- Email: [email protected]
In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells
Accepted Manuscript In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells Francieli Capote-Bonato, Karina Mayumi Sakita, Admilton Gonçalves de Oliveira, Junior, Patrícia de Souza Bonfim-Mendonça, Leandro Zuccolotto Crivellenti, Melyssa Negri, Terezinha Inez Estivalet Svidzinski PII:
S0882-4010(18)30774-5
DOI:
10.1016/j.micpath.2018.09.029
Reference:
YMPAT 3179
To appear in:
Microbial Pathogenesis
Received Date: 2 May 2018 Revised Date:
14 August 2018
Accepted Date: 15 September 2018
Please cite this article as: Capote-Bonato F, Sakita KM, de Oliveira Junior AdmiltonGonç, BonfimMendonça PatríSouza, Crivellenti LZ, Negri M, Estivalet Svidzinski TI, In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells, Microbial Pathogenesis (2018), doi: https:// doi.org/10.1016/j.micpath.2018.09.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro interaction of Candida tropicalis biofilm formed on catheter with human
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cells
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Francieli Capote-Bonato1, Karina Mayumi Sakita1, Admilton Gonçalves de Oliveira
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Junior2, Patrícia de Souza Bonfim-Mendonça1, Leandro Zuccolotto Crivellenti3,
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Melyssa Negri1, Terezinha Inez Estivalet Svidzinski1*.
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Department of Clinical Analysis (DCA), State University of Maringá, Paraná, Brazil
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Department of Microbiology (DM), State University of Londrina, Paraná, Brazil
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Department of Animal Science (DAS), Franca University, São Paulo, Brazil
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* Corresponding author: Terezinha Inez Estivalet Svidzinski
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Divisão de Micologia Médica – Departamento de Análises Clínicas e Biomedicina –
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Universidade Estadual de Maringá – Paraná - Brazil. Av. Colombo, 5790, CEP: 87020-
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900, Maringá, PR., Brazil. Phone: +55 44 3011-4809. Fax: +55 44 3011-4860. E-mail:
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[email protected]
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Abstract
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Candida tropicalis has emerged as one of the major Candida non-C. albicans
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species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis
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pathogenic mechanism has yet not been fully defined. The current study aimed to
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demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on
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catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms
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were produced on small catheter fragments (SCF) and were mainly composed by
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blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces
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(HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of
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SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous
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form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of
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blastoconidia involved by extracellular matrix. Total biomass and metabolic activity
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from the remaining biofilm on SCF surface, after direct contact with human cells,
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exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular
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matrix components, after contact with human cells. Thus, we described for the first time
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an easy and simple in vitro model with catheter, which could be a powerful tool for
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future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.
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Keywords
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Candida tropicalis; virulence; catheter; biofilm; yeast-hyphal transition.
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1. Introduction
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In the latest decades, the number of opportunistic fungal infections caused by
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Candida species remains high in humans [1]. Candidemia is the main invasive fungal
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disease among hospitalized patients and the frequency of Candida non-C. albicans
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(CNCA) species as causative agent has increased [2]. For this reason, current research
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interest involves the understanding and characterization of emerging CNCA species.
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In terms of epidemiology and virulence, C. tropicalis appears as the second most
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frequently isolated species of CNCA in South America and Asia and the third most
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commonly isolated in the USA and Europe [3–7]. In particular, infections by C.
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tropicalis are frequent in cancer patients and associated with a higher mortality rate [8].
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An important step for a successful colonization and infection depends essentially
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on the ability of the pathogen to adhere and form biofilm to host surfaces [9–11].
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Biofilm is a microbial community attached to biological or inert surfaces, such as
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medical devices, embedded by extracellular polymeric substances, composed by
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carbohydrates, hexosamines, uronic acids, proteins and nucleic acids [12,13]. Biofilms
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are assumed to be the most important virulence factor for pathogenicity in Candida
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species [12]. The ability to produce biofilms is developed by microorganisms in order to
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protect themselves against host defense mechanisms and antifungal drugs. Therefore,
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biofilm formation plays an important role as a virulence factor, which contributes to the
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pathogenesis of candidiasis. Most of our knowledge related to fungal biofilm formation
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is based on studies carried out in C. albicans. Recently, investigations showed that C.
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tropicalis is a strong biofilm producer [9,14]. Despite that, several biological
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mechanisms involved in C. tropicalis biofilm formation are not yet fully understood.
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Since C. tropicalis is able to colonize and infect several human anatomically
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distinct sites [10], it is possible that these yeast cells could damage or compromise the
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function of internal organs, such as bladder, kidneys and blood vessels, by their ability
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to form biofilms on biotic and abiotic surfaces (medical devices, such as catheters). C. tropicalis cells also have the ability to detached from biofilms and colonize
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human cells causing injury and reduction of cellular metabolic activity [15]. However,
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studies involving biofilms, pre-formed on catheters, and their interaction with animal
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cells are still limited.
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Different methods may be used to investigate biofilm formation and its behavior.
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These methods include crystal violet staining, 2,3-(2-methoxy-4-nitro-5- sulphophenyl)-
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5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and
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scanning electronic microscopy
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investigate yeast catheter-associated infections are limited. Most recently, a successful
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catheter-associated biofilm infection induced in a murine model investigated different
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aspects of candiduria [17]. However, it is still not clear whether yeast biofilm cells
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suffer alterations due to the contact to host cells. Thus, the present study used a simple
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and easy-to-execute method to evaluate the interaction between C. tropicalis biofilm
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organized in a piece of catheter with two different human cell lines.
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[10,14,16]. Both in vitro and in vivo models to
2. Materials and Methods
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All the necessary steps to investigate the maturity of C. tropicalis biofilm
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formed on a fragment of catheter and the in vitro biofilm-dynamics to interact with
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human cells are shown in the flowchart (Fig. 1).
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2.1. Catheter for biofilm formation
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In order to obtain C. tropicalis biofilms, sterile polyurethane catheters (24G,
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Safelet Cath®, Nipro Medical Industries, Ltd., Japan), were aseptically cut into small
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fragments (0.5 cm) and designated as SCF.
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2.2. Strains and Biofilm formation
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To evaluate the interactions between C. tropicalis biofilm and human cells the
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reference strain ATCC 750. This strain was maintained in glycerol stock at -80 °C,
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which belongs to the collection of the Medical Mycology Laboratory from Universidade
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Estadual de Maringá. Strain from frozen stock was culture onto Petri dishes containing
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Sabouraud Dextrose Agar medium (SDA; Difco, Detroit, MI, USA) for 24 h at 35 °C.
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The biofilm production on SCF was performed according to Capote-Bonato et al. [17].
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Briefly small catheter fragments (SCF) 0.5 cm in length were placed in contact with 200
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µl of a suspension containing 1×107 yeasts/ml, inside to wells of a 96-well microplate
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(Kasvi®, K12-096). In these conditions, according Negri et al., 2010 [18] and Estivil et
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al., 2011 [9] the biofilm appears to be consistent and at high concentrations [9,18]. After
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incubation, the biofilms formed on catheter pieces had their medium culture refreshed
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with 100 µL of RPMI 1640 medium and were further incubated for another 24 h under
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the same conditions previously described. After maturation periods of 48 and 72 h were
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reached, SCF were gently removed, and the biofilms attached were quantified.
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2.3. Quantification of in vitro mature C. tropicalis biofilms on SCF
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Aiming the characterization of the biofilm produced by C. tropicalis on SCF was
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evaluated after 48 and 72 h regarding three parameters: The number of cultivable cells
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established by CFU (colony forming unit) counts, the in situ total metabolic activity of
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biofilm, by 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-
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tetrazolium hydroxide (XTT) reduction assay were determined and the biofilm biomass
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quantification by crystal violet staining, according to the protocol of Silva et al. [16]. The amount of total biofilm biomass at 48 and 72 h was estimated by crystal
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violet staining methodology [16] with some adaptations. Briefly, after maturation, the
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medium was removed, and SCF with C. tropicalis biofilms were washed with PBS to
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remove non-adherent yeast cells. SCF were immersed in 100 µL of methanol (100%)
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for 15 min. After that, SCF were air-dry at room temperature. Biofilms were stained
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with 100 µL of 1% (w/v in water) crystal violet (CV) for 5 min. SCF were removed
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with sterile forceps and transferred to another 96-well polystyrene microtiter plate,
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where were washed twice with 100 µL of ultrapure water. Then, SCF were exposed to
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33% (v/v) of acetic acid. Absorbance values were measured with a microtiter plate
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reader at 570 nm (ASYS Expert Plus, Biochrom, Cambridge, UK) and expressed in
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absorbance per unit area of the SCF. Uninoculated SCF were used as controls
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performing the same procedures described above.
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2.4. Interaction of C. tropicalis biofilm with human cells
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To verify if there was any type of interaction between C. tropicalis biofilm and
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human cells, 48 h mature biofilms formed on SCF were placed in direct contact with
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epithelial (HeLa) and endothelial (HUVEC) human cells. First, the aforementioned
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human lines were subcultured in RPMI medium supplemented with 10% fetal bovine
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serum (FBS). After a confluent epithelial/endothelial monolayer was formed, cells were
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enzymatically detached, and the cell concentration was adjusted to 2 x 105 cells mL-1.
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Subsequently, total of 200 µL of this suspension were transferred to each well of a new
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96-well bottom-flat microtiter plate and incubated at 37 °C for 24 h in 5% CO2
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atmosphere. After 24 h incubation, the culture medium was removed, and the wells
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were washed once with PBS. Then, RPMI medium was added, but this time without
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FBS. Subsequently, mature C. tropicalis biofilms (48 h) formed on SCF were carefully
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washed twice with PBS and placed in contact with HeLa and HUVEC cells for 24 h at
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37 °C in the presence of 5% CO2.
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2.5. Quantification of mature C. tropicalis biofilm after contact with biotic surfaces
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After 24 h of co-incubation of mature C. tropicalis biofilms on SCF with human
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cells, SCF were removed and washed twice with PBS to remove unattached yeast cells.
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Then, each SCF was transferred to an Eppendorf tube containing 1 mL of PBS to
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determine: fungal load (CFU), total biomass (CV) and metabolic activity (XTT), as
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previously described. Medium was removed, and human cells were washed twice with
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PBS. Then, cells were detached enzymatically with 25% trypsin-EDTA solution
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(Gibco/Invitrogen, Grand Island, NE, USA), resuspended in PBS, and vortexed for 30 s
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in order to disaggregate yeast cells from human cells. Ten-fold serial dilutions in PBS
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from the obtained yeast suspension were made. A total of 20 µL of each dilution were
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plated onto SDA and incubated at 37 °C for 24 h. The results were expressed as the
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number of CFU per unit area (Log10 CFU/cm2) to estimate the ability of C. tropicalis
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mature biofilm cells to adhere to human cells (CFU/mL). We used as controls SCF and
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human cells that were not inoculated with yeast cells.
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2.6. Scanning electron microscopy of mature biofilm and humans cells after co-
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incubation
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Microscopic evaluations of both human cell surfaces after contact with C.
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tropicalis biofilm and SCF surfaces were performed. All samples were sent to the
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Laboratory of electron microscopy and microanalysis of Universidade Estadual de
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Londrina (UEL/SETI) for the realization of scanning electron microscopy (SEM).
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After 24 h of co-incubation with human cells, SCF were removed. Then, SCF
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and microtiter plates containing human cells were washed twice with PBS, and air-dried
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at room temperature. After that, they were fixed by immersion in 1 mL of 2.5%
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glutaraldehyde (Merck KGaA, Darmstadt, HE, Germany), and 2% paraformaldehyde
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diluted in 0.1 M cacodylate buffer (Sigma-Aldrich, St. Louis, MO, USA) solution (pH
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7.2) for 2 h. Then post-fixation in 1% OsO4, the samples were dehydrated in an ethanol
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gradient (70, 80, 90 and 100%) and critical point dried with CO2 (BALTEC, DPC-030),
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coated with gold (BALTEC, SDC-050 Sputter Coater) and photographed under a SEM
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microscope (FEI Quanta 200) [19,20]. 72 h-mature C. tropicalis biofilm formed on SCF
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(without contact with human cells) and human cell lines (without yeast) were
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considered as controls. Triplicate samples were examined and at least 10 fields were
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observed for each replicate and the most representative images were selected.
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2.7. Statistical analysis
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All assays were performed in triplicate, and the results are shown as means ± SD
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of three independent experiments. The analyses were performed using GraphPad Prism
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version 5.0 software package for Windows (GraphPad Software, La Jolla California
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USA, Graph Pad Software, San Diego, CA, USA). A one-way analysis of variance
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(ANOVA), followed by the Bonferroni’s post-test, was used to evaluate the biofilm
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maturity (48 h and 72 h) and to investigate a possible interaction between mature SCF
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biofilm and human cells. Significance was set at p < 0.05.
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3. Results and Discussion
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In this study, we proposed a simplified technique to evaluate experimentally
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reproducing C. tropicalis biofilm-based on catheter. Our results showed that C.
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tropicalis cells from the biofilm formed on catheter are able to migrate to the human
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cells surface. Furthermore, morphological changes of yeast cells occurred during this
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process.
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The ability of C. tropicalis to form biofilm on SCF was confirmed and our
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results were similar to others published studies [14,21,22]. In addition, a significant
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increase in the number of yeasts, biomass and metabolic activity of mature biofilm on
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SCF was observed (p < 0.05) (48 h and 72 h) (Table 1). Interestingly, there is a relation
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between to an increased extracellular material and metabolic activity with biofilm
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maturation over time, factors that unhappily appear to be also associated with increased
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biofilm resistance [23]. Fig. 2 illustrates the morphological aspects of C. tropicalis cells
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organized in 48 and 72h-mature biofilms on catheter. Through the observation of many
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microscopic fields by SEM, it was possible to detect an increase of C. tropicalis biofilm
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over time. However, it is important to highlight that the culture media can profoundly
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affect C. tropicalis biofilm development, and consequently, these findings may vary
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according to biofilm-forming conditions that yeast cells are exposed [24].
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As verified in Table 2, C. tropicalis yeast cells organized in SCF biofilm
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suffered alterations after contact with human cells. Although not statistically significant,
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there was a trend which suggested that the number of CFUs from biofilms on SCF
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surface, after contact with HeLa (5.101±0.190) (p=0.06) and HUVEC (5.103±0.200)
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(p=0.08), reduced in comparison to CFU count of 72-h mature C. tropicalis SCF
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biofilm (before contact with human cells) (5.552±0.032). These findings, visualized by
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(Fig. 2B and Fig. 3A and B) respectively, suggest that this weak reduction of yeast cells
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(CFUs) on the catheter may be related to their migration from the catheter to the human
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cell surfaces. Thus, we can hypothesize that this phenomenon (migration) is actually a
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simulation of what occurs with patients undergoing catheterization. Therefore, it is
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reasonable to assume that detached yeasts from mature biofilm could reach host cells
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and other human body sites by dissemination, as it has been previously described by
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different studies for C. albicans and C. tropicalis [17,21,25].
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On the other hand, according to Table 2 the mature biofilm (72 h), after contact
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with both human cells showed a significant reduction in total biomass and metabolic
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activity (p < 0.05). This reduction in the metabolic activity is possibly due to many cells
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become dormant when biofilm is matured, these are metabolically inactive cells, namely
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persistent cells, which are high resistance to antifungals [26]. In addition, was observed
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also a difference in CV and metabolic activity among HeLa and HUVEC cells (Table
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2). Candida invasion into endothelial cells occurs as in hyphal as yeast form, thus it is
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possible the yeast could be endocytosed before of morphogenic transformation, in the
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subsequent trans-endothelial cell migration happened [27]. Besides, the different
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behavior among cells-yeast is common, Negri et al. [28] showed that C. tropicalis
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adhere significantly better in intestinal cells (Caco-2) than to urinary cells (TCC-SUP)
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or cervical cells (HeLa). These data endorsed our finds that adhesion is specie specific
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and also tissue specific. CV staining was used to assess total biomass quantification
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(pseudohyphae, hyphae and yeasts cells into biofilm and matrix components). Since
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only CFUs’ number did not change statistically significant, this fact could suggest that
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matrix components were actually the ones to suffer some type of modulation on their
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structures (Table 2) and the yeast morphology (Fig 2B and Fig 3A and B). Recent
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studies [7,29] have showed Candida spp. matrix adaptation in response to antifungal
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stress, resulting in decrease of protein components and increase of carbohydrates
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molecules, such as β-1,3 glucans, to promote the thickness of the biofilm and higher
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protection.
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It is known that C. tropicalis biofilm is highly influenced by environmental
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conditions and further they are able to colonize human cells and tissues in vitro and in
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vivo studies, as well as other Candida species [10,15,17,24,27,30,31]. Also, C.
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tropicalis is able to affect the virulence profile of C. albicans by reducing the biofilm
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formation by decreasing viable cell counts, metabolic activity and hyphal growth in co-
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incubation environment [30]. However, few studies about biofilm after contact with a
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biotic surface, such as human cells and tissues had been performed.
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In relation to morphological aspects, important alterations (between SCF’s and
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human cells’ surfaces) were observed. Firstly, yeast cells of mature C. tropicalis biofilm
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on SCF were primarily composed by budding cells (Fig. 2B); whereas after the contact
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with the tested human cell lines, these yeasts appeared predominantly in the filamentous
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form (Fig. 3A, 3B). This important morphological alteration could be attributed to the
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interaction between the biofilm and the cellular environment. In addition, it is important
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to mention that yeast-to-hyphal transition is highly relevant to some pathogenic yeasts
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and is considered one of the most important steps for the establishment of candidiasis
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[4]. In this case, it was confirmed that C. tropicalis cells organized in biofilm switched
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to filamentous form and showed high ability to interact with human cells. The present study also evaluated the yeast cells that migrated from the biofilm,
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formed on SCF, to the human cells. For that, quantitative and qualitative evaluations of
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the fresh biofilm formed on human cell surfaces were assessed by CFU, which showed
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a similar number of viable yeast cells (count 5.440 Log10/cm2 for HeLa cells and 5.524
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Log10/cm2 for HUVEC cells). Visual observation of human cell surfaces by SEM
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revealed that yeasts cells from C. tropicalis biofilm, originally attached to SCF, were
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able to form a fresh biofilm on both human cell lines as demonstrated in Figure 3 (3C,
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3D).
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Uppuluri et al. [25], using a flow biofilm model, observed the phenomenon of C.
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albicans biofilm dispersion. Likewise, the authors noticed that dispersed C. albicans
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were predominantly in the yeast form (blastoconidia), while true hyphae were observed
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inside the biofilm biomass. Similar to C. albicans, yeast-to-hyphal transition is also an
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important step for C. tropicalis in order to promote adhesion and invasion of cells and
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tissues. C. tropicalis has developed strategies to damage the epithelial cell barrier that
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consequently leads to invasion of such cells and even tissues [10,15]. Therefore, it may
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be necessary the occurrence of hyphae elongation in order to initiate an active invasion
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on human cells. As a result, hyphae elongation is able to push yeast cells inside of
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epithelial cell [32]. It is noteworthy that blastoconidia can also facilitate the division,
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and consequently, the distribution of yeasts in this environment [33].
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Although previous studies have found out that C. tropicalis has actually the
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ability to invade human tissue [10], it is still unclear whether this phenomenon occurs
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during the initial phase of contact between biofilm and human cellular surface.
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4. Conclusion
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The present study described a simple and easy in vitro model, based on well-
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known techniques, which allowed to reproduce a mature C. tropicalis biofilm on SCF
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and, evaluate its interaction with human cells. Our results indicated that this
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methodology allowed to characterize morphological aspects of C. tropicalis biofilms
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under different circumstances. It was demonstrated that C. tropicalis organized in
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mature biofilm on SCF suffer a morphology transition, from blastoconidia to the
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filamentous form. Besides, also was observed change regarding to extracellular matrix
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components after contact between this biofilm and human cells, with reduction of total
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biomass and metabolic activity. These facts have been hypothesized to be due to the
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migration of yeasts from the biofilm, and its availability for possible adhesion in other
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sites. Consequently, this in vitro model revealed to be a useful alternative assessment
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tool for future studies, especially those involving animal models, since it provides great
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contribution in understanding the mechanisms of C. tropicalis biofilms. Deep insights
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on pathogenesis of C. tropicalis are essential in order to the diseases caused by yeast in
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catheterized patients.
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Acknowledgements
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The authors thank the personnel of Universidade Estadual de Maringá,
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Laboratory of electron microscopy and microanalysis, Universidade Estadual de
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Londrina (UEL/SETI) and Universidade de Franca their excellent technical assistance.
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We also thank Dr. Pedrina Gonçalves Vidigal for review of the English text.
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Funding Information
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This study received financial support from CAPES, CNPq, Fundação Araucária
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and FAPESP. Bonato holds a scholarship from CAPES. Svidzinski holds a fellowship
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from CNPq.
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Conflict of Interest
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Figure legends
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Fig. 1 - Description and evaluation of biofilm production and interaction with human
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cell lines. Mature C. tropicalis (ATCC 750) biofilms on catheter (A) without contact
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with human cells and (B) after contact with human cells. Abbreviations: SCF- Small
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Catheter Fragment; CV - Cristal Violet; XTT -2,3 – bis (2–methoxy–4–nitro–5-
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sulfophenyl) -2H– tetrazolium– 5- carboxanilide; CFU - Colony Forming Units; SEM -
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Scanning Electron Microscopy.
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Fig. 2 - Scanning electron microscopy (SEM) images of mature C. tropicalis biofilm on
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catheter, observed at magnification x 6000. (A) Biofilm formed in 48 h; (B) Biofilm
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formed in 72 h (Fig. 1 A).
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Fig. 3 - Scanning electron microscopy images of mature C. tropicalis biofilms on SCF
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and human cells surfaces. Surface of SCF containing mature C. tropicalis biofilms after
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contact with Hela (A) and HUVEC (B) human cells, original magnification x 800.
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Human cell surfaces HeLa (C) and HUVEC (D) showed predominant adhesion of
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blastoconidia and dense extracellular matrix, original magnification x 5000.
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Biofilm maturity (h)
CV§
XTT||
(Log10/cm2)
(Abs570nm/cm2)
(Abs490nm/cm2)
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4.261±0.194
0.151±0.030
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5.552±0.032*
0.291±0.030*
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CFU‡
0.111±0.082
0.349±0.040*
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*Significant increase between 48 and 72 h (p<0.05). Abs: Absorbance; †SD: Standard deviation; ‡CFU: Colony forming units per cm2; §CV: Crystal violet; ||XTT: 2,3 – bis
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Mature biofilm
on catheter Without
After contact with human
contact
cells
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(Mean±SD†)
HeLa
HUVEC
5.103±0.200
5.552±0.032
5.101±0.190
CV§ (Abs570nm/cm2)
0.291±0.029
0.170±0.004* 0.099±0.022*
XTT|| (Abs490nm/cm2)
0.349±0.040
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CFU‡ (Log10/cm2)
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*Represents a significant decrease after contact with human cells (p<0.05). Abs: Absorbance; †SD: Standard deviation; ‡CFU: Colony forming units; §CV: Crystal violet; ||
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C. tropicalis was able to form biofilm on small catheter fragments of 0.5 cm length. Yeast cells from mature biofilm migrated and formed a fresh one on human cell surface A simple methodology to investigate C. tropicalis migration from biofilms is proposed C. tropicalis biofilm formed on catheter was mainly composed by filamentous form. The fresh C. tropicalis biofilm formed on human cells had mainly blastoconidia.
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S0882-4010(18)30774-5
DOI:
10.1016/j.micpath.2018.09.029
Reference:
YMPAT 3179
To appear in:
Microbial Pathogenesis
Received Date: 2 May 2018 Revised Date:
14 August 2018
Accepted Date: 15 September 2018
Please cite this article as: Capote-Bonato F, Sakita KM, de Oliveira Junior AdmiltonGonç, BonfimMendonça PatríSouza, Crivellenti LZ, Negri M, Estivalet Svidzinski TI, In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells, Microbial Pathogenesis (2018), doi: https:// doi.org/10.1016/j.micpath.2018.09.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro interaction of Candida tropicalis biofilm formed on catheter with human
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cells
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Francieli Capote-Bonato1, Karina Mayumi Sakita1, Admilton Gonçalves de Oliveira
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Junior2, Patrícia de Souza Bonfim-Mendonça1, Leandro Zuccolotto Crivellenti3,
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Melyssa Negri1, Terezinha Inez Estivalet Svidzinski1*.
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Department of Clinical Analysis (DCA), State University of Maringá, Paraná, Brazil
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Department of Microbiology (DM), State University of Londrina, Paraná, Brazil
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Department of Animal Science (DAS), Franca University, São Paulo, Brazil
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* Corresponding author: Terezinha Inez Estivalet Svidzinski
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Divisão de Micologia Médica – Departamento de Análises Clínicas e Biomedicina –
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Universidade Estadual de Maringá – Paraná - Brazil. Av. Colombo, 5790, CEP: 87020-
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900, Maringá, PR., Brazil. Phone: +55 44 3011-4809. Fax: +55 44 3011-4860. E-mail:
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[email protected]
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Abstract
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Candida tropicalis has emerged as one of the major Candida non-C. albicans
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species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis
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pathogenic mechanism has yet not been fully defined. The current study aimed to
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demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on
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catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms
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were produced on small catheter fragments (SCF) and were mainly composed by
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blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces
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(HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of
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SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous
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form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of
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blastoconidia involved by extracellular matrix. Total biomass and metabolic activity
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from the remaining biofilm on SCF surface, after direct contact with human cells,
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exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular
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matrix components, after contact with human cells. Thus, we described for the first time
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an easy and simple in vitro model with catheter, which could be a powerful tool for
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future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.
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Keywords
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Candida tropicalis; virulence; catheter; biofilm; yeast-hyphal transition.
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1. Introduction
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In the latest decades, the number of opportunistic fungal infections caused by
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Candida species remains high in humans [1]. Candidemia is the main invasive fungal
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disease among hospitalized patients and the frequency of Candida non-C. albicans
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(CNCA) species as causative agent has increased [2]. For this reason, current research
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interest involves the understanding and characterization of emerging CNCA species.
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In terms of epidemiology and virulence, C. tropicalis appears as the second most
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frequently isolated species of CNCA in South America and Asia and the third most
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commonly isolated in the USA and Europe [3–7]. In particular, infections by C.
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tropicalis are frequent in cancer patients and associated with a higher mortality rate [8].
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An important step for a successful colonization and infection depends essentially
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on the ability of the pathogen to adhere and form biofilm to host surfaces [9–11].
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Biofilm is a microbial community attached to biological or inert surfaces, such as
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medical devices, embedded by extracellular polymeric substances, composed by
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carbohydrates, hexosamines, uronic acids, proteins and nucleic acids [12,13]. Biofilms
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are assumed to be the most important virulence factor for pathogenicity in Candida
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species [12]. The ability to produce biofilms is developed by microorganisms in order to
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protect themselves against host defense mechanisms and antifungal drugs. Therefore,
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biofilm formation plays an important role as a virulence factor, which contributes to the
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pathogenesis of candidiasis. Most of our knowledge related to fungal biofilm formation
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is based on studies carried out in C. albicans. Recently, investigations showed that C.
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tropicalis is a strong biofilm producer [9,14]. Despite that, several biological
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mechanisms involved in C. tropicalis biofilm formation are not yet fully understood.
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Since C. tropicalis is able to colonize and infect several human anatomically
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distinct sites [10], it is possible that these yeast cells could damage or compromise the
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function of internal organs, such as bladder, kidneys and blood vessels, by their ability
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to form biofilms on biotic and abiotic surfaces (medical devices, such as catheters). C. tropicalis cells also have the ability to detached from biofilms and colonize
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human cells causing injury and reduction of cellular metabolic activity [15]. However,
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studies involving biofilms, pre-formed on catheters, and their interaction with animal
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cells are still limited.
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Different methods may be used to investigate biofilm formation and its behavior.
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These methods include crystal violet staining, 2,3-(2-methoxy-4-nitro-5- sulphophenyl)-
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5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and
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scanning electronic microscopy
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investigate yeast catheter-associated infections are limited. Most recently, a successful
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catheter-associated biofilm infection induced in a murine model investigated different
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aspects of candiduria [17]. However, it is still not clear whether yeast biofilm cells
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suffer alterations due to the contact to host cells. Thus, the present study used a simple
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and easy-to-execute method to evaluate the interaction between C. tropicalis biofilm
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organized in a piece of catheter with two different human cell lines.
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[10,14,16]. Both in vitro and in vivo models to
2. Materials and Methods
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All the necessary steps to investigate the maturity of C. tropicalis biofilm
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formed on a fragment of catheter and the in vitro biofilm-dynamics to interact with
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human cells are shown in the flowchart (Fig. 1).
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2.1. Catheter for biofilm formation
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In order to obtain C. tropicalis biofilms, sterile polyurethane catheters (24G,
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Safelet Cath®, Nipro Medical Industries, Ltd., Japan), were aseptically cut into small
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fragments (0.5 cm) and designated as SCF.
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2.2. Strains and Biofilm formation
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To evaluate the interactions between C. tropicalis biofilm and human cells the
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reference strain ATCC 750. This strain was maintained in glycerol stock at -80 °C,
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which belongs to the collection of the Medical Mycology Laboratory from Universidade
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Estadual de Maringá. Strain from frozen stock was culture onto Petri dishes containing
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Sabouraud Dextrose Agar medium (SDA; Difco, Detroit, MI, USA) for 24 h at 35 °C.
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The biofilm production on SCF was performed according to Capote-Bonato et al. [17].
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Briefly small catheter fragments (SCF) 0.5 cm in length were placed in contact with 200
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µl of a suspension containing 1×107 yeasts/ml, inside to wells of a 96-well microplate
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(Kasvi®, K12-096). In these conditions, according Negri et al., 2010 [18] and Estivil et
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al., 2011 [9] the biofilm appears to be consistent and at high concentrations [9,18]. After
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incubation, the biofilms formed on catheter pieces had their medium culture refreshed
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with 100 µL of RPMI 1640 medium and were further incubated for another 24 h under
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the same conditions previously described. After maturation periods of 48 and 72 h were
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reached, SCF were gently removed, and the biofilms attached were quantified.
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2.3. Quantification of in vitro mature C. tropicalis biofilms on SCF
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Aiming the characterization of the biofilm produced by C. tropicalis on SCF was
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evaluated after 48 and 72 h regarding three parameters: The number of cultivable cells
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established by CFU (colony forming unit) counts, the in situ total metabolic activity of
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biofilm, by 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-
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tetrazolium hydroxide (XTT) reduction assay were determined and the biofilm biomass
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quantification by crystal violet staining, according to the protocol of Silva et al. [16]. The amount of total biofilm biomass at 48 and 72 h was estimated by crystal
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violet staining methodology [16] with some adaptations. Briefly, after maturation, the
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medium was removed, and SCF with C. tropicalis biofilms were washed with PBS to
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remove non-adherent yeast cells. SCF were immersed in 100 µL of methanol (100%)
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for 15 min. After that, SCF were air-dry at room temperature. Biofilms were stained
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with 100 µL of 1% (w/v in water) crystal violet (CV) for 5 min. SCF were removed
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with sterile forceps and transferred to another 96-well polystyrene microtiter plate,
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where were washed twice with 100 µL of ultrapure water. Then, SCF were exposed to
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33% (v/v) of acetic acid. Absorbance values were measured with a microtiter plate
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reader at 570 nm (ASYS Expert Plus, Biochrom, Cambridge, UK) and expressed in
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absorbance per unit area of the SCF. Uninoculated SCF were used as controls
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performing the same procedures described above.
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2.4. Interaction of C. tropicalis biofilm with human cells
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To verify if there was any type of interaction between C. tropicalis biofilm and
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human cells, 48 h mature biofilms formed on SCF were placed in direct contact with
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epithelial (HeLa) and endothelial (HUVEC) human cells. First, the aforementioned
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human lines were subcultured in RPMI medium supplemented with 10% fetal bovine
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serum (FBS). After a confluent epithelial/endothelial monolayer was formed, cells were
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enzymatically detached, and the cell concentration was adjusted to 2 x 105 cells mL-1.
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Subsequently, total of 200 µL of this suspension were transferred to each well of a new
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96-well bottom-flat microtiter plate and incubated at 37 °C for 24 h in 5% CO2
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atmosphere. After 24 h incubation, the culture medium was removed, and the wells
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were washed once with PBS. Then, RPMI medium was added, but this time without
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FBS. Subsequently, mature C. tropicalis biofilms (48 h) formed on SCF were carefully
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washed twice with PBS and placed in contact with HeLa and HUVEC cells for 24 h at
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37 °C in the presence of 5% CO2.
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2.5. Quantification of mature C. tropicalis biofilm after contact with biotic surfaces
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After 24 h of co-incubation of mature C. tropicalis biofilms on SCF with human
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cells, SCF were removed and washed twice with PBS to remove unattached yeast cells.
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Then, each SCF was transferred to an Eppendorf tube containing 1 mL of PBS to
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determine: fungal load (CFU), total biomass (CV) and metabolic activity (XTT), as
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previously described. Medium was removed, and human cells were washed twice with
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PBS. Then, cells were detached enzymatically with 25% trypsin-EDTA solution
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(Gibco/Invitrogen, Grand Island, NE, USA), resuspended in PBS, and vortexed for 30 s
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in order to disaggregate yeast cells from human cells. Ten-fold serial dilutions in PBS
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from the obtained yeast suspension were made. A total of 20 µL of each dilution were
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plated onto SDA and incubated at 37 °C for 24 h. The results were expressed as the
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number of CFU per unit area (Log10 CFU/cm2) to estimate the ability of C. tropicalis
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mature biofilm cells to adhere to human cells (CFU/mL). We used as controls SCF and
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human cells that were not inoculated with yeast cells.
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2.6. Scanning electron microscopy of mature biofilm and humans cells after co-
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incubation
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Microscopic evaluations of both human cell surfaces after contact with C.
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tropicalis biofilm and SCF surfaces were performed. All samples were sent to the
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Laboratory of electron microscopy and microanalysis of Universidade Estadual de
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Londrina (UEL/SETI) for the realization of scanning electron microscopy (SEM).
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After 24 h of co-incubation with human cells, SCF were removed. Then, SCF
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and microtiter plates containing human cells were washed twice with PBS, and air-dried
184
at room temperature. After that, they were fixed by immersion in 1 mL of 2.5%
185
glutaraldehyde (Merck KGaA, Darmstadt, HE, Germany), and 2% paraformaldehyde
186
diluted in 0.1 M cacodylate buffer (Sigma-Aldrich, St. Louis, MO, USA) solution (pH
187
7.2) for 2 h. Then post-fixation in 1% OsO4, the samples were dehydrated in an ethanol
188
gradient (70, 80, 90 and 100%) and critical point dried with CO2 (BALTEC, DPC-030),
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coated with gold (BALTEC, SDC-050 Sputter Coater) and photographed under a SEM
190
microscope (FEI Quanta 200) [19,20]. 72 h-mature C. tropicalis biofilm formed on SCF
191
(without contact with human cells) and human cell lines (without yeast) were
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considered as controls. Triplicate samples were examined and at least 10 fields were
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observed for each replicate and the most representative images were selected.
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2.7. Statistical analysis
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All assays were performed in triplicate, and the results are shown as means ± SD
198
of three independent experiments. The analyses were performed using GraphPad Prism
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version 5.0 software package for Windows (GraphPad Software, La Jolla California
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USA, Graph Pad Software, San Diego, CA, USA). A one-way analysis of variance
201
(ANOVA), followed by the Bonferroni’s post-test, was used to evaluate the biofilm
202
maturity (48 h and 72 h) and to investigate a possible interaction between mature SCF
203
biofilm and human cells. Significance was set at p < 0.05.
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3. Results and Discussion
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In this study, we proposed a simplified technique to evaluate experimentally
208
reproducing C. tropicalis biofilm-based on catheter. Our results showed that C.
209
tropicalis cells from the biofilm formed on catheter are able to migrate to the human
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cells surface. Furthermore, morphological changes of yeast cells occurred during this
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process.
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The ability of C. tropicalis to form biofilm on SCF was confirmed and our
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results were similar to others published studies [14,21,22]. In addition, a significant
214
increase in the number of yeasts, biomass and metabolic activity of mature biofilm on
215
SCF was observed (p < 0.05) (48 h and 72 h) (Table 1). Interestingly, there is a relation
216
between to an increased extracellular material and metabolic activity with biofilm
217
maturation over time, factors that unhappily appear to be also associated with increased
218
biofilm resistance [23]. Fig. 2 illustrates the morphological aspects of C. tropicalis cells
219
organized in 48 and 72h-mature biofilms on catheter. Through the observation of many
220
microscopic fields by SEM, it was possible to detect an increase of C. tropicalis biofilm
221
over time. However, it is important to highlight that the culture media can profoundly
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affect C. tropicalis biofilm development, and consequently, these findings may vary
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according to biofilm-forming conditions that yeast cells are exposed [24].
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As verified in Table 2, C. tropicalis yeast cells organized in SCF biofilm
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suffered alterations after contact with human cells. Although not statistically significant,
226
there was a trend which suggested that the number of CFUs from biofilms on SCF
227
surface, after contact with HeLa (5.101±0.190) (p=0.06) and HUVEC (5.103±0.200)
228
(p=0.08), reduced in comparison to CFU count of 72-h mature C. tropicalis SCF
229
biofilm (before contact with human cells) (5.552±0.032). These findings, visualized by
230
(Fig. 2B and Fig. 3A and B) respectively, suggest that this weak reduction of yeast cells
231
(CFUs) on the catheter may be related to their migration from the catheter to the human
232
cell surfaces. Thus, we can hypothesize that this phenomenon (migration) is actually a
233
simulation of what occurs with patients undergoing catheterization. Therefore, it is
234
reasonable to assume that detached yeasts from mature biofilm could reach host cells
235
and other human body sites by dissemination, as it has been previously described by
236
different studies for C. albicans and C. tropicalis [17,21,25].
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On the other hand, according to Table 2 the mature biofilm (72 h), after contact
238
with both human cells showed a significant reduction in total biomass and metabolic
239
activity (p < 0.05). This reduction in the metabolic activity is possibly due to many cells
240
become dormant when biofilm is matured, these are metabolically inactive cells, namely
241
persistent cells, which are high resistance to antifungals [26]. In addition, was observed
242
also a difference in CV and metabolic activity among HeLa and HUVEC cells (Table
243
2). Candida invasion into endothelial cells occurs as in hyphal as yeast form, thus it is
244
possible the yeast could be endocytosed before of morphogenic transformation, in the
245
subsequent trans-endothelial cell migration happened [27]. Besides, the different
246
behavior among cells-yeast is common, Negri et al. [28] showed that C. tropicalis
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adhere significantly better in intestinal cells (Caco-2) than to urinary cells (TCC-SUP)
248
or cervical cells (HeLa). These data endorsed our finds that adhesion is specie specific
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and also tissue specific. CV staining was used to assess total biomass quantification
250
(pseudohyphae, hyphae and yeasts cells into biofilm and matrix components). Since
251
only CFUs’ number did not change statistically significant, this fact could suggest that
252
matrix components were actually the ones to suffer some type of modulation on their
253
structures (Table 2) and the yeast morphology (Fig 2B and Fig 3A and B). Recent
254
studies [7,29] have showed Candida spp. matrix adaptation in response to antifungal
255
stress, resulting in decrease of protein components and increase of carbohydrates
256
molecules, such as β-1,3 glucans, to promote the thickness of the biofilm and higher
257
protection.
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It is known that C. tropicalis biofilm is highly influenced by environmental
259
conditions and further they are able to colonize human cells and tissues in vitro and in
260
vivo studies, as well as other Candida species [10,15,17,24,27,30,31]. Also, C.
261
tropicalis is able to affect the virulence profile of C. albicans by reducing the biofilm
262
formation by decreasing viable cell counts, metabolic activity and hyphal growth in co-
263
incubation environment [30]. However, few studies about biofilm after contact with a
264
biotic surface, such as human cells and tissues had been performed.
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In relation to morphological aspects, important alterations (between SCF’s and
266
human cells’ surfaces) were observed. Firstly, yeast cells of mature C. tropicalis biofilm
267
on SCF were primarily composed by budding cells (Fig. 2B); whereas after the contact
268
with the tested human cell lines, these yeasts appeared predominantly in the filamentous
269
form (Fig. 3A, 3B). This important morphological alteration could be attributed to the
270
interaction between the biofilm and the cellular environment. In addition, it is important
271
to mention that yeast-to-hyphal transition is highly relevant to some pathogenic yeasts
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and is considered one of the most important steps for the establishment of candidiasis
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[4]. In this case, it was confirmed that C. tropicalis cells organized in biofilm switched
274
to filamentous form and showed high ability to interact with human cells. The present study also evaluated the yeast cells that migrated from the biofilm,
276
formed on SCF, to the human cells. For that, quantitative and qualitative evaluations of
277
the fresh biofilm formed on human cell surfaces were assessed by CFU, which showed
278
a similar number of viable yeast cells (count 5.440 Log10/cm2 for HeLa cells and 5.524
279
Log10/cm2 for HUVEC cells). Visual observation of human cell surfaces by SEM
280
revealed that yeasts cells from C. tropicalis biofilm, originally attached to SCF, were
281
able to form a fresh biofilm on both human cell lines as demonstrated in Figure 3 (3C,
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3D).
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Uppuluri et al. [25], using a flow biofilm model, observed the phenomenon of C.
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albicans biofilm dispersion. Likewise, the authors noticed that dispersed C. albicans
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were predominantly in the yeast form (blastoconidia), while true hyphae were observed
286
inside the biofilm biomass. Similar to C. albicans, yeast-to-hyphal transition is also an
287
important step for C. tropicalis in order to promote adhesion and invasion of cells and
288
tissues. C. tropicalis has developed strategies to damage the epithelial cell barrier that
289
consequently leads to invasion of such cells and even tissues [10,15]. Therefore, it may
290
be necessary the occurrence of hyphae elongation in order to initiate an active invasion
291
on human cells. As a result, hyphae elongation is able to push yeast cells inside of
292
epithelial cell [32]. It is noteworthy that blastoconidia can also facilitate the division,
293
and consequently, the distribution of yeasts in this environment [33].
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Although previous studies have found out that C. tropicalis has actually the
295
ability to invade human tissue [10], it is still unclear whether this phenomenon occurs
296
during the initial phase of contact between biofilm and human cellular surface.
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4. Conclusion
299
The present study described a simple and easy in vitro model, based on well-
301
known techniques, which allowed to reproduce a mature C. tropicalis biofilm on SCF
302
and, evaluate its interaction with human cells. Our results indicated that this
303
methodology allowed to characterize morphological aspects of C. tropicalis biofilms
304
under different circumstances. It was demonstrated that C. tropicalis organized in
305
mature biofilm on SCF suffer a morphology transition, from blastoconidia to the
306
filamentous form. Besides, also was observed change regarding to extracellular matrix
307
components after contact between this biofilm and human cells, with reduction of total
308
biomass and metabolic activity. These facts have been hypothesized to be due to the
309
migration of yeasts from the biofilm, and its availability for possible adhesion in other
310
sites. Consequently, this in vitro model revealed to be a useful alternative assessment
311
tool for future studies, especially those involving animal models, since it provides great
312
contribution in understanding the mechanisms of C. tropicalis biofilms. Deep insights
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on pathogenesis of C. tropicalis are essential in order to the diseases caused by yeast in
314
catheterized patients.
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Acknowledgements
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The authors thank the personnel of Universidade Estadual de Maringá,
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Laboratory of electron microscopy and microanalysis, Universidade Estadual de
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Londrina (UEL/SETI) and Universidade de Franca their excellent technical assistance.
321
We also thank Dr. Pedrina Gonçalves Vidigal for review of the English text.
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Funding Information
324
This study received financial support from CAPES, CNPq, Fundação Araucária
326
and FAPESP. Bonato holds a scholarship from CAPES. Svidzinski holds a fellowship
327
from CNPq.
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Conflict of Interest
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Figure legends
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Fig. 1 - Description and evaluation of biofilm production and interaction with human
457
cell lines. Mature C. tropicalis (ATCC 750) biofilms on catheter (A) without contact
458
with human cells and (B) after contact with human cells. Abbreviations: SCF- Small
459
Catheter Fragment; CV - Cristal Violet; XTT -2,3 – bis (2–methoxy–4–nitro–5-
460
sulfophenyl) -2H– tetrazolium– 5- carboxanilide; CFU - Colony Forming Units; SEM -
461
Scanning Electron Microscopy.
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Fig. 2 - Scanning electron microscopy (SEM) images of mature C. tropicalis biofilm on
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catheter, observed at magnification x 6000. (A) Biofilm formed in 48 h; (B) Biofilm
465
formed in 72 h (Fig. 1 A).
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Fig. 3 - Scanning electron microscopy images of mature C. tropicalis biofilms on SCF
468
and human cells surfaces. Surface of SCF containing mature C. tropicalis biofilms after
469
contact with Hela (A) and HUVEC (B) human cells, original magnification x 800.
470
Human cell surfaces HeLa (C) and HUVEC (D) showed predominant adhesion of
471
blastoconidia and dense extracellular matrix, original magnification x 5000.
472
ACCEPTED MANUSCRIPT Table 1. Characterization of Candida tropicalis biofilm formed on SCFs Characterization of mature biofilms on catheter (Mean±SD†)
Biofilm maturity (h)
CV§
XTT||
(Log10/cm2)
(Abs570nm/cm2)
(Abs490nm/cm2)
48
4.261±0.194
0.151±0.030
72
5.552±0.032*
0.291±0.030*
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CFU‡
0.111±0.082
0.349±0.040*
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*Significant increase between 48 and 72 h (p<0.05). Abs: Absorbance; †SD: Standard deviation; ‡CFU: Colony forming units per cm2; §CV: Crystal violet; ||XTT: 2,3 – bis
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(2–methoxy–4–nitro–5-sulfophenyl) -2H– tetrazolium– 5- carboxanilide.
ACCEPTED MANUSCRIPT Table 2. Characterization of yeast biofilm on catheter after contact with human cells. Assay to characterization of biofilm
Mature biofilm
on catheter Without
After contact with human
contact
cells
RI PT
(Mean±SD†)
HeLa
HUVEC
5.103±0.200
5.552±0.032
5.101±0.190
CV§ (Abs570nm/cm2)
0.291±0.029
0.170±0.004* 0.099±0.022*
XTT|| (Abs490nm/cm2)
0.349±0.040
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CFU‡ (Log10/cm2)
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0.063±0.048* 0.140±0.176*
*Represents a significant decrease after contact with human cells (p<0.05). Abs: Absorbance; †SD: Standard deviation; ‡CFU: Colony forming units; §CV: Crystal violet; ||
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XTT: 2,3 - bis (2-methoxy-4-nitro-5-sulfophenyl) -2H- tetrazolium-5- carboxanilide.
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT Highlights
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C. tropicalis was able to form biofilm on small catheter fragments of 0.5 cm length. Yeast cells from mature biofilm migrated and formed a fresh one on human cell surface A simple methodology to investigate C. tropicalis migration from biofilms is proposed C. tropicalis biofilm formed on catheter was mainly composed by filamentous form. The fresh C. tropicalis biofilm formed on human cells had mainly blastoconidia.
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