
MN, Macêdo DPC, et al. (2017) A new approach by
optical coherence tomography for elucidating
biofilm formation by emergent Candida species.
PLoS ONE 12(11): e0188020. https://doi.org/
10.1371/journal.pone.0188020
Editor: David R. Andes, University of Wisconsin
Medical School, UNITED STATES
Received: June 27, 2017
Accepted: October 29, 2017
Published: November 16, 2017
Copyright: ©2017 Leite de Andrade et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The author Rejane Neves received
financial support from FACEPE/Brazil to acquire
reagents and the necessary materials and Melyna
Leite-Andrade received a scholarship from CNPq/
Brazil. This study was also funded by CAPES and
the National Institute of Photonics (to REdA). The
funders had no role in study design, data collection

use of medical devices including catheter and biofilm formation [2,3]. In particular, systemic
infections by emergent yeasts as Candida ciferrii,C.famata,C.guilliermondii,C.haemulonii,
C.lusitaniae, and C.pelliculosa, had increased the mortality rate, especially when associated
with severe underlying pathologies and the use of medical devices [4].
Interestingly, the majority of microorganisms survive in nature because of their community
style of life as biofilm ecosystems [5]. Biofilms are structured microbial communities attached
to either biotic or abiotic surface embedded in an exopolymeric matrix constituted mainly by
carbohydrates, hexosamines, uronic acids, proteins and nucleic acids [6]. The biofilm architec-
ture maintenance may be ensured by the formation of canals and columns, which allow the
passage of nutrients and oxygen for the entire microbial community.
The biofilm formation is essential for yeasts protection against host defense mechanisms
and commonly used antifungal drugs. Biofilm formation in implant devices is also an impor-
tant medical concern, leading to clinical failure [7,8]. During the last decades, unusual yeast
species have emerged as agents of invasive candidiasis and strong cause of death [9].
Classically, C.albicans develop highly structured biofilms with multiple cell types as budding
yeast-form cells, pseudohyphae and true hyphae encased in an extracellular matrix. Commonly,
Candida non-albicans biofilms form extracellular matrix but do not produce true hyphae [10].
Thus, its formation is an important feature for yeast virulence, and studies regarding this complex
structure by emergent Candida species are still incipient [11,12]. Various techniques may be
used in the biofilm detection commonly SEM and metabolic activity evaluation by XTT (2,3-Bis-
(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide inner salt) [12,13].
Although rarely applied, imaging techniques may be an option in the detection of fungal biofilm.
Thus, optical coherence tomography (OCT) is a well-established, low-coherence interferometric
technique that performs high-resolution, ultrafast, noninvasive, and cross-sectional tomographic
imaging, This optical technique evaluates interference patterns of backscattering light to build
images, in depth, of biological structures as Candida albicans biofilm [14,15]. In this context, the
purpose of our study was to evaluate the potential use of OCT on analyzing the ability of emer-
gent Candida species (Candida haemulonii,C.lusitaniae,C.pelliculosa,C.guilliermondii,C.
famata and C.ciferrii) to develop well structured biofilms with cell viability and architecture.
Results
Emergent Candida strains
The emergent Candida species included in the present study were as follows: Candida haemu-
lonii (3 strains), C.lusitaniae (3), C.pelliculosa (3), C.guilliermondii (4), C.famata (1) and C.
ciferri (1). All yeast cultures evaluated in this study were previously isolated from critically ill
patients, identified by MALDI TOF-MS and then were kept in the URM Culture Collection,
Pernambuco, Brazil.
Quantitative analyses of biofilms: Oxidative activity and optical
coherence tomography (OCT)
During the oxidative activity with the colorimetric assays based on XTT reduction, we
observed that the emergent Candida strains used in the study were able to form an active bio-
film. Quantitative XTT analyses revealed that mature stages with highest metabolic activities
occurred at 48 hours of incubation (Fig 1).
Fig 1. shows the mean OD 492 nm for each strain, for biofilms formed after 24 and 48
hours of incubation. C.guilliermondii strains did not present a significant variation for biofilm
formation detected by XTT activity, exhibiting a homogenous quantitative pattern. This
Optical coherence tomography and biofilm by emergent Candida species
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and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.

characteristic was not verified among the other emergent strains, such as C.pelliculosa,C.hae-
mulonii and C.lusitaniae, which presented considerable differences in optical densities caused
by XTT activity values for the strains within the same species.
Samples were analyzed using OCT, which exhibited the extension of changes in the cathe-
ters discs. In the OCT images, red shades represent higher scattering areas. Pixel intensity dis-
tributions (A-scan) of selected regions are also presented, indicating that the amplitude of the
scattered light decrease at deeper areas under the biofilm surface (Fig 2). The OCT results are
independently among Candida strains and no species-specific pattern.
The correspondence between the XTT and the measured OCT values are shown in Fig 3.
There was a significant positive correlation between oxidative activity and optical coherence
tomography in biofilm development (Pearson correlation test, r = 0.8752, p<0.0001). Fur-
thermore, correspondence in results were visually demonstrated by SEM through observation
of cells attachment, multilayer and morphologic characteristics (Fig 4).
Qualitative analyses of biofilms: Evaluation of architecture in catheter
discs
During the first 24 hours of incubation, C.lusitaniae strains exhibited intense biofilm forma-
tion, followed by C.famata,C.haemulonii and C.albicans reference strain. However, the other
species (C.ciferri,C.pelliculosa and C.guilliermondii) showed sparse yeast cells adhered to
catheter discs’ surface, typical of initial stage of the biofilm formation. Nevertheless, in 48
hours of exposure, all yeast strains showed a mature biofilm on the surface of the discs.
Discussion
The Candida species evaluated in this study are rarely described in clinical cases of invasive
fungal infections. However, when this condition occurs, these yeasts are commonly isolated
from the bloodstream of patients with poor prognostic [16–17].
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.
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Optical coherence tomography and biofilm by emergent Candida species
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The fact that these emergent yeast species are able to form biofilms may be clinically rele-
vant. In fact, Tumbarello et al. [18] found that patients with candidemia caused by non-bio-
film-forming Candida spp. strains had better outcomes. In contrast, the biofilm-forming
strains showed a significant association with poor prognoses.
In addition, Iturrieta-Gonzalez et al. [19] described that Trichosporon, another emergent
yeast genus, showed levels of biofilm formation similar or greater than those described for
Candida spp. and that biofilm-forming cells were at least 1,000 times more resistant to antifun-
gals than their planktonic counterparts. These data reinforce the likely importance of biofilm
formation by the rare Candida species used in the present study.
The evaluation using the XTT reduction and the biofilms images demonstrated the meta-
bolic viability inside matrix in all tested yeasts, with significant differences between species and
within different strains of the same species. In contrast, C.guilliermondii isolates showed no
significant intra-specific variations, demonstrating a homogenous pattern of viability for ses-
sile cells present in the biofilms. Interestingly, the high XTT OD 492nm obtained for C.lusita-
niae (URM 1812) biofilm is in agreement with the literature because emergent yeasts may
form viable multicellular structures in inert surfaces as catheters and invasive devices or colo-
nize valves in humans [12].
Based on light scattering, OCT images showed the extension of changes inside the catheter
structure, indicating the pathogenicity of these emerging yeast species. A clear image
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.
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difference can be observed among the samples with and without (control) biofilm (Fig 2). The
presence of the biofilm enhances light scattering of the sample, increasing the brightness of
undersurface areas of the OCT biofilm images. Scattering can be attributed to the biofilm
microstructure on the catheter. Thus, it was observed that the OCT values are higher for those
isolates with the high XTT activity. Few studies have been developed using these techniques to
clarify structure and metabolism of Candida biofilms [12,15]. This is the first study to explore
OCT technique to evaluate emergent clinical yeasts. The OCT technique offers advantages
once it clearly proved to be non-invasive, real-time, privileging in-situ analysis of biofilm lay-
ers. Moreover this biofilm keep its original structure without the performance of a destructive
process enabling the assessment of biofilm roughness and surface area [20].
Through of the scanning electron microscopy, it was visually verified that all biofilm forma-
tion stages occurred in the yeasts tested within 48 hours. Thus, with a significant increase of
cell proliferation with amorphous material, representing the extracellular matrix and featuring
a mature biofilm. Interestingly, rare yeast cells were adhered to the surface of the catheter disc
during the first 24h in C.ciferri,C.pelliculosa and C.guilliermondii.C.lusitaniae (URM 1812)
showed intense cell adhesion at this time of evaluation. This species showed a higher number
of yeast cells adhered to the discs and intercellular adhesion. In addition, we observed in
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).
https://doi.org/10.1371/journal.pone.0188020.g003
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Optical coherence tomography and biofilm by emergent Candida species
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scanning electron microscopy that the biofilm of all species was composed of blastoconidia,
but did not present filamentous forms. All emergent Candida biofilms appeared as discontinu-
ous layers of blastoconidia anchored to the surface and rich in extracellular matrix but not
hyphae, which was in accordance with the findings of Silva et al. [21] after evaluating C.glab-
rata,C.parapsilosis and C.tropicalis biofilm. As demonstrated by Chandra et al. [22] both
RPMI as YNB media are used to form well-structured biofilms, however YNB enhances bud-
ding reproduction and RPMI stimulates filamentation. Their data indicate that biofilm growth
was not morphology specific.
Sparse investigations upon C.albicans biofilm architecture can be found in the literature
[23]. However, currently, there are no studies characterizing the structures of some emergent
yeasts. Moreover, we observed that C.ciferrii developed a classic biofilm, with cell viability,
indicating the pathogenic potential of this species. Then, with microscopy, we confirmed that
in addition to C.albicans, which was used in the trials as control species, emerging Candida
species are also able to form biofilms in their entirety, showing the architecture in all stages. In
addition, the chemical nature of the catheter, a material made up of polystyrene, polyurethane
or polyvinyl chloride enable biofilm formation [24].
The knowledge of pathogenicity mechanisms of yeast biofilms is crucial for the develop-
ment of new antifungal therapies and diagnostic strategies.
Materials and methods
Candida strains
Fifteen Candida clinical isolates obtained from the Micoteca URM Culture Collection, Medical
Mycology Laboratory (MML) from Federal University of Pernambuco and Laboratory of
Medical and Molecular Mycology (UFRN), Federal University of Rio Grande do Norte were
analyzed in this study. In addition, C.albicans ATCC 90028 was used as reference strain. The
strain descriptions are summarized in Table 1.
Emergent Candida species identification by MALDI-TOF MS
Homogenous inoculum of yeast cells were grown and maintained on Yeast Extract Peptone
Dextrose Agar medium (YEPD). Incubations were performed at 20h and strains were grown
aerobically at 37˚C according to Lima-Neto et al. [25]. In order to avoid changes in the protein
expression pattern, the culture conditions and growth time were standardized as described
above. One single colony was directly deposited onto a 196- position target plate (Bruker Dal-
tonik GmbH), in duplicate for each strain.
Aliquots of 1μL of 70% formic acid were added and mixed gently with yeasts. When
the liquid medium was almost evaporated, the preparation was overlaid with 1μL of satu-
rated matrix solution {75mg/ml of α-cyano-4-hydroxycinnamic acid (CHCA) in ethanol/
water/acetonitrile [1:1:1] with 0.03% trifluoroacetic acid (TFA)}. The isolates were depos-
ited per plate in duplicate, and the matrix-sample was crystallized by air-drying at 25˚C
for 5 minutes.
The equipment used was MALDI TOF Autoflex III Mass Spectrometer (Bruker Daltonics
Inc., USA/Germany) composed of a Nd:YAG (neodymium-doped yttrium aluminium garnet;
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.
https://doi.org/10.1371/journal.pone.0188020.g004
Optical coherence tomography and biofilm by emergent Candida species
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Nd:Y3Al5O12) laser of 1064nm, set to a 66% power. The mass range from 2,000 to 20,000 Da
was recorded using a linear mode with a delay of 104ns and an acceleration voltage of +20 kV.
The resulting peak lists were exported to the software MALDI Biotyper™3.1 (Bruker Daltonics,
Bremem, Germany) where the final identifications were achieved.
Quantitative analyses of biofilms
Inoculum and biofilm development for oxidative activity. Yeast strains were cultured
aerobically at 37˚C for 18h on Sabouraud Dextrose Agar (SDA) and then inoculated in Yeast
Nitrogen Base (YNB) broth (Difco Laboratories, Detroit, MI, USA) supplemented with 50mM
glucose. After 18h of incubation, cells were harvested, washed twice with PBS (pH 7.2) and
resuspended in YNB supplemented with 100mM glucose. Candida strains suspensions were
prepared to a concentration of 107 cells/mL evaluated using a spectrophotometer Genesis 10S
UV-Vis (Thermo Scientific) at 530nm, corresponding to 80% transmittance.
Candida species biofilm formation was performed as described by Silva et al. [13]. Briefly,
biofilms were grown in commercially available pre-sterilized, polystyrene, flat-bottomed
96-well microtiter plates (TPP; Trasadingen, Switzerland). Aliquots of 100μL of standard cell
suspensions of yeasts (107 cells/mL) were transferred into each well and incubated for 1.5h
(adhesion phase) at 37˚C at 75rpm. After the adhesion phase, cell suspensions were gently
aspirated and each well was washed twice with PBS to carefully remove any remaining plank-
tonic cells. In order to allow the growth of biofilm (biofilm phase), 200μL of YNB supple-
mented with 100mM glucose was added to each well. The plates were incubated for 24 and
48h at 37˚C at 75rpm in a shaking TE-424 (Tecnal). After 24h incubation, the medium was
aspirated and, biofilms were washed twice with PBS followed by addition of 200μL of YNB
medium. All assays were performed in triplicate.
Table 1. Descriptions of the strains analyzed in the study obtained in the collection of culture URM,
Laboratory of Medical Mycology (MML) of the federal university of pernambuco and laboratory of
Medical and Molecular Mycology (UFRN), federal university of Rio Grande do Norte.
Microorganism Strain number Substrate of origin
C.ciferri 02UFRN Blood
C.famata 05MML Blood
C.guilliermondii 39MML Blood
C.guilliermondii 63MML Blood
C.guilliermondii 129MML Blood
C.guilliermondii 825MML Blood
C.haemulonii 123MML Blood
C.haemulonii 347MML Blood
C.haemulonii URM6554 Nail
C.lusitaniae 01UFRN Blood
C.lusitaniae URM1812 -
C.lusitaniae URM2101 -
C.pelliculosa URM6345 Blood
C.pelliculosa URM6346 Blood
C.pelliculosa URM6384 Blood
C.albicans ATCC 90028*-
-: Substrate of origin uninformed.
*Reference strain
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Oxidative activity assay. The 2,3—Bis—(2—Methoxy—4—Nitro—5—Sulfophenyl) - 2H
- Tetrazolium—5 Carboxanilide (XTT) (Sigma-Aldrich Corp.) was dissolved in PBS at a final
concentration of 1mg/mL.
The solution was filter-sterilized and stored frozen at -70˚C until use. Menadione solution
(0.4mM; Sigma-Aldrich Corp.) was prepared immediately before each assay. For each assay,
XTT solution was thawed on ice and mixed with menadione solution at a volume ratio of 20:1.
Biofilms were washed twice with 200μL of PBS to remove no adherent cells.
Subsequently, 158μL of PBS with or without glucose at different concentrations, 40μL of
XTT and 2μL of menadione were transferred to each well of 96-well plates. The plates were
covered with aluminum foil and incubated in the dark at 37˚C for 3h. Thereafter, 100μL of the
solution was transferred to each well of new 96-well plates. The colorimetric changes were
measured at 492nm wavelength using a microtiter plate reader (Spectra-MAX 340; Molecular
Devices Ltd., Sunnyvale, CA, USA).
Inoculum and biofilm development for optical coherence tomography (OCT) assay.
The evaluation of biofilm formation stages was followed using catheter discs. The tests for bio-
film formation were developed according Vandenbosch et al. [26]. The strains were grown on
Sabouraud dextrose at 37˚C for 16h, and then were centrifuged and the supernatant removed
for washing the cells with a 0.85% saline solution. Cells were resuspended in 1ml saline solu-
tion (Novolab, Geraardsbergen, Belgium) and inocula were subsequently diluted in YNB
medium supplemented with 50mM glucose to obtain an optical density of 0.07 at 600nm.
After standardization of fungal inoculum, 1ml of a 1:100 dilution in YNB was added to each
well containing catheter discs. The microtiter plates were incubated for 1h at 37˚C. Subse-
quently, the discs were washed (three times) with 1mL saline solution to remove non-adherent
cells aseptically transferred to a new well. Subsequently, 1mL of diluted YNB was added with a
final glucose concentration of 0.2mM, and plates were incubated for 24h and 48h at 37˚C for
further analysis by OCT.
Optical coherence tomography imaging analysis. Analyzes were performed using OCT
technique based on the evaluation of the light scattered by the object, and provides in-depth
information of the structures. A 2D image was obtained by the combination of depth-resolved
back-scatter light intensity profiles (A-scans) along the section of interest on the sample. In
this work, a commercial spectral optical coherence tomography system (Ganymede from
Thorlabs Inc.) was used. The image system exploits a super luminescent diode as light source,
emitting infrared light, with wavelength centered at 930nm and spectral width of 100nm. With
an A-scan rate of 29kHz, this system can produce 29 frames per second with 512 lines per
frame and an axial resolution of 5 micrometers. A theoretical model based on an exponential
decay fitting is used to describe light intensity distribution (A-scan) in the sample. The light
scattering depth of the biofilm (OCT extension value) was determined, and quantified as the
inverse of the exponential decay constant of the theoretical fitting.
Qualitative analyses of biofilms
Evaluation of architecture in catheter discs by scanning electron microscopy. The inoc-
ulum preparations and biofilm formation were performed as prior described in OCT assays
according to Vandenbosch et al. [26]. The biofilms formed on the catheter discs were fixed in
2.5% glutaraldehyde in 0.1M Sodium Cacodylate buffer for two hours. After fixation, the disks
were washed with 0.1M Cacodylate buffer three times for 10 minutes for removal of the entire
fixative.
Subsequently, post-fixation was performed in a ratio of 1:1 osmium 2% + 0.1M Cacodylate
buffer for 30min, allowing a higher contrast of the material.
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Two washes were carried out for 10min in 0.1M Cacodylate buffer and distilled water. Sub-
sequently, the samples were submitted to serial dehydration with 30%, 50%, 70%, 90% and
100% acetone for 5 minutes and dried at the critical point. The material was assembled in
metal stubs containing carbon tape and silver ink (which serve as an electrons conductor) and
then metallized by bombarding with gold, and analyzed using a FEI (Quanta 200 FEG). The
processing of the samples followed the proposed protocol Duarte et al. [27].
Ethics statement
The Candida isolates were obtained after review board approval to use the samples from the
Micoteca URM Culture Collection, Medical Mycology Laboratory (MML) from Federal Uni-
versity of Pernambuco and Laboratory of Medical and Molecular Mycology (UFRN), Federal
University of Rio Grande do Norte. All Candida samples were anonymized in accordance to
the ethics statement.
Statistical analysis
Assays were carried out in triplicate for each strain. Statistical analysis was calculated using
GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) software. The results from
the XTT colorimetric assay were statistically evaluated by analysis of variance with ANOVA
and Tukey’s test, with significance level p<0.05. The presented OCT average values and the
corresponding standard deviations were obtained by the evaluation of 75 A-scan of each
image. The relationship between quantitative analyses of biofilms was evaluated by the Pearson
correlation test (p0.05).
Acknowledgments
The authors would like to thank the Department of Mycology from Federal University of Per-
nambuco, for their technical support and the Technological Development Program in Materi-
als for Health (PDTIS) of FIOCRUZ, for the Electron Microscopy Service from Technology
Platforms Nucleus/Aggeu Magalhães Research Center (FIOCRUZ-PE), CNPq and CAPES for
financial support.
Author Contributions
Conceptualization: Melyna Chaves Leite de Andrade, Rejane Pereira Neves.
Data curation: Melyna Chaves Leite de Andrade, Rejane Pereira Neves.
Formal analysis: Henrique Jonh Pereira Neves, Ildnay de Souza Lima Brandão.
Funding acquisition: Melyna Chaves Leite de Andrade, Rejane Pereira Neves.
Investigation: Melyna Chaves Leite de Andrade, Marcos Andre Soares de Oliveira, Franz de
Assis Graciano dos Santos, Pamella de Brito Ximenes Vilela, Michellangelo Nunes da Silva,
Reginaldo Gonc¸alves de Lima Neto, Guilherme Maranhão Chaves, Renato Evangelista de
Araujo.
Methodology: Melyna Chaves Leite de Andrade, Marcos Andre Soares de Oliveira, Renato
Evangelista de Araujo, Rejane Pereira Neves.
Project administration: Danielle Patrı
´cia Cerqueira Macêdo, Rejane Pereira Neves.
Resources: Melyna Chaves Leite de Andrade, Reginaldo Gonc¸alves de Lima Neto, Renato
Evangelista de Araujo, Rejane Pereira Neves.
Optical coherence tomography and biofilm by emergent Candida species
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Software: Henrique Jonh Pereira Neves, Ildnay de Souza Lima Brandão.
Supervision: Rejane Pereira Neves.
Validation: Melyna Chaves Leite de Andrade, Danielle Patrı
´cia Cerqueira Macêdo, Reginaldo
Gonc¸alves de Lima Neto, Renato Evangelista de Araujo, Rejane Pereira Neves.
Visualization: Melyna Chaves Leite de Andrade, Franz de Assis Graciano dos Santos, Pamella
de Brito Ximenes Vilela, Michellangelo Nunes da Silva.
Writing – original draft: Melyna Chaves Leite de Andrade, Danielle Patrı
´cia Cerqueira
Macêdo, Reginaldo Gonc¸alves de Lima Neto, Rejane Pereira Neves.
Writing – review & editing: Melyna Chaves Leite de Andrade, Danielle Patrı
´cia Cerqueira
Macêdo, Reginaldo Gonc¸alves de Lima Neto, Guilherme Maranhão Chaves, Renato Evan-
gelista de Araujo, Rejane Pereira Neves.
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