Next Article in Journal
Synergistic Activation of VDR-RXR Heterodimers by Vitamin D and Rexinoids in Human Kidney and Brain Cells
Previous Article in Journal
Targeting Glucose Metabolism: A Novel Therapeutic Approach for Parkinson’s Disease
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cells
Volume 13
Issue 22
10.3390/cells13221877
cells-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces

by
Enrique Bravo
1,
Marion Arce
2,
David Herrera
1 and
Mariano Sanz
1,*
1
ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) Research Group, Faculty of Dentistry, Complutense University, 28040 Madrid, Spain
2
Department of Conservative Dentistry, Faculty of Dentistry, University of Chile, Santiago 8380544, Chile
*
Author to whom correspondence should be addressed.
Cells 2024, 13(22), 1877; https://doi.org/10.3390/cells13221877
Submission received: 4 October 2024 / Revised: 6 November 2024 / Accepted: 12 November 2024 / Published: 13 November 2024
(This article belongs to the Special Issue Novel Insights into the Biofilms)
Browse Figures
Versions Notes

Abstract

:
The aim of this in vitro study was to evaluate the effect of xanthohumol and thymol on the impact of Candida albicans on the structure, size and cell viability of subgingival biofilms formed on dental implant surfaces. The structure and microbial biomass of biofilms developed after 72 h, treated and untreated with both extracts, were compared by scanning electron microscopy (SEM) and confocal laser microscopy (CLSM). Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total microorganisms of each of the biofilm-forming strains in each condition. A general linear model was used to compare and validate the CLSM and qPCR results. The presence of xanthohumol and thymol during biofilm development inhibited the filamentous growth of C. albicans. The biofilm incubated with xanthohumol had significantly lower bacterial biomass and cell viability than the biofilm not exposed to the extract (p < 0.05). In contrast, these global parameters showed no differences when the biofilm was incubated with thymol. In the presence of xanthohumol, there was a decrease in counts and cell viability of Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Thymol treatment reduced the viability of F. nucleatum and P. gingivalis. The presence of these vegetable extracts during the development of a dynamic in vitro multispecies biofilm model inhibited the filamentous growth of C. albicans, partially reversing the effect that the fungus exerted on the structure, size and vitality of periodontopathogenic bacteria.

1. Introduction

Dental implants are a widely accepted tool to rehabilitate total or partial edulism [1,2], having demonstrated high long-term success rates, although with a high incidence of complications, mainly peri-implant diseases. In the World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions (2017), these diseases were classified as peri-implant mucositis and peri-implantitis, with prevalences ranging from 43 to 47% and 20 to 22%, respectively [3]. Both are inflammatory conditions resulting from the accumulation of biofilms on the surface of dental implants or the associated prosthetic components, which, in the case of peri-implantitis, are associated with progressive bone destruction that, if left untreated, may result in implant loss [4,5,6]. These submarginal biofilms consist of microbial communities within an extracellular matrix. The complex polymicrobial communities consist primarily of bacteria but may include viruses, protozoa, and fungi [7,8,9,10]. In the case of biofilms, composed of both bacteria and fungi, complex inter-kingdom relationships can be established, either favoring bacterial colonization or increasing the intrinsic virulence of these biofilms [11,12,13].
Candida albicans, a facultative dimorphic anaerobic fungus, is the most frequent eukaryotic biofilm-forming pathogenic microorganism in the oral cavity [14]. Although the data from clinical studies are heterogeneous, there is a consistently higher prevalence of C. albicans in biofilms from subjects with periodontitis than those from subjects with good periodontal health [15]. Similarly, the few studies evaluating the prevalence of C. albicans in peri-implant diseases have reported a higher prevalence in patients with peri-implantitis [16,17,18,19].
The pathogenicity of C. albicans is mainly related to its filamentous growth. The development of pseudohyphae is associated with the secretion of hydrolytic enzymes, basically lipases, proteases, and hemolysins, that trigger the chronic inflammatory response associated with the infected tissue [20,21], which, if unresolved, may lead to the alveolar bone resorption characteristic of peri-implantitis [22,23]. A predominance of Gram-negative bacteria associated with the anaerobic environment and nutrient selection associated with the submarginal pocket conditions favors C. albicans growth through hyphae to the detriment of yeast growth [24]. A recent study from our research group has demonstrated that C. albicans growth through pseudohyphae favors the anchorage of bacteria during biofilm maturation, increasing their viability and progression on dental implant surfaces [25]. Furthermore, several in vitro studies have demonstrated the association between fungus and periodontopathogenic bacteria, leading to dysbiotic biofilms and increasing their virulence [11,26,27,28,29,30,31,32].
Therefore, therapies aimed at inhibiting or blocking C. albicans filamentation may facilitate the onset and/or progression of periodontal and peri-implant diseases. In this context, so-called phytochemicals (natural substances of plant origin) exhibiting inhibitory properties in the process of C. albicans filamentation could be considered antifungal agents. These extracts have also shown activity against periodontopathogenic bacteria [33,34,35].
One of these phytochemicals of interest is thymol, an aromatic polyphenol (C10H14O) (Figure 1) found in the leaves of plants such as oregano (Origanum vulgare), thyme (Thymus vulgaris) or basil (Ocimum basilicum). Its mechanism of action includes the suppression of cellulase and peptinase enzyme activities, linked to the hyphal formation process, thus preventing the filamentous growth of the fungi [36,37].
A second phytochemical of interest is xanthohumol, a prenylated flavonoid (C21H22O5) (Figure 2) from the female flowers of the hop plant (Humulus lupulus) that exhibits antiseptic, anti-inflammatory, antidiuretic, and antioxidant properties [38]. The mechanism of action of xanthohumol is related to its inhibition of quorum sensing and de-stabilization of the microbial cell wall [39], with demonstrated activity against C. albicans biofilms by limiting their ability to adhere and form filaments [40,41].
Thus, this in vitro investigation aimed to determine whether inhibition of C. albicans pseudohyphal growth using thymol and xanthohumol would impact biofilm growth and pathogenicity.

2. Materials and Methods

2.1. Microbial Strains and Culture Conditions

Bacterial strains Streptococcus oralis CECT 907T, Actinomyces naeslundii ATCC 19039, Veillonella parvula NCTC 11810, Fusobacterium nucleatum DMSZ 20482, Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans DSMZ 8324 were grown on blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg/L haemin (Sigma, St. Louis, MO, USA) and 1.0 mg/L menadione (Merck, Darmstadt, Germany) at 37 °C for 24–72 h under anaerobic conditions (10% H2, 10% CO2 and N2 balance). The fungal strain Candida albicans SC 5314 was grown on yeast-peptone-glucose (YPD) agar plates (2% glucose (Panreac, Barcelona, Spain), 2% peptone (Life Technologies, Detroit, MI, USA), 1% peptone yeast extract (Life Technologies, Detroit, MI, USA) and 2% agar (Becton, Dickinson and Company, Sparks, MD, USA) at 37 °C for 24 h under aerobic conditions.

2.2. Minimum Inhibitory Concentration (MIC) of Xanthohumol and Thymol Against the Microorganisms Included in the Biofilm Model

To select sublethal concentrations of thymol and xanthohumol for their application in the biofilm model, MICs against each bacterium and C. albicans were determined [42].
Isolated colonies of each microorganism were grown in a protein-enriched BHI-modified medium adjusted to pH 7.2 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), which was supplemented with 2.5 g/L mucin (Oxoid), 1.0 g/L yeast extract (Oxoid), 0.1 g/L cysteine (Sigma), 2.0 g/L sodium bicarbonate (Merck), 5.0 mg/L haemin (Sigma), 1.0 mg/L menadione (Merck) and 0.25% (v/v) glutamic acid (Sigma) at 37 °C under anaerobic conditions (10% H2, 10% CO2 and N2 equilibrium). Isolated C. albicans colonies were grown in a liquid YPD medium under aerobic conditions at 37 °C.
The exponential growth phase was detected spectrophotometrically, with cultures always below an optical density (OD550 nm) of 1.2. Once exponential growth was reached, 200 μL of each inoculum was transferred to a 24-well microplate, reaching a final concentration of 106 colony-forming units (CFU)/mL. Then, xanthohumol (NATECO® GmbH & Co, Wolnzach, Germany) and thymol (Sigma-Aldrich®, Steinheim, Germany) were added at final concentrations of 6.25, 12.5, 25, 50, 100, 250, 500, and 1000 µM in both cases, using phosphate-buffer saline (PBS) as a negative control. These microplates were incubated for 24 h at 37 °C under anaerobic conditions. The MICs of each extract were determined on blood agar plates for the bacteria and YPD plates for C. albicans, on which 100 µL of each suspension was seeded. The plates were incubated for 72 h at 37 °C under anaerobic conditions. The lowest concentrations of thymol and xanthohumol extracts showing visible inhibition of microbial growth were considered the MICs of each extract for each strain, thus selecting a lower concentration range to ensure no effect on cell viability.
All tests were carried out in triplicate, with the respective controls for contamination.

2.3. Agar Invasive Assays of C. albicans in the Presence of Xanthohumol and Thymol

To evaluate the inhibitory effect of thymol and xanthohumol on the pseudohyphal growth of C. albicans, agar invasiveness assays were performed in the presence of both substances [43].
Isolated C. albicans colonies were grown in a liquid YPD medium for 24 h at 37 °C under aerobic conditions. The exponential growth phase was detected spectrophotometrically, with the culture always below an optical density (OD550 nm) of 1.2. Once this exponential growth was reached, 10 μL droplets were plated at an optical density equivalent to 104 CFU/mL on YPG agar plates (2% galactose (Panreac, Barcelona, Spain), 2% peptone (Life Technologies, Detroit, MI, USA), 1% peptone yeast extract (Life Technologies, Detroit, MI, USA) and 2% agar (Becton, Dickinson and Company, Sparks, MD, USA). The absence of glucose and the presence of galactose in this growth medium stimulated filamentous growth of C. albicans [44]. To evaluate the inhibitory effect on filamentation, these culture plates that contained xanthohumol and thymol at concentrations of 0, 6.25, 12.5, 25, 50 and 100 µM and 0, 62.5, 125, 250, 500 and 1000 µM, respectively, were grown at 37 °C for 48 h under aerobic conditions. After incubation, the inoculation sites were washed by exposing the plates to dripping tap water, and the invasiveness of the culture grown on the agar was assessed in each case. The minimum concentration of each extract at which a reduction in C. albicans invasion into the agar was observed was established as optimal for use in the biofilm model.
Tests were carried out in triplicate.

2.4. In Vitro Dynamic Multispecies Biofilm Model

A previously validated multispecies dynamic biofilm in vitro model was applied on dental implant surfaces [35,45]. The system consists of a sterile vessel, where the modified BHI medium flows into the bioreactor using a peristaltic pump. The bioreactor (Lambda Minifor© bioreactor, LAMBDA Laboratory Instruments, Sihlbruggstrasse, Switzerland) maintained the culture medium in the selected stable conditions of the oral cavity (temperature of 37 °C, a pH of 7.2, and an anaerobic atmosphere (10% H2, 10% CO2 and N2 balance).
To prepare the bacterial and fungal pre-inoculum, a pure culture of each strain was grown for 24 h under the conditions described above. Microbial growth was measured spectrophotometrically, and a microbial mixture of 106 colony-forming units (CFU)/mL of each bacterium and 104 CFU/mL of C. albicans was obtained.
The vessel was inoculated with 5 mL of the microbial mixture and incubated for 12 h under the described conditions. When the multispecies culture grew to the exponential phase, a second peristaltic pump was activated, with a constant flow rate of 30 mL/h, to start continuous culture and transfer the culture to the Robbins device, inside which the dental implants on which the biofilm developed were placed (Straumann® Tissue Level Standard, Straumann Institute AG, Basel, Switzerland). Implant size was 8 mm in length and 3.3 mm in diameter, with the patented SLA surface: moderately rough, sand-sprayed and acid-etched surface. They were kept for 72 h under the conditions described in the interior of the Robbins device until the formation of mature biofilms.
To evaluate the effect of thymol and xanthohumol on the growth and filamentation of C. albicans and, consequently, on sub-marginal biofilm formation, 72 h mature biofilms were developed under the following conditions:
  • Negative control: biofilms were developed for 72 h using the six bacterial strains of the model.
  • Positive control: the six bacterial strains of the model and C. albicans included in biofilms for 72 h to generate the dysbiotic event.
  • Xanthohumol treatment: The six bacterial strains model and C. albicans were included in biofilms for 72 h. Xanthohumol was injected (12.5 µM) into the Robbins device at 12 h intervals.
  • Thymol treatment: the six-bacterial-strain model and C. albicans were included in biofilms for 72 h. Thymol was injected (250 µM) into the Robbins device at 12 h intervals.
For each mentioned condition, three implants were analyzed by scanning electron microscopy (SEM) (n = 3), three by confocal laser scanning microscopy (CLSM) (n = 3), and nine by real-time quantitative polymerase chain reaction (qPCR) (n = 9).

2.5. Scanning Electron Microscopy (SEM)

Following the optimized protocol for analyzing biofilm samples by SEM [45], the implants were removed from the Robbins device and were washed, dried, and coated with gold before analysis. The samples were analyzed using a JSM 6400 electron microscope (JSM6400, JEOL, Tokyo, Japan).
This analysis was conducted at the National Centre of Electron Microscopy (In-stalación Científico-Técnico singular; ICTS) at the Moncloa Campus of the Complutense University of Madrid (Madrid, Spain).

2.6. Confocal Laser Scanning Microscopy (CSLM)

Samples were extracted, washed, and stained using the LIVE/DEAD® BacLightTM bacterial viability kit solution and 3% Calcofluor White for bacteria and C. albicans, respectively, following the protocols reported by Blanc et al. and Bravo et al. [25,45].
A Leica® LCS SP8 STED 3X inverted microscope (Mannheim, Germany) was used for noninvasive confocal imaging of biofilms. The COMSTAT 2.1 software (www.comstat.dk (accessed on April, May, June and July 2024)) was used to calculate the biomass in micrometres3/micrometres2 (µm3/µm2) and the roughness coefficient (Ra*) of the CLSM acquisitions.
This analysis was performed at the Biological Research Centre Margarita Salas (Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas—CIB-CSIC), located at the Moncloa Campus of the Complutense University of Madrid (Madrid, Spain).

2.7. Quantitative Polymerase Chain Reaction (qPCR)

Samples were extracted, washed, and disaggregated from the implants following the method described by Sánchez et al. [46]. Fractions of 200 µL of each sample were dissociated, treating one of them with propidium monoazide (PMA) to quantify each species’ viability percentage in each condition [25].
DNA was obtained using the commercial kit MolYsisComplete5 (Molzym GmbH & CoKG, Bremen, Germany), following the manufacturer’s instructions (the protocol for microbial DNA extraction will be followed from step 6, avoiding preliminary steps). Primers and probes selected were provided by Life Technologies Invitrogen (Carlsbad, CA, USA), Applied Biosystems (Carlsbad, CA, USA), and Roche (Roche Diagnostic GmbH, Mann-heim, Germany).
The amplification reaction was performed in a total mix volume of 10 μL. Reaction mixtures included 5 μL of MasterMix 2x (LC 480 Probes Master, Roche), optimal primer and probe concentrations, and 2.5 μL of DNA extracted from the samples. The negative control was 2.5 μL of sterile water (non-template control (NTC)) (Roche) [45,47].
The amplification program used included an initial cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min in a LightCycler® 480 II thermal cycler (Roche Diagnostic GmbH, Mannheim, Germany). LightCycler480 Multiwell Plate 384 White microplates (Roche) were used.
Each DNA sample was analyzed in duplicate. The provided software determined the quantification cycle (Cq) values (LC 480 Software 1.5, Roche). For cell quantification, the data reported was extrapolated with previously designed standard curves with the Cq values generated in qPCR vs. log CFU/mL. The software (LC 480 Software 1.5, Roche) automatically generated a correlation between Cq values and CFU/mL.

2.8. Statistical Analysis

The primary outcome variable was the counts of viable and total bacteria present in the biofilm of each condition, measured by qPCR and expressed as CFU/mL. The secondary outcome variable was the microbial biomass obtained by CLSM and expressed in µm3/µm2. Data are shown as means and standard deviations (SDs). The Shapiro–Wilk good-ness-of-fit test was applied to assess data normality. When the two sets of data compared showed a normal distribution, a T-test with Welch’s correction was executed. When at least one of the two groups did not show a normal distribution, a Mann–Whitney test was used. Comparisons that reported p-values < 0.05 were considered statistically significant.
GraphPad Prism software was used for all data analysis.

3. Results

3.1. MICs of Thymol and Xanthohumol on Planktonic Microorganisms

For xanthohumol, the MICs were set at 25 µM for S. oralis, V. parvula, A. naeslundii, and F. nucleatum, 50 µM for P. gingivalis and C. albicans, and 100 µM for A. actinomycetemcomitans.
For thymol, the MICs were set at 500 µM for S. oralis, A. naeslundii, V. parvula, F. nucleatum, A. actinomycetemcomitans, and C. albicans, and 1 mM for P. gingivalis.

3.2. Agar Invasive Assays of C. albicans in the Presence of Thymol and Xanthohumol

The ability of xanthohumol and thymol to inhibit filamentous growth of C. albicans was tested by assessing the fungus’s ability to invade agar at increasing concentrations of both substances.
As shown in Figure 3, the MICs of xanthohumol and thymol on C. albicans filamentation were 12.5 and 250 µM, respectively. The surface yeast growth was not altered at these concentrations of both extracts.

3.3. SEM Analysis

Figure 4 shows the biofilms developed after 72 in the presence of C. albicans treated with thymol and xanthohumol, without treatment (positive control), and in the absence of fungus (negative control).
C. albicans hyphae developed during the biofilm’s maturation resulted in architecture with increased robustness and solidity (Figure 4B). The filaments acted as anchoring support for bacteria, mainly fusiform forms (most probably F. nucleatum) and coccobacilli (P. gingivalis and A. actinomycetemcomintans) (Figure 4F).
Treatments with thymol and, to a greater extent, with xanthohumol prevented the filamentous development of C. albicans, resulting in less compact biofilms, where bacterial cells aggregated without the yeast support (Figure 4G,H). In fact, biofilms developed in the presence of the fungus and treated with both extracts (Figure 4C,D) depicted a similar appearance to the mature biofilm without C. albicans (Figure 4A,E).

3.4. CLSM Analysis

Figure 5 and Figure 6 show the impact of xanthohumol and thymol treatment on biofilms developed in the presence of C. albicans. These mature biofilms, in the presence of C. albicans (positive control), demonstrated higher bacterial biomass compared with the negative control biofilms (6.77 µm3/µm2, SD = 1.32 and 3.39 µm3/µm2, SD = 2.19; respectively). Furthermore, as shown in Figure 5A,B, the bacterial viabilities of the biofilms developed with and without C. albicans were 60.33% (SD = 17.55%), and 31.12% (SD = 12.83%), respectively, demonstrating statistically significant differences (p < 0.05). Figure 5E shows the biomass corresponding to C. albicans, which was 7.26 µm3/µm2 (SD = 1.64).
When these mixed biofilms were treated with xanthohumol during their development (Figure 5C), the bacterial density and cell viability were lower compared to non-treated biofilms (3.38 µm3/µm2, SD = 1.61 and 25.55%, SD = 9.2%; respectively). Similarly, the fungal biomass was significantly lower than the controls, being 3.48 µm3/µm2 (SD = 0.8), (Figure 5F). The same biofilms incubated with thymol (Figure 5D) demonstrated similar patterns, with reduced bacterial density (4.64 µm3/µm2, SD = 4.23) and a lower percentage of cell viability (44.63%, SD = 12.91%) when compared with the untreated biofilm. Similarly, the density of C. albicans also decreased (5.88 µm3/µm2, SD = 3.5) after thymol treatment (Figure 5G).
In addition, the roughness coefficients (Ra*) of biofilms developed with C. albicans and incubated with xanthohumol (2.34, SD = 0.47) and thymol (2.58, SD = 0.39) were significantly higher than those grown in the absence of the extracts (2.04 SD = 0.22) and similar to those developed without the fungus (2.47, SD = 0.72).

3.5. Quantitative and Qualitative Analysis by qPCR

Biofilms developed in the presence of C. albicans demonstrated significantly higher counts of F. nucleatum and P. gingivalis, a pattern also observed to a lesser extent on A. actinomycetemcomitans. Similarly, the cell viability of S. oralis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was also significantly higher than in biofilms grown without the fungus (Figure 7 and Table S1).
The mixed biofilm treatment with xanthohumol significantly decreased the amount of F. nucleatum and P. gingivalis. Cell viability of F. nucleatum, P. gingivalis, and A. actinomycetemcomitans was also significantly reduced in the same biofilms incubated with xanthohumol. The viability ratio of these bacteria was similar to that obtained in biofilms developed without the fungus and treatment. This effect was observed to a more limited extent for S. oralis.
The impact of thymol on biofilms with C. albicans resulted in lower counts of P. gingivalis and lower cell viability of F. nucleatum and P. gingivalis, reductions that were statistically significant when compared to the untreated mixed biofilms. No effect of thymol was observed on A. actinomycetemcomitans and the effect on P. gingivalis was significantly reduced compared to that exerted by xanthohumol.
The treatments with both substances had no quantitative impact on C. albicans, A. naeslundii, and V. parvula.

4. Discussion

The results of the present in vitro study have clearly shown that the treatment with phytochemicals, mainly xanthohumol and, to a lesser extent, thymol, inhibited the development of pseudohyphae during the biofilm growth of C. albicans, which significantly influenced the maturation of a validated in vitro dynamic multispecies biofilm model using periodontopathogenic bacterial strains developed on dental implant surfaces. These treatments impacted the biofilm size and the cell viability of F. nucleatum, P. gingivalis, A. actinomycetemcomitans, and S. oralis bacterial strains.
The SEM images (Figure 4) revealed the positive interaction between the C. albicans filaments and the biofilm-forming bacteria, resulting in larger and well-structured biofilm architectures. In fact, the hyphal formation favored by the conditions of the peri-implant sulcus/pocket (37 °C, presence of Gram-negative bacteria, and anaerobic environment) is the main virulence factor of C. albicans [24] and these filamentous forms are associated with the secretion of hydrolytic enzymes such as proteases, lipases, and hemolysins and the production of inflammatory cytokines that favor the unresolved chronic inflammatory condition [20,48]. Furthermore, the presence of C. albicans has been linked to the etiopathology of peri-implant diseases through their dysbiotic effect by increasing the quantity and cell viability of periodontopathogenic bacteria [25] and generating positive inter-microbial aggregates and interactions (Figure 5 and Figure 7).
The treatment with phytochemicals, namely xanthohumol, prevented these complexes’ formation during the biofilm maturation on the implant surfaces (Figure 4). This antifungal activity is based on the inhibition of mycelial growth and spore germination by interfering with the regulation of cell wall synthesis and cell wall stability [49]. This study also reports the additional ability of xanthohumol to inhibit the morphological transition from yeast to filamentous form in C. albicans, both in pure cultures and mixed biofilms (Figure 4). It should be noted that the application of xanthohumol doses below the MIC did not affect fungal viability (Figure 3 and Figure 7), but impacted the filamentation regulation, probably mediated by a transcriptional regulatory network (TRN) leading to the inhibition of the Ras1-Cyr1/cAMP-PKA pathway, which was involved in hyphal synthesis [50]. TRN comprises nine transcription factors with prion-binding domains (PrLDs) at the ROB19465 allele [51,52]. The inhibition of the filamentous growth in the xanthohumol-incubated biofilms led to smaller biofilms (Figure 5), probably by diminishing the bacterial adhesion and migratory motility mediated by the C. albicans hyphae [11]. In addition, xanthohumol has a potential inhibitory effect on quorum-sensing, which may decrease the accumulation of extracellular polymeric substances (EPSs) generated by C. albicans within the biofilm matrix, thus resulting in biofilms with a less complex architecture [53].
Similarly, the treated complex biofilms, compared with untreated controls, resulted in significant reductions in the viability of F. nucleatum, P. gingivalis, A. actinomycetemcomitans, and, to a lesser extent, S. oralis (Figure 7 and Table S1). Thus, the increase in these bacteria reported with biofilms grown in the presence of C. albicans was partially or completely reversed when the biofilm was incubated with xanthohumol. By inhibiting the filamentous growth, there could be a reduced interaction between the fungal cell wall protein FLO9 and the bacterial adhesin radD of F. nucleatum, thus affecting their cell survival and, consequently, limiting the mechanical bridging role that C. albicans may play between primary colonizers and late colonizers, such as P. gingivalis and A. actinomycetemcomitans [29,30,54]. Similarly, the absence of C. albicans filaments may prevent the formation of the anaerobic environments generated by the hyphae, thus limiting the viability of P. gingivalis and A. actinomycetemcomitans [27,55]. Along the same lines, the interlin InlJ, involved in the expression of P. gingivalis genes related to the interaction with C. albicans hyphae, and the adhesins Als3, associated with the interaction with the fungal Sap6 and Sap 9 proteases, may have their activity reduced, decreasing the aggregation of the bacteria [26,55]. These molecular interactions may also occur at the level of the initial colonizers, although the impact of xanthohumol was lesser on S. oralis. A possible mechanism may be the inhibition of the specific, dual, affinity-based interaction between the fungal adhesins Als1, Als2, Als3, and Hwp1 and the cell wall receptors SspA and SspB [56], which may be related to the smaller overall biofilm size observed by CLSM. Furthermore, the inhibition of filament formation may also reduce the interactions between cocci and the gtfR glucan-binding domain in the C. albicans cell wall [57].
Thymol also inhibited C. albicans filamentous growth similar to xanthohumol (Figure 3 and Figure 4) through the interaction with the enzymatic activities involved in hyphal cell wall formation [36,37]. However, the overall impact of thymol on the size and viability of multispecies biofilms developed with C. albicans was notably less than that observed with xanthohumol (Figure 5). In fact, when these mixed biofilms were incubated with thymol, it only affected the vitality of F. nucleatum and P. gingivalis, and always to a lesser extent compared with xanthohumol (Figure 7 and Table S1). This is depicted in Figure 6, with mixed biofilms incubated with xanthohumol having a clear lesser overall biomass than biofilms developed in thymol.
In the present work, the use of sublethal concentrations of xanthohumol and thymol, which did not influence the growth of the bacterial strains, prevented the filamentous development of C. albicans, which resulted in a decrease in the size and viability of the resulting biofilms. Thus, the ratio of viable anaerobic periodontopathogenic bacteria in the biofilm decreased with the treatments. In addition, the roughness of the biofilms incubated in the presence of the extracts increased significantly, compared to the control biofilms.
In a global context where resistance to antibiotic and antifungal agents is increasing [58], the results of the present study indicate the potential of phytochemicals as an alternative in the prevention and treatment of peri-implant and periodontal diseases. Indeed, thymol and xanthohumol, applied at appropriate concentrations, have shown a strong bactericidal effect against F. nucleatum, P. gingivalis, A. actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus and Treponema denticola [33,34], against multispecies oral biofilms containing C. albicans [59], against S. mutans [60] and against multispecies biofilms formed by the same bacterial strains used in this study [35].
Some limitations of this in vitro study should also be noted: (1) the in vitro biofilm model used is intended to represent the biological diversity of the oral cavity, but natural supramarginal and subgingival biofilms consist of hundreds of microbial species; (2) although the used dynamic biofilm model mimics the oral cavity conditions, there are variables that cannot be reproduced, including different behaviors and immune responses of patients, what makes it difficult to replicate in vivo the conditions of this in vitro research; (3) working with live microorganisms is associated with a higher than desirable variability, which should be considered when interpreting the results, especially in the biofilms incubated with thymol.
However, the qualitative and quantitative results reported in the present study, as well as their statistical processing, suggest that the exacerbation of the quantity and viability of the periodontopathogenic bacteria F. nucleatum, P. gingivalis, and A. actinomycetemcomitans, in biofilms developed in the presence of C. albicans, is due to the filamentous growth of the fungus and that this effect can be controlled by inhibiting the formation of these fungal hyphae. These findings highlight the possibility of using therapies with dual action, being antimicrobial at the appropriate lethal concentrations and, at the same time, interfering with critical biofilm intermicrobial interactions.
In conclusion, xanthohumol and, to a lesser extent, thymol have been shown to inhibit, in vitro, the pseudohyphal growth and filamentation of C. albicans, thus reverting the dysbiotic effect that C. albicans exerts on the growth of complex biofilms on implant surfaces.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cells13221877/s1: Table S1: Counts (expressed as mean and standard deviation (SD) and 95% confidence intervals (CIs)) of total and live microbial species (colony-forming units (CFUs)/mL) determined by quantitative polymerase chain reaction (qPCR) in control negative biofilms, developed without Candida albicans (C−), positive control mixed biofilms, developed with C. albicans (C+), mixed biofilms developed with C. albicans and incubated with xanthohumol (X) and mixed biofilms developed with C. albicans and incubated with thymol (T) (n = 9), using specific primers and probes directed to the 16S rRNA gene of bacterial strains and ITS2 region of C. albicans.

Author Contributions

E.B., D.H. and M.S. designed the study. E.B. performed the experiments and analyzed and interpreted the collected data. E.B. and M.A. performed the statistical analysis. E.B. drafted the manuscript. M.A., D.H. and M.S. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study has been self-funded by the ETEP Research group (University Complutense of Madrid, Spain).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors would like to acknowledge the technical support of M.G. Elvira and M.T. Seisdedos at the Confocal and Fluorescence Microscopy Service of Margarita Salas Biological Research Center (Superior Center for Scientific Research), A.M. Vicente at the ICTS National Centre of Electron Microscopy (University Complutense, Madrid, Spain). and R. Ayuso at the Platform of Mechanic Workshops (University Complutense, Madrid, Spain). We also thank J. Arroyo and R. García at the Faculty of Pharmacy (University Complutense, Madrid, Spain) for the loan of the SC 5314 strain of Candida albicans and xanthohumol.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Buser, D.; Janner, S.F.; Wittneben, J.G.; Bragger, U.; Ramseier, C.A.; Salvi, G.E. 10-year survival and success rates of 511 titanium implants with a sandblasted and acid-etched surface: A retrospective study in 303 partially edentulous patients. Clin. Implant. Dent. Relat. Res. 2012, 14, 839–851. [Google Scholar] [CrossRef] [PubMed]
  2. Buser, D.; Sennerby, L.; De Bruyn, H. Modern implant dentistry based on osseointegration: 50 years of progress, current trends and open questions. Periodontol. 2000 2017, 73, 7–21. [Google Scholar] [CrossRef] [PubMed]
  3. Herrera, D.; Berglundh, T.; Schwarz, F.; Chapple, I.; Jepsen, S.; Sculean, A.; Kebschull, M.; Papapanou, P.N.; Tonetti, M.S.; Sanz, M.; et al. Prevention and treatment of peri-implant diseases-The EFP S3 level clinical practice guideline. J. Clin. Periodontol. 2023, 50 (Suppl. 26), 4–76. [Google Scholar] [CrossRef] [PubMed]
  4. Derks, J.; Tomasi, C. Peri-implant health and disease. A systematic review of current epidemiology. J. Clin. Periodontol. 2015, 42 (Suppl. 16), S158–S171. [Google Scholar] [CrossRef]
  5. Burgers, R.; Gerlach, T.; Hahnel, S.; Schwarz, F.; Handel, G.; Gosau, M. In vivo and in vitro biofilm formation on two different titanium implant surfaces. Clin. Oral. Implants Res. 2010, 21, 156–164. [Google Scholar] [CrossRef]
  6. Ferreira Ribeiro, C.; Cogo-Muller, K.; Franco, G.C.; Silva-Concilio, L.R.; Sampaio Campos, M.; de Mello Rode, S.; Claro Neves, A.C. Initial oral biofilm formation on titanium implants with different surface treatments: An in vivo study. Arch. Oral. Biol. 2016, 69, 33–39. [Google Scholar] [CrossRef]
  7. Kato, A.; Imai, K.; Sato, H.; Ogata, Y. Prevalence of Epstein-Barr virus DNA and Porphyromonas gingivalis in Japanese peri-implantitis patients. BMC Oral. Health 2017, 17, 148. [Google Scholar] [CrossRef]
  8. Sahrmann, P.; Gilli, F.; Wiedemeier, D.B.; Attin, T.; Schmidlin, P.R.; Karygianni, L. The Microbiome of Peri-Implantitis: A Systematic Review and Meta-Analysis. Microorganisms 2020, 8, 661. [Google Scholar] [CrossRef]
  9. Samaranayake, L.; Matsubara, V.H. Normal Oral Flora and the Oral Ecosystem. Dent. Clin. N. Am. 2017, 61, 199–215. [Google Scholar] [CrossRef]
  10. Montelongo-Jauregui, D.; Srinivasan, A.; Ramasubramanian, A.K.; Lopez-Ribot, J.L. An In Vitro Model for Candida albicans(-)Streptococcus gordonii Biofilms on Titanium Surfaces. J. Fungi 2018, 4, 66. [Google Scholar] [CrossRef]
  11. Ren, Z.; Jeckel, H.; Simon-Soro, A.; Xiang, Z.; Liu, Y.; Cavalcanti, I.M.; Xiao, J.; Tin, N.N.; Hara, A.; Drescher, K.; et al. Interkingdom assemblages in human saliva display group-level surface mobility and disease-promoting emergent functions. Proc. Natl. Acad. Sci. USA 2022, 119, e2209699119. [Google Scholar] [CrossRef] [PubMed]
  12. Nett, J.E.; Andes, D.R. Contributions of the Biofilm Matrix to. J. Fungi 2020, 6, 21. [Google Scholar] [CrossRef]
  13. Scherlach, K.; Hertweck, C. Chemical Mediators at the Bacterial-Fungal Interface. Annu. Rev. Microbiol. 2020, 74, 267–290. [Google Scholar] [CrossRef] [PubMed]
  14. Ghannoum, M.A.; Jurevic, R.J.; Mukherjee, P.K.; Cui, F.; Sikaroodi, M.; Naqvi, A.; Gillevet, P.M. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 2010, 6, e1000713. [Google Scholar] [CrossRef] [PubMed]
  15. Canabarro, A.; Valle, C.; Farias, M.R.; Santos, F.B.; Lazera, M.; Wanke, B. Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis. J. Periodontal Res. 2013, 48, 428–432. [Google Scholar] [CrossRef]
  16. Leonhardt, A.; Renvert, S.; Dahlen, G. Microbial findings at failing implants. Clin. Oral. Implants Res. 1999, 10, 339–345. [Google Scholar] [CrossRef]
  17. Schwarz, F.; Becker, K.; Rahn, S.; Hegewald, A.; Pfeffer, K.; Henrich, B. Real-time PCR analysis of fungal organisms and bacterial species at peri-implantitis sites. Int. J. Implant. Dent. 2015, 1, 9. [Google Scholar] [CrossRef]
  18. Alrabiah, M.; Alshagroud, R.S.; Alsahhaf, A.; Almojaly, S.A.; Abduljabbar, T.; Javed, F. Presence of Candida species in the subgingival oral biofilm of patients with peri-implantitis. Clin. Implant. Dent. Relat. Res. 2019, 21, 781–785. [Google Scholar] [CrossRef]
  19. Alsahhaf, A.; Al-Aali, K.A.; Alshagroud, R.S.; Alshiddi, I.F.; Alrahlah, A.; Abduljabbar, T.; Javed, F.; Vohra, F. Comparison of yeast species in the subgingival oral biofilm of individuals with type 2 diabetes and peri-implantitis and individuals with peri-implantitis without diabetes. J. Periodontol. 2019, 90, 1383–1389. [Google Scholar] [CrossRef]
  20. Mayer, F.L.; Wilson, D.; Hube, B. Candida albicans pathogenicity mechanisms. Virulence 2013, 4, 119–128. [Google Scholar] [CrossRef]
  21. De-La-Torre, J.; Quindos, G.; Marcos-Arias, C.; Marichalar-Mendia, X.; Gainza, M.L.; Eraso, E.; Acha-Sagredo, A.; Aguirre-Urizar, J.M. Oral Candida colonization in patients with chronic periodontitis. Is there any relationship? Rev. Iberoam. Micol. 2018, 35, 134–139. [Google Scholar] [CrossRef] [PubMed]
  22. Martin, R.; Wachtler, B.; Schaller, M.; Wilson, D.; Hube, B. Host-pathogen interactions and virulence-associated genes during Candida albicans oral infections. Int. J. Med. Microbiol. 2011, 301, 417–422. [Google Scholar] [CrossRef] [PubMed]
  23. Dongari-Bagtzoglou, A.; Kashleva, H.; Villar, C.C. Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells. Med. Mycol. 2004, 42, 531–541. [Google Scholar] [CrossRef] [PubMed]
  24. Chow, E.W.L.; Pang, L.M.; Wang, Y. From Jekyll to Hyde: The Yeast-Hyphal Transition of Candida albicans. Pathogens 2021, 10, 859. [Google Scholar] [CrossRef] [PubMed]
  25. Bravo, E.; Arce, M.; Ribeiro-Vidal, H.; Herrera, D.; Sanz, M. The Impact of Candida albicans in the Development, Kinetics, Structure, and Cell Viability of Biofilms on Implant Surfaces—An In Vitro Study with a Validated Multispecies Biofilm Model. Int. J. Mol. Sci. 2024, 25, 3277. [Google Scholar] [CrossRef]
  26. Bartnicka, D.; Karkowska-Kuleta, J.; Zawrotniak, M.; Satala, D.; Michalik, K.; Zielinska, G.; Bochenska, O.; Kozik, A.; Ciaston, I.; Koziel, J.; et al. Adhesive protein-mediated cross-talk between Candida albicans and Porphyromonas gingivalis in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment. Sci. Rep. 2019, 9, 4376. [Google Scholar] [CrossRef]
  27. de Jongh, C.A.; Bikker, F.J.; de Vries, T.J.; Werner, A.; Gibbs, S.; Krom, B.P. Porphyromonas gingivalis interaction with Candida albicans allows for aerobic escape, virulence and adherence. Biofilm 2024, 7, 100172. [Google Scholar] [CrossRef]
  28. Guo, Y.; Wang, Y.; Wang, Y.; Jin, Y.; Wang, C. Heme Competition Triggers an Increase in the Pathogenic Potential of Porphyromonas gingivalis in Porphyromonas gingivalis-Candida albicans Mixed Biofilm. Front. Microbiol. 2020, 11, 596459. [Google Scholar] [CrossRef]
  29. Wu, T.; Cen, L.; Kaplan, C.; Zhou, X.; Lux, R.; Shi, W.; He, X. Cellular Components Mediating Coadherence of Candida albicans and Fusobacterium nucleatum. J. Dent. Res. 2015, 94, 1432–1438. [Google Scholar] [CrossRef]
  30. Delaney, C.; Alapati, S.; Alshehri, M.; Kubalova, D.; Veena, C.L.R.; Abusrewil, S.; Short, B.; Bradshaw, D.; Brown, J.L. Investigating the role of Candida albicans as a universal substrate for oral bacteria using a transcriptomic approach: Implications for interkingdom biofilm control? APMIS 2023, 131, 601–612. [Google Scholar] [CrossRef]
  31. Bachtiar, E.W.; Bachtiar, B.M.; Jarosz, L.M.; Amir, L.R.; Sunarto, H.; Ganin, H.; Meijler, M.M.; Krom, B.P. AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation. Front. Cell Infect. Microbiol. 2014, 4, 94. [Google Scholar] [CrossRef] [PubMed]
  32. Xu, H.; Sobue, T.; Bertolini, M.; Thompson, A.; Dongari-Bagtzoglou, A. Streptococcus oralis and Candida albicans Synergistically Activate mu-Calpain to Degrade E-cadherin From Oral Epithelial Junctions. J. Infect. Dis. 2016, 214, 925–934. [Google Scholar] [CrossRef] [PubMed]
  33. Lauritano, D.; Pazzi, D.; Iapichino, A.; Gaudio, R.M.; Di Muzio, M.; Lo Russo, L.; Pezzetti, F. Evaluation of the efficacy of a new oral gel containing carvacrol and thymol for home oral care in the management of chronic periodontitis using PCR analysis: A microbiological pilot study. J. Biol. Regul. Homeost. Agents 2016, 30, 129–134. [Google Scholar] [PubMed]
  34. Idir, F.; Van Ginneken, S.; Coppola, G.A.; Grenier, D.; Steenackers, H.P.; Bendali, F. Origanum vulgare ethanolic extracts as a promising source of compounds with antimicrobial, anti-biofilm, and anti-virulence activity against dental plaque bacteria. Front. Microbiol. 2022, 13, 999839. [Google Scholar] [CrossRef] [PubMed]
  35. Alonso-Espanol, A.; Bravo, E.; Ribeiro-Vidal, H.; Virto, L.; Herrera, D.; Alonso, B.; Sanz, M. The Antimicrobial Activity of Curcumin and Xanthohumol on Bacterial Biofilms Developed over Dental Implant Surfaces. Int. J. Mol. Sci. 2023, 24, 2335. [Google Scholar] [CrossRef]
  36. Priya, A.; Nivetha, S.; Pandian, S.K. Synergistic Interaction of Piperine and Thymol on Attenuation of the Biofilm Formation, Hyphal Morphogenesis and Phenotypic Switching in Candida albicans. Front. Cell Infect. Microbiol. 2022, 11. [Google Scholar] [CrossRef]
  37. Ranjbar, A.; Ramezanian, A.; Shekarforoush, S.; Niakousari, M.; Eshghi, S. Antifungal activity of thymol against the main fungi causing pomegranate fruit rot by suppressing the activity of cell wall degrading enzymes. Lwt-Food Sci. Technol. 2022, 161, 113303. [Google Scholar] [CrossRef]
  38. Rozalski, M.; Micota, B.; Sadowska, B.; Stochmal, A.; Jedrejek, D.; Wieckowska-Szakiel, M.; Rozalska, B. Antiadherent and antibiofilm activity of Humulus lupulus L. derived products: New pharmacological properties. Biomed. Res. Int. 2013, 2013, 101089. [Google Scholar] [CrossRef]
  39. Leoni, L.; Landini, P. Microbiological methods for target-oriented screening of biofilm inhibitors. Methods Mol. Biol. 2014, 1147, 175–186. [Google Scholar] [CrossRef]
  40. Leonida, M.D.; Belbekhouche, S.; Benzecry, A.; Peddineni, M.; Suria, A.; Carbonnier, B. Antibacterial hop extracts encapsulated in nanochitosan matrices. Int. J. Biol. Macromol. 2018, 120, 1335–1343. [Google Scholar] [CrossRef]
  41. Maresso, A.W.; Schneewind, O. Sortase as a target of anti-infective therapy. Pharmacol. Rev. 2008, 60, 128–141. [Google Scholar] [CrossRef] [PubMed]
  42. Ribeiro-Vidal, H.; Sanchez, M.C.; Alonso-Espanol, A.; Figuero, E.; Ciudad, M.J.; Collado, L.; Herrera, D.; Sanz, M. Antimicrobial Activity of EPA and DHA against Oral Pathogenic Bacteria Using an In Vitro Multi-Species Subgingival Biofilm Model. Nutrients 2020, 12, 2812. [Google Scholar] [CrossRef] [PubMed]
  43. Naseem, S.; Douglas, L.M.; Konopka, J.B. Candida albicans Agar Invasion Assays. Bio Protoc. 2020, 10, e3730. [Google Scholar] [CrossRef] [PubMed]
  44. Brown, D.H., Jr.; Giusani, A.D.; Chen, X.; Kumamoto, C.A. Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene. Mol. Microbiol. 1999, 34, 651–662. [Google Scholar] [CrossRef] [PubMed]
  45. Blanc, V.; Isabal, S.; Sanchez, M.C.; Llama-Palacios, A.; Herrera, D.; Sanz, M.; Leon, R. Characterization and application of a flow system for in vitro multispecies oral biofilm formation. J. Periodontal Res. 2014, 49, 323–332. [Google Scholar] [CrossRef]
  46. Sanchez, M.C.; Llama-Palacios, A.; Fernandez, E.; Figuero, E.; Marin, M.J.; Leon, R.; Blanc, V.; Herrera, D.; Sanz, M. An in vitro biofilm model associated to dental implants: Structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces. Dent. Mater. 2014, 30, 1161–1171. [Google Scholar] [CrossRef]
  47. He, Z.X.; Zhao, H.H.; Wang, F.K. PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model. Open Life Sci. 2020, 15, 677–682. [Google Scholar] [CrossRef]
  48. Samaranayake, L.P.; McCourtie, J.; MacFarlane, T.W. Factors affecting th in-vitro adherence of Candida albicans to acrylic surfaces. Arch. Oral. Biol. 1980, 25, 611–615. [Google Scholar] [CrossRef]
  49. Garcia-Gamboa, R.; Dominguez-Simi, M.; Gradilla-Hernandez, M.S.; Bravo, J.; Moya, A.; Ruiz-Alvarez, B.; Gonzalez-Avila, M. Anticandidal and Antibiofilm Effect of Synbiotics including Probiotics and Inulin-Type Fructans. Antibiotics 2022, 11, 1135. [Google Scholar] [CrossRef]
  50. Wang, X.; He, H.; Liu, J.; Xie, S.; Han, J. Inhibiting roles of farnesol and HOG in morphological switching of. Am. J. Transl. Res. 2020, 12, 6988–7001. [Google Scholar]
  51. Glazier, V.E.; Kramara, J.; Ollinger, T.; Solis, N.V.; Zarnowski, R.; Wakade, R.S.; Kim, M.J.; Weigel, G.J.; Liang, S.H.; Bennett, R.J.; et al. The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism. mBio 2023, 14, e0152123. [Google Scholar] [CrossRef] [PubMed]
  52. Ganser, C.; Staples, M.I.; Dowell, M.; Frazer, C.; Dainis, J.; Sircaik, S.; Bennett, R.J. Filamentation and biofilm formation are regulated by the phase-separation capacity of network transcription factors in Candida albicans. PLoS Pathog. 2023, 19, e1011833. [Google Scholar] [CrossRef] [PubMed]
  53. Kim, D.; Sengupta, A.; Niepa, T.H.; Lee, B.H.; Weljie, A.; Freitas-Blanco, V.S.; Murata, R.M.; Stebe, K.J.; Lee, D.; Koo, H. Candida albicans stimulates Streptococcus mutans microcolony development via cross-kingdom biofilm-derived metabolites. Sci. Rep. 2017, 7, 41332. [Google Scholar] [CrossRef] [PubMed]
  54. Thurnheer, T.; Karygianni, L.; Flury, M.; Belibasakis, G.N. Fusobacterium Species and Subspecies Differentially Affect the Composition and Architecture of Supra- and Subgingival Biofilms Models. Front. Microbiol. 2019, 10, 1716. [Google Scholar] [CrossRef] [PubMed]
  55. Sztukowska, M.N.; Dutton, L.C.; Delaney, C.; Ramsdale, M.; Ramage, G.; Jenkinson, H.F.; Nobbs, A.H.; Lamont, R.J. Community Development between Porphyromonas gingivalis and Candida albicans Mediated by InlJ and Als3. mBio 2018, 9. [Google Scholar] [CrossRef]
  56. Holmes, A.R.; Gilbert, C.; Wells, J.M.; Jenkinson, H.F. Binding properties of Streptococcus gordonii SspA and SspB (antigen I/II family) polypeptides expressed on the cell surface of Lactococcus lactis MG1363. Infect. Immun. 1998, 66, 4633–4639. [Google Scholar] [CrossRef]
  57. Souza, J.G.S.; Bertolini, M.; Thompson, A.; Mansfield, J.M.; Grassmann, A.A.; Maas, K.; Caimano, M.J.; Barao, V.A.R.; Vickerman, M.M.; Dongari-Bagtzoglou, A. Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans. ISME J. 2020, 14, 1207–1222. [Google Scholar] [CrossRef]
  58. Okeke, I.N.; de Kraker, M.E.A.; Van Boeckel, T.P.; Kumar, C.K.; Schmitt, H.; Gales, A.C.; Bertagnolio, S.; Sharland, M.; Laxminarayan, R. The scope of the antimicrobial resistance challenge. Lancet 2024, 403, 2426–2438. [Google Scholar] [CrossRef]
  59. Loaiza Oliva, M.; Morales Uchima, S.M.; Puerta Suarez, J.; Mesa Arango, A.C.; Martinez Pabon, M.C. Lippia origanoides derivatives in vitro evaluation on polymicrobial biofilms: Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans. Arch. Oral. Biol. 2023, 148, 105656. [Google Scholar] [CrossRef]
  60. Tagashira, M.; Uchiyama, K.; Yoshimura, T.; Shirota, M.; Uemitsu, N. Inhibition by hop bract polyphenols of cellular adherence and water-insoluble glucan synthesis of mutans streptococci. Biosci. Biotechnol. Biochem. 1997, 61, 332–335. [Google Scholar] [CrossRef]
Cells 13 01877 g001
Figure 1. Chemical structure of thymol.
Figure 1. Chemical structure of thymol.
Cells 13 01877 g001
Cells 13 01877 g002
Figure 2. Chemical structure of xanthohumol.
Figure 2. Chemical structure of xanthohumol.
Cells 13 01877 g002
Cells 13 01877 g003
Figure 3. Growth of Candida albicans at increasing concentrations of xanthohumol (A) and thymol (B). Images obtained before washing show superficial yeast growth, and images obtained after washing show filamentous growth into the agar of the same culture.
Figure 3. Growth of Candida albicans at increasing concentrations of xanthohumol (A) and thymol (B). Images obtained before washing show superficial yeast growth, and images obtained after washing show filamentous growth into the agar of the same culture.
Cells 13 01877 g003
Cells 13 01877 g004
Figure 4. Images obtained by scanning electron microscopy (SEM), with 2500× magnification, of negative control biofilms (in the absence of Candida albicans), positive control biofilms (in the presence of C. albicans), biofilms developed with C. albicans and treated with xanthohumol and biofilms developed with C. albicans and treated with thymol ((A), (B), (C) and (D), respectively) (scale bar = 20 µm). Figures (E–H) show the same biofilm clusters with 5000× amplification (scale bar = 10 µm).
Figure 4. Images obtained by scanning electron microscopy (SEM), with 2500× magnification, of negative control biofilms (in the absence of Candida albicans), positive control biofilms (in the presence of C. albicans), biofilms developed with C. albicans and treated with xanthohumol and biofilms developed with C. albicans and treated with thymol ((A), (B), (C) and (D), respectively) (scale bar = 20 µm). Figures (E–H) show the same biofilm clusters with 5000× amplification (scale bar = 10 µm).
Cells 13 01877 g004
Cells 13 01877 g005
Figure 5. Images obtained with confocal laser scanning microscopy (CLSM) on negative control biofilms (in the absence of Candida albicans), positive control biofilms (in the presence of C. albicans), biofilms developed with C. albicans and treated with xanthohumol and biofilms developed with C. albicans and treated with thymol, developed at 72 h ((A), (B), (C) and (D), respectively). Images (E), (F), and (G) show C. albicans on biofilms developed in the absence and presence of xanthohumol and thymol, respectively. (scale bar = 200 µm). LIVE/DEAD® BackLight Kit was used to stain live bacteria (green), dead bacteria (red), and implant surfaces (white). Calcofluor White (CFW) was used to stain C. albicans (blue).
Figure 5. Images obtained with confocal laser scanning microscopy (CLSM) on negative control biofilms (in the absence of Candida albicans), positive control biofilms (in the presence of C. albicans), biofilms developed with C. albicans and treated with xanthohumol and biofilms developed with C. albicans and treated with thymol, developed at 72 h ((A), (B), (C) and (D), respectively). Images (E), (F), and (G) show C. albicans on biofilms developed in the absence and presence of xanthohumol and thymol, respectively. (scale bar = 200 µm). LIVE/DEAD® BackLight Kit was used to stain live bacteria (green), dead bacteria (red), and implant surfaces (white). Calcofluor White (CFW) was used to stain C. albicans (blue).
Cells 13 01877 g005
Cells 13 01877 g006
Figure 6. Bacterial biomass (grey bar) and Candida albicans biomass (blue bar) [expressed as microbial biomass of biofilm (µm3/µm2)] of biofilms obtained by quantification of images of confocal laser scanning microscopy (CLSM). The internal cyclogram shows the proportion of viable (green) and dead (red) bacterial cells at each biofilm cluster. * p < 0.05 and ** p < 0.01: statistically significant differences when comparing negative and positive control biofilms. † p < 0.05 and †† p < 0.01: statistically significant differences when comparing positive control biofilms with biofilms incubated in the presence of xanthohumol and thymol. ^ p < 0.05: statistically significant differences when comparing between biofilms incubated with xanthohumol and thymol.
Figure 6. Bacterial biomass (grey bar) and Candida albicans biomass (blue bar) [expressed as microbial biomass of biofilm (µm3/µm2)] of biofilms obtained by quantification of images of confocal laser scanning microscopy (CLSM). The internal cyclogram shows the proportion of viable (green) and dead (red) bacterial cells at each biofilm cluster. * p < 0.05 and ** p < 0.01: statistically significant differences when comparing negative and positive control biofilms. † p < 0.05 and †† p < 0.01: statistically significant differences when comparing positive control biofilms with biofilms incubated in the presence of xanthohumol and thymol. ^ p < 0.05: statistically significant differences when comparing between biofilms incubated with xanthohumol and thymol.
Cells 13 01877 g006
Cells 13 01877 g007
Figure 7. Amounts (expressed as mean and standard deviation (SD) of total and live microbial species (colony-forming units (CFUs)/mL)) determined by quantitative polymerase chain reaction (qPCR) in control negative biofilms developed without C. albicans (C−), positive control mixed biofilms developed with C. albicans (C+), mixed biofilms developed with C. albicans and incubated with xanthohumol (X) and mixed biofilms developed with C. albicans and incubated with thymol (T) (n = 9), using specific primers and probes directed to the 16S rRNA gene of bacterial strains and ITS2 region of C. albicans. * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.001: statistically significant differences when comparing negative control biofilms (C−) with positive control biofilms (C+) or mixed biofilms incubated with xanthohumol (X) or thymol (T). † p < 0.05, †† p < 0.01 and †††† p < 0.001: statistically significant differences when comparing positive control biofilms (C+) whit biofilms incubated with xanthohumol (X) or thymol (T). ^ p < 0.05 and ^^^ p < 0.005: statistically significant differences when comparing between biofilms incubated with xanthohumol (X) and thymol (T). Comparisons between groups were performed considering viable cells and total cells. Figure corresponding to Supplementary Table S1.
Figure 7. Amounts (expressed as mean and standard deviation (SD) of total and live microbial species (colony-forming units (CFUs)/mL)) determined by quantitative polymerase chain reaction (qPCR) in control negative biofilms developed without C. albicans (C−), positive control mixed biofilms developed with C. albicans (C+), mixed biofilms developed with C. albicans and incubated with xanthohumol (X) and mixed biofilms developed with C. albicans and incubated with thymol (T) (n = 9), using specific primers and probes directed to the 16S rRNA gene of bacterial strains and ITS2 region of C. albicans. * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.001: statistically significant differences when comparing negative control biofilms (C−) with positive control biofilms (C+) or mixed biofilms incubated with xanthohumol (X) or thymol (T). † p < 0.05, †† p < 0.01 and †††† p < 0.001: statistically significant differences when comparing positive control biofilms (C+) whit biofilms incubated with xanthohumol (X) or thymol (T). ^ p < 0.05 and ^^^ p < 0.005: statistically significant differences when comparing between biofilms incubated with xanthohumol (X) and thymol (T). Comparisons between groups were performed considering viable cells and total cells. Figure corresponding to Supplementary Table S1.
Cells 13 01877 g007
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Bravo, E.; Arce, M.; Herrera, D.; Sanz, M. The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces. Cells 2024, 13, 1877. https://doi.org/10.3390/cells13221877

AMA Style

Bravo E, Arce M, Herrera D, Sanz M. The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces. Cells. 2024; 13(22):1877. https://doi.org/10.3390/cells13221877

Chicago/Turabian Style

Bravo, Enrique, Marion Arce, David Herrera, and Mariano Sanz. 2024. "The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces" Cells 13, no. 22: 1877. https://doi.org/10.3390/cells13221877

APA Style

Bravo, E., Arce, M., Herrera, D., & Sanz, M. (2024). The Effect of Xanthohumol and Thymol on Candida albicans Filamentation and Its Impact on the Structure, Size, and Cell Viability of Biofilms Developed over Implant Surfaces. Cells, 13(22), 1877. https://doi.org/10.3390/cells13221877

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop