Real-time Assessment of Interspecies Candida Biofilm Formation
Pedro M.D.S. Abrantes1, Kauthar Behardien1, Charlene W.J. Africa1, *
Identifiers and Pagination:Year: 2022
E-location ID: e187428582211230
Publisher ID: e187428582211230
Article History:Received Date: 11/08/2022
Revision Received Date: 04/10/2022
Acceptance Date: 24/10/2022
Electronic publication date: 30/12/2022
Collection year: 2022
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Candida infections are responsible for increased morbidity and mortality in immunocompromised patients, with Candida having the ability to form highly structured biofilms that protect them from the external environment and the action of antifungals. Few studies have reported on real-time interactions between Candida species.
This study aimed to investigate the interspecies biofilm changes of oral Candida species using the xCELLigence system and the CV staining technique.
This study demonstrated the mixed biofilm interactions of Candida species using an impedance-based biofilm monitoring system and crystal violet staining.
When using the xCELLigence system, the maximum cell index increased in most mixed biofilms, except for the C. glabrata/C. parapsilosis and C. albicans combinations. Bulk biofilm formation measured by CV staining was the highest in C. albicans and C. tropicalis combinations and the lowest in the C. glabrata/C. parapsilosis combination. Pseudohyphae were observed in C. albicans and C. glabrata in combination with C. tropicalis or C. parapsilosis.
This study is the first to report on the real-time interactions of Candida species using the xCELLigence system and suggests that the presence of specific species influences the biofilm formation of commonly isolated Candida species.
Infections by commensal yeasts of the Candida genus range from superficial mucosal infections to disseminated infections. Candidiasis is the most frequent of the opportunistic infections, occurring in patients with underlying disease or those on broad-spectrum antibiotics or immunosuppressive therapy. Oral candidiasis usually presents with yellowish-white plaques on the oral mucosa (oral thrush), while the erythema-tous form of candidiasis, associated with denture wear or poor denture hygiene , consists of bright red spots on the tongue or palate. Candida is also involved in the development of angular cheilitis and has been associated with oral cancer  and dental caries . It is present in virtually all HIV-positive patients with severe immunosuppression, having the ability to spread to other body sites and resulting in increased patient morbidity and mortality.
Topical polyene antifungals such as amphotericin B and nystatin or imidazole drugs such as miconazole and clotrimazole are used to manage localised oral Candida infections . However, these infections often recur. In denture-associated erythematous candidiasis, the most common form of candidiasis, the localised reddening of the mucosa beneath the fitting surface of dentures can be treated with either antifungals, denture cleansing agents, natural extracts or denture base resin coatings, which may reduce the attachment of Candida albicans (C. albicans) to dentures .
Cases requiring systemic antifungal therapy are treated with either triazoles, which have fewer side effects than other drug classes , the more recently developed echinocandins, which are used to treat infections by the inherently azole-resistant species Candida glabrata (C. glabrata) and Candida krusei (C. krusei) [7, 8] or the fluorinated pyrimidine 5-flucytosine, which has considerable side effects and is often ineffective when used on its own . Due to its low cost, fluconazole is routinely administered for candidiasis in healthcare facilities on the African continent when topical antifungal treatment is ineffective and for the treatment of oropharyngeal or oesophageal candidiasis . However, the resistance to this drug is increasing . Drug-resistant, non-albicans species have emerged and are increasing due to severe patient immunosuppression or the indiscriminate use of broad-spectrum antimicrobials .
Distinct Candida species express different virulence factors that allow them to attach to the host tissues, evade the action of innate immune responses and survive exposure to antifungals [11, 12]. Furthermore, Candida species can form polymicrobial biofilms, including those composed of multiple combinations of Candida species . Biofilms provide microbial communities with metabolic cooperation and protection from the external environment and antimicrobial drugs , with different Candida species demonstrating differences in biofilm formation, morphology, characteristics of the extracellular matrix (ECM) and the ability to confer antifungal resistance [15, 16].
Biofilm formation involves the processes of adhesion, morphologic modifications, increases in cell number and the production of the ECM, which facilitates the adhesion between cells in the biofilm and the environment or substrate they colonise . C. albicans is known to have a higher biofilm-forming ability than non-albicans species, and its biofilms were shown to be affected when grown with Candida rugosa , Candida tropicalis (C. tropicalis) and C. krusei . Most previously published studies on Candida biofilms employed either the 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, which measures metabolic cell activity  or the crystal violet (CV) staining technique, an assay that measures the uptake of crystal violet by already formed biofilms . However, the XTT reduction assay has been criticised for having a low detection limit and being difficult to apply in polymicrobial biofilms (due to different metabolic rates of organisms), while the CV staining technique can lead to an overestimation or underestimation of biofilm biomass . These methodologies are also not able to measure different biofilm formation parameters over time.
The xCELLigence real-time cell analyser is an impedance-based monitoring system that allows for real-time monitoring of changes in cell number, size, adhesion and extra-cellular polymeric substances (EPS) biofilm formation and continuous and automated data analysis. This system, which has been used to assess the biofilm formation abilities of C. albicans [23, 24], relies on microtiter plates with gold microelectrodes through which impedance signals (expressed as cell index, CI) are measured and assesses the cell adhesion /EPS formation dynamics over time. When biofilm development occurs, the CI values initially increase as the biofilm adheres and proliferates on the bottom of the E-plate wells, with a peak CI being consistent with the start of the biofilm maturation stage and a decrease taking place when the biofilm initiates the detachment phase . A declining slope after the peak CI value has been suggested as a measure of biofilm formation . As the polysaccharides that hold the biofilm together are broken down by enzymes, releasing the cells from the biofilm matrix, the electrical signals are also further affected by extracellular components and physical appendages of microorganisms, which are consistent with the death phase . There is a paucity of data related to the real-time mixed Candida species biofilm dynamics using this method.
This pilot study investigates the interspecies biofilm changes of oral Candida species using the xCELLigence system and the CV staining technique.
2. MATERIALS AND METHODS
2.1. Organisms used in this Study
Candida type strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and National Collection of Pathogenic Fungi (NCPF, Public Health Culture Collections, England) and included Candida albicans ATCC 90028, Candida dubliniensis NCPF 3949a, Candida glabrata ATCC 26512, Candida tropicalis ATCC 950, Candida krusei ATCC 2159 and Candida parapsilosis ATCC 22019. These six isolates were revived by growth in 10 ml Sabouraud dextrose broth (Cat. no. CMO147, Oxoid, UK), followed by incubation at 37 °C for 3–5 days. Purity of growth was confirmed by microscopy and subculture of single colonies previously grown on Sabouraud dextrose agar (Cat. no. 84088, Sigma-Aldrich, USA) and incubated aerobically at 37 °C for 24-48 h. Species differentiation was confirmed by growth on Fluka chromogenic Candida identification agar (Cat. no. 94382; Sigma-Aldrich, St. Louis, MI, USA) and Oxoid chromogenic Candida agar (Cat. no. CM1002A; Oxoid, Hampshire, UK) at 30 °C for 24–72 h.
2.2. Real-time Monitoring of Biofilm Formation
Prior to conducting this study, preliminary toxicity tests were performed by growing the organisms in the presence of other species’ sterile supernatants, as previously described , to assess whether any organisms induced toxicity against the others. The adhesion changes of the individual isolates were tested in yeast peptone dextrose (YPD) broth (Cat. no. Y1375, Sigma-Aldrich, USA), a medium known to promote Candida adherence and biofilm formation [24, 28, 29] with 100 µl of a 1:20 dilution of a 0.5 McFarland standard suspension and 100 µl of YPD broth added to the wells of an E-plate 16 (Cat. no. 05469830001, ACEA Biosciences, USA). When testing the mixed growth of two species, C. albicans, C. dubliniensis and C. glabrata were grown in the presence of the less commonly isolated oral species C. tropicalis, C. krusei and C. parapsilosis. Fifty microliters (50 µl) of a 1:20 dilution of a 0.5 McFarland standard suspension of each organism was added to an individual E-plate 16 well, resulting in a total volume of 100 µl cell suspension and a further 100 µl of YPD broth added to the well. Sterile distilled water was placed in the surrounding evaporation-control troughs, and the plate was inserted into the RTCA plate analyser (Cat. no. 05469759001, ACEA Biosciences, USA), previously placed in a 37°C incubator. An experimental procedure was logged on the RTCA software package (Cat. no. 05454433001, ACEA Biosciences, USA), with impedance readings set to take place at 15-minute intervals for 48 h. The real-time CI values for the full duration of the experiments were then plotted in individual graphs. Experiments were duplicated, and sterility controls were included in every tested plate.
2.3. Bulk Biofilm Morphology
Bulk biofilm morphology was observed by growing the organisms on 12-well cell culture plates (Cat. no. 3513, Corning, USA). Two millilitres (2 ml) of 0.5 McFarland standard Roswell Park Memorial Institute (RPMI) 1640 medium with L-glutamine (Cat. no. R6504, Sigma-Aldrich, USA) suspensions of the Candida isolates were dispensed into 12-well plates, followed by shaking at 37°C for 90 min at 75 rpm to allow the cells to adhere to the bottom of the wells. The medium was subsequently aspirated, and the wells were gently washed with 2 ml phosphate buffered saline (PBS) pH 7.4 (Cat. no. P3813, Sigma-Aldrich, USA), followed by the addition of fresh RPMI-1640 medium and shaking at 37°C for 24 and 48 h at 75 rpm. For the 48 h growth, the medium was replenished after 24 h. Controls with the medium but no cultures were included. The medium was then aspirated, and the wells were washed with 2 ml PBS to remove unattached cells. Fluorescence microscopy (FM) was used to observe both the monospecies and mixed-species biofilm morphology. A volume of 100 µl 0.05% (vol/vol) calcofluor white stain (Cat. no. 18909, Sigma-Aldrich, USA) that binds to chitin in fungal cell walls was added to the wells in a dark room together with 50 µl of a 10% potassium hydroxide solution, for better observation of fungal structures. The formed biofilms were observed under ultraviolet (UV) light at 40 X magnification using an Eclipse Ts2-FL epi-fluorescence inverted microscope (Nikon Inc., Japan) with an excitation of 300 to 440 nm, with pictures taken at 6-20 ms exposure.
2.4. CV Staining
For the CV staining assay, individual colonies of freshly grown Candida cultures were transferred to sterile test tubes containing filter-sterilised RPMI-1640 medium and MOPS (N-Morpholino-propanesulfonic acid) (Cat. no. M1254, Sigma-Aldrich, USA) 0.165 M, but without sodium bicarbonate (pH 7.0). After adjusting the inoculum to a 0.5 McFarland standard suspension, suspensions of individual cultures were added to a 96-well microtiter plate (200 µl for individual species and 100 µl for each species for mixed growth). Experiments for each condition were repeated four times and included sterility and negative controls in each tested plate.
The plates were sealed using sealing membranes (Cat. no. P2SEA002Z, Lasec, South Africa) and shaken at 37 °C for 90 min at 75 rpm in a shaking incubator to allow the cells to adhere to the well surfaces. The medium was subsequently aspirated, and the wells were washed with 150 µl PBS to remove loosely adherent cells. After PBS aspiration, 150 µl of fresh RPMI-1640 medium was added to the wells, followed by incubation at 37°C for 66 h at 75 rpm. The medium was aspirated daily, and 150 µl fresh RPMI-1640 medium was added to the wells. The medium was then aspirated, and the wells were washed twice with 200 µl PBS and air-dried for 45 min, followed by staining with 110 µl of 0.4% aqueous CV solution for 45 min, washed four times with 200 µl sterile distilled water and de-stained for 45 min in 200 µl of a 95% ethanol solution, to provide a decolourisation solution which serves as a proxy for the number of cells within the biofilm [30, 31]. A volume of 100 µl of the solution was then transferred from each well to a new plate, and the absorbance was read at OD595 using a FLUOstar Omega spectrophotometer plate reader (BMG Lab-Tech, Germany). Absorbance values for the controls were subtracted from test well values to minimise background interference. The mean absorbance values of all replicate tests were used to compare the biofilm formation results.
3.1. Real-time Biofilm Formation
No toxicity-associated growth reduction was observed with any combination of species and supernatants. All tested species formed biofilms after 7-10 h except for Candida parapsilosis (C. parapsilosis), which had negative CI values when grown on its own (Fig. 1).
Peak CI values occurred between 15-17 h for individual species, except for C. glabrata (11 h). C. albicans had the highest CI values of all tested species, followed by C. glabrata. The highly virulent C. tropicalis and the inherently azole-resistant C. krusei also resulted in positive CI values (indicative of adhesion and biofilm formation) when grown independently. The adhesion of mixed species over time was higher than in monocultures, except for the C. albicans and C. parapsilosis combination (Fig. 1). Although the peak CI values for most combined Candida species were higher than those of individual species, the maximum CI for C. albicans was reduced when in the presence of any of the three species tested, while C. glabrata had a noticeably lower maximum CI when combined with C. parapsilosis (Table 1).
|Mean Individual and Combined Species Maximum CI-Values|
|Ct (0.67)||Ck (0.85)||Cp (-0.17)|
3.2. Bulk Biofilm Morphology Evaluation using FM
When growing the monospecies Candida biofilms, increased biofilm density between 24 h and 48 h was most notable in C. albicans, Candida dubliniensis (C. dubliniensis), C. glabrata and C. tropicalis, which showed a marked increase in biofilm mass after 48 h (shown as confluent growth in the well surface and increased fluorescence) (Fig. 2). When grown independently, Candida parapsilosis failed to demonstrate a noticeable increase in biofilm mass between 24 and 48 h.
When observing the mixed species biofilms, increased cell density indicated the interactions between C. albicans with C. tropicalis and C. parapsilosis. C. parapsilosis combinations with C. dubliniensis and C. glabrata also demonstrated increased cell density. The C. tropicalis combinations with C. glabrata and C. dubliniensis also resulted in a notable increase in cell density.
Candida albicans and C. parapsilosis appeared to form germ tubes and filamentous structures (pseudohyphae and hyphae) as part of their monospecies biofilms, while extensive filamentous forms were prominent in the C. tropicalis monoculture biofilm. In the case of the mixed biofilms, pseudohyphae were extensive in the C. albicans combinations with C. tropicalis and C. parapsilosis, as well as the C. glabrata combinations with the same two species (Fig. 2). A transparent mass surrounding large clusters of cells was also observed, indicating EPS formation.
3.3. Bulk Biofilm Quantification using CV Staining
The 66-hour bulk biofilm production absorbance values (A595) in individual wells ranged between 0.89 and 3.33. When looking at the bulk biofilm production of individual species, C. albicans formed the greatest mass of biofilm, followed by C. tropicalis and the other species (C. albicans > C. tropicalis > C. krusei > C. parapsilosis > C. dubliniensis > C. glabrata). The biofilm production of combined species was the highest for C. albicans/C. tropicalis followed by the mixed biofilm combinations of these and other species (Ca/Ct > Cg/Ct > Cd/Ct > Ca/Cp > Ca/Ck > Cg/Ck > Cd/Ck > Cd/Cp > Cg/Cp).
When using both the novel xCELLigence system and the conventional CV staining method, C. albicans demonstrated the highest absorbance/impedance values when compared to the other species studied, supporting previous studies which have described it as the most notorious biofilm former of all pathogenic yeasts [14, 32]. Most xCELLigence mixed growth curves appeared to follow similarly (albeit increased) adhesion, maturation and detachment phase curves, an interpretation of biofilm formation first described by Gutiérrez et al. .
C. parapsilosis is less likely to produce biofilms, and when it does, the biofilm tends to be thinner and less structured than C. albicans , probably due to C. parapsilosis biofilms having less extracellular material compared to C. albicans, C. glabrata and C. tropicalis biofilms . When using the CV method in our study, C. parapsilosis formed less bulk biofilm than C. albicans, C. tropicalis or C. krusei when grown on their own, and although its presence resulted in biofilm formation when combined with C. albicans, it had much lower biofilm abilities when combined with the other species. Although C. parapsilosis did not form biofilms when grown using the xCELLigence system, it increased CI values over time when combined with C. dubliniensis and C. glabrata. These results could be explained by the fact that the xCELLigence system, which provides continuous monitoring of biofilm formation and automated data analysis, takes into consideration various factors such as changes in cell number and size, adhesion and EPS formation (as opposed to only the bulk biofilm formation measured by CV staining) and that in biofilms, because matrix assembly is coordinated extracellularly, synergistic activity between species may restore matrix structure and function when species lacking a particular carbohydrate can be complemented by neighbouring cells lacking a different carbohydrate .
Although an increase in biofilm mass over time was not observed for C. parapsilosis, fluorescence microscopy allowed for the observation of increased cell density when this organism was grown with the other species, demonstrating interspecies synergism when C. parapsilosis was present. This technique allowed for the observation of cell density and filamentation. Our results concur with previous studies showing extensive filamentation in C. albicans biofilm formations with C. tropicalis and C. parapsilosis [35, 36] and C. glabrata [37, 38]. This switch to pseudohyphal and hyphal morphologies has been associated with increased invasion, penetration and growth in between host epithelial cells , endothelial cell damage  and the destruction of immune cells following phagocytosis . It can therefore be inferred that the extensive pseudohyphae observed in the aforementioned species combinations increase Candida virulence in affected hosts, leading to increased patient morbidity.
A limitation of this study is that we did not include biofilm-defectant mutants of Candida . Homozygous insertions in NUP85, MDS3, KEM1 and SUV3 were reported to block biofilm development in C. albicans in the early stage of biofilm formation [ 42 ] and suggested that Candida mutants are defective in the production of hyphae, which are thought to anchor the biofilm structure. This study provides the basis for further studies to explore the effect of mutant strains on mixed biofilm formation using this system.
Since adhesion is the first step towards the colonisation and invasion of host cells during the infectious process, the ability of the xCELLigence system to (i) monitor, in real-time, the adhesion and formation stages of Candida biofilms, (ii) assess other biofilm formation parameters and (iii) reveal the distinct phases of biofilm formation  could provide a distinct advantage over other biofilm assessment techniques (such as the CV staining method, which only quantifies the bulk biofilm), making it the method of choice for the assessment of interspecies biofilm formation.
The results of this study show that C. albicans, C. dubliniensis and C. glabrata enhance the biofilm formation of C. tropicalis, C. krusei and C. parapsilosis, all of which have different antifungal susceptibility profiles. This is important for the consideration and application of antifungal drugs for the treatment of resistant Candida biofilms. The authors are satisfied that the study's objective was achieved while also recognising the need for further studies to address the nature of the adhesins involved and the morphological changes that may occur.
LIST OF ABBREVIATIONS
|CV||= Crystal Violet|
|EPS||= Extra-cellular Polymeric Substances|
|YPD||= Yeast Peptone Dextrose|
Conceptualization, C.A.; methodology, C.A. and P.A.; investigation, P.A. and K.B.; writing—original draft preparation, P.A.; writing—review and editing, C.A.; visualization, P.A. and K.B.; supervision, C.A. and P.A.; funding acquisition, C.A. and P.A. All authors gave their final approval and agreed to be accountable for all aspects of the work.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONFLICT OF INTEREST
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
AVAILABILITY OF DATA AND MATERIALS
All data is included in the manuscript, and there is no additional data to deposit.
The National Research Foundation of South Africa partially supported this study financially. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors, and therefore, the NRF does not accept any liability in regards thereto.
The authors gratefully acknowledge ACEA Biosciences (San Diego, CA) and Anatech Analytical Technology (Johannesburg, South Africa) for the use of the xCELLigence RTCA equipment, Christelle Klopper from Anatech Instruments for her excellent technical advice and Prof. David Fisher from the Neurobiology Research Group, Department of Medical Biosciences, UWC, for the use of the fluorescence microscope.
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