# Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans

Guillaume Hamion, Willy Aucher, Anne Mercier, Frederic Tewes, Maëlenn Menard, Joanne Bertaux, Marion Girardot, Christine Imbert

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## ► To cite this version:

Guillaume Hamion, Willy Aucher, Anne Mercier, Frederic Tewes, Maëlenn Menard, et al. Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans. International Journal of Antimicrobial Agents, 2024, 63 (6), pp.107166. 10.1016/j.ijantimicag.2024.107166. hal-04542854

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Submitted on 9 Jul 2024

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# Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm *Staphylococcus aureus–Candida albicans*

<figure><img src="image_3.png" /></figure>

Guillaume Hamion<sup>a,*</sup>, Willy Aucher<sup>a</sup>, Anne Mercier<sup>a</sup>, Frederic Tewes<sup>b</sup>, Maëlenn Menard<sup>a</sup>, Joanne Bertaux<sup>a</sup>, Marion Girardot<sup>a</sup>, Christine Imbert<sup>a</sup>

## ARTICLE INFO

**Article history:**

Received 24 October 2023

Accepted 28 March 2024

Editor: Stefania Stefani

**Keywords:**

Betulinic acid

*Staphylococcus aureus*

*Candida albicans*

Interkingdom biofilm

Bi-species biofilm

Antibiofilm

Cell surface hydrophobicity

## ABSTRACT

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom *Staphylococcus aureus–Candida albicans* occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species *S. aureus–C. albicans* biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature *S. aureus–C. albicans* biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in *C. albicans*, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in *S. aureus–C. albicans* physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of *S. aureus–C. albicans* biofilm-related infections.

© 2024 The Author(s). Published by Elsevier Ltd.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## 1. Introduction

Polymicrobial biofilm-associated infections represent a challenge in human diseases [1–3]. Among them are the *Staphylococcus aureus–Candida albicans* biofilm-associated infections reported, in particular, in patients with bloodstream infections, cystic fibrosis, urinary tract infections and periodontitis [4–6]. Although *S. aureus* and *C. albicans* have long been recognised as opportunistic bacterial and yeast pathogens, respectively [7,8], their interactions within biofilms can lead to increased drug tolerance, virulence and immune evasion, leading to failure of conventional antibiotic and antifungal treatments [5,9–11]. Due to the complexity of the *S. aureus–C. albicans* biofilm-associated infections (and, largely, to bacterial–fungal biofilms) and the limited availability of therapeutic options, curative strategies depend on the urgent

development of novel therapies or drugs belonging to new classes of antimicrobials, acting on new targets through new mechanisms of action. In this context, plant specialised metabolites have been recognised as an alternative natural source of molecules for the treatment of bacterial and fungal infections [12]. Among phytochemical classes providing bioactive compounds, triterpenoids encompass structurally diverse molecules such as betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid; $C_{30}H_{48}O_3$). This naturally occurring pentacyclic triterpenoid derived from lupane, is widely described in the stem bark of plants belonging to the Betulaceae family, but is also widespread among plants from other families [13]. We recently identified betulinic acid in *Ludwigia grandiflora*, an invasive macrophyte belonging to the Onagraceae family [14]. Betulinic acid falls under the scope of a wide range of applications, such as anti-inflammatory properties, antimalarial and antihelmintic activities [15]. Also, it is well known for its anti-HIV-1 activity and cytotoxicity against diverse tumour cell lines. Betulinic acid causes apoptosis through the induction of changes in the mitochondrial membrane potential (ΔΨm), the production of reactive oxygen species and the opening of mitochondrial permeability

<footnote><sup>a</sup>Ecology and Biology of Interactions, University of Poitiers, UMR CNRS 7267, Poitiers, France</footnote>

<footnote><sup>b</sup>Pharmacology of Antimicrobial Agents and Ant bioresistance, University of Poitiers, INSERM U1070, Poitiers, France</footnote>

<footnote>* Corresponding author: Guillaume Hamion, Ecology and Biology of Interactions, University of Poitiers, UMR CNRS 7267, F-86000 Poitiers, France.
E-mail address: guillaume.hamion@yahoo.com (G. Hamion).</footnote>
transition pores [16]. As a result, mitochondrial apoptogenic factors are released and contribute to caspase activation and DNA fragmentation. Betulinic acid is cytotoxic against melanoma cells and other tumour cell types, including brain tumour and neuroectodermal cells. It is currently classified as a mitocan, an anticancer drug acting on mitochondria, with selective cytotoxicity in mammalian cells [17–19].

More recently, promising antimicrobial and antibiofilm activities of betulinic acid were reported against reference and clinical strains of S. aureus and C. albicans [14,20–27]. In contrast to the well-defined mechanism of anticancer action, molecular pathways targeted by betulinic acid resulting in antibiofilm activity are poorly described [23,26]. Thus, this antibiofilm activity requires more in-depth investigations to make it a candidate compound to combat polymicrobial biofilm-associated infections.

The aims of the present study were to evaluate the antibiofilm properties of betulinic acid against interkingdom biofilms formed by S. aureus and C. albicans and to increase the understanding of its mechanism of action, using a multiphase approach.

## 2. Materials and methods

### 2.1. Chemicals and reagents

Analytical grade betulinic acid and betulin were purchased from Sigma-Aldrich Chemical Corporation (Saint Louis, MO, USA), as were the other reagents used in this study, including dimethyl sulfoxide (DMSO), Tween®80, ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulfate and amphotericin B.

Betulinic acid and betulin were prepared as 1.25 mg/mL stock solutions in DMSO and stored at 4°C for use within 24 h. Betulinic acid was used at 25 µg/mL (55 µM) throughout the present study, based on its pharmacological and chemical profiles (solubility, cell toxicity and activity parameters), as previously reported [14].

A 100 mg/mL Tween®80 stock solution was prepared in DMSO. Phosphate-buffered saline (PBS) (Gibco, New York, NY, USA) used for cell washes was sterilised again through final 0.1 µm pore-size filters.

(Note: all mentioned concentrations are final concentrations.)

### 2.2. Microbial strains and culture conditions

For mono- and bi-species culture and biofilm experiments with betulinic acid, S. aureus ATCC® 29213TM and C. albicans ATCC® 28367TM strains were grown overnight at 37°C in brain heart infusion (BHI) medium (BD Difco, Sparks, MD, USA), at 80 rpm for S. aureus and in static conditions for C. albicans, with some exceptions. Then, both cell types were washed with 10 mL PBS by centrifugation at 5000 × g for 5 min and enumerated by reading absorbance at 600 nm for S. aureus cultures (a value of 1 corresponding to 16.4 × 10⁹ CFU/mL) and using Fast-Read 102® counting chambers for C. albicans [14].

### 2.3. Spatial analyses of 24 h-old bi-species biofilms treated with betulinic acid

Microscopic observations were conducted to investigate bi-species biofilms treated with betulinic acid. For fluorescent microscopy, bi-species biofilms were prepared in 1.2 mL BHI (cell density adjusted at 10⁶ CFU/mL for each species) in wells from 24-well polystyrene bottom Vision plates™ (4titude Ltd, Dorking, UK) previously precoated with foetal bovine serum (FBS, dialysed, US origin, Gibco). The 24 h-old biofilms were treated with DMSO (2% v/v, used as the solvent control throughout our study) and with betulinic acid 25 µg/mL, after one wash with PBS, for an additional

incubation of 24 h at 37°C, as described above. Then, adhered cells were washed once with PBS and stained with 250 µL calcofluor white 0.1% (Calcofluor White reagent droppers, Becton Dickinson, Franklin Lakes, NJ, USA) and 20 µL Syto9 1.67 µM in 1 mL PBS, followed by a 45 min incubation in the dark at room temperature. Images were recorded using an Axio Observer A1 coupled with an apotome (Carl Zeiss, Marly-le-Roi, France) for automated three-dimensional acquisition. The microscope was equipped with a mercury lamp and both Zeiss Filters Set 44 and Set 49. Images were analysed with Zen software (Zeiss). At least five localisations from each well were analysed.

Bi-species biofilms were also grown on sterile polycarbonate coupons (diameter 13 mm; thickness: biofouling 3514 mm; Bio-Surface Technologies Corporation, Bozeman, MO, USA) deposited in 24-well microplates as previously described [14]. DMSO and betulinic acid were added at concentrations mentioned above. Coupons coated with biofilms were dried and frozen at –140°C (Leica EM VCM Vacuum Cryo Manipulation System), sublimed and coated with platinum (Leica EM ACE600 High Vacuum Coater). Images were recorded with a scanning electron microscope (FEI Teneo Volume Scope; FEI Company, Hillsboro, OR, USA) at –100°C and between 5 and 10 kV. At least five localisations from each well were analysed.

### 2.4. Quantification of 24 h-old mono- and bi-species biofilms treated with betulinic acid

Biofilms were produced in 96-well non-treated polystyrene microtitre plates (Costar, Corning, NY, USA), using the protocol described by Hamion et al. [14]. Briefly, S. aureus and C. albicans were inoculated at 10⁶ CFU/mL in 200 µL BHI, for mono- and bi-species biofilms, using a 1:1 ratio. After incubation at 37°C for 2 h, culture medium was removed to eliminate non-adherent cells, replaced by 200 µL fresh BHI medium and incubation was then extended for an additional 24 h at 37°C. Then, the obtained preformed 24 h-old biofilms were treated with betulinic acid 25 µg/mL (test conditions) or DMSO 2% (control) for an additional incubation of 24 h at 37°C. The obtained biofilms were washed with 200 µL PBS, avoiding disturbing adhered cells, and then vigorously scratched using a sterile folded cone in 100 µL PBS. The obtained microbial suspensions were diluted one-tenth in PBS and aggregated cells were dispersed by sonication for 10 min, followed by vortex mixing for 30 s. Microbial suspensions were stained with 1–2 µL 334 µM Syto9 (LIVE/DEAD™ Viability/Cytotoxicity Kit, Invitrogen, Carlsbad, CA, USA). Finally, cell quantification was carried out using a CytoFlex (Beckman Coulter, Brea, CA, USA) with parameters previously described [14].

### 2.5. Quantification of bi-species biofilms on betulinic acid-impregnated surfaces

Biofilms were developed on surfaces (96-well non-treated polystyrene microplates) pre-impregnated for 3 h, at room temperature with: (a) 30 µL DMSO, (b) Tween®80 stock solution 100 mg/mL or (c) betulinic acid stock solution 1.25 mg/mL. Three conditions were applied: (i) S. aureus and C. albicans cells were added in wells after the removal of the impregnation solutions but without washing first; (ii) impregnated surfaces were washed with 200 µL PBS before they were seeded with microorganisms and (iii) surfaces were seeded with microbial cells as the impregnation was carried out.

The non-ionic surfactant Tween®80, known for its ability to reduce bacterial attachment and inhibit biofilm formation, was applied as a control [28]. In all conditions, microbial cells were added at 10⁷ CFU/mL and incubated at 37°C for 2 h in liquid BHI medium. Planktonic cells were then removed by washing with 200 µL PBS
and the adhered biomass was quantified using a crystal violet assay [14].

## 2.6. Quantification of C. albicans mono-species biofilm formed from a yeast preculture treated with betulinic acid

To test the capacity of betulinic acid to interfere with cell-cell and/or cell-surface interactions, yeasts were treated with betulinic acid before adhesion. Briefly, yeasts at 10⁶ CFU/mL were incubated for 24 h at 37°C in liquid BHI supplemented with DMSO 2% or betulinic acid 25 µg/mL. Yeasts were then washed twice with PBS (centrifugation 5000 × g, 5 min), suspended at 10⁶ CFU/mL and used to form a biofilm for 24 h at 37°C as described above. Adhered cells were quantified using a crystal violet staining test.

## 2.7. Yeast-to-hyphae transition of C. albicans in mono- and bi-species biofilms treated with betulinic acid

The capacity of betulinic acid to interfere with C. albicans filamentation was evaluated. Yeasts grown in liquid BHI were washed in PBS and suspended at 10⁶ CFU/mL in RPMI (RPMI 1640 supplemented with GlutaMAXTM, Gibco, Invitrogen) with 10% FBS in the presence of (i) DMSO 2%, (ii) betulinic acid 25 µg/mL or (iii) EDTA 25 mM, and incubated for 3 h at 37°C. EDTA (control) caused filamentation inhibition as reported by Gil et al. [29]. Cells were then collected and 10 µL were loaded on a Fast-Read 102® counting chamber (Biosigma, Cantarana, Italy): the yeast form/hyphae proportion was determined in each condition by enumeration by microscopic count.

Moreover, S. aureus and C. albicans cultures were also prepared in RMPI instead of BHI, in order to favour the yeast-to-hyphae transition of C. albicans as soon as the early growth phase, before bi-species biofilm production. S. aureus and C. albicans at 10⁶ CFU/mL were incubated in 96-well non-treated polystyrene microtitre plates, in RPMI for 24 h at 37°C. Then, the 24 h-old biofilms were treated with DMSO 2% or betulinic acid 25 µg/mL for 24 h. Adhered cells were assessed using a crystal violet staining test.

## 2.8. Cell surface hydrophobicity (CSH) and membrane integrity of C. albicans treated with betulinic acid

The C. albicans CSH assay was based on the microbial adhesion to hydrocarbon (MATH) technique [30]. Briefly, freshly washed yeast cultures were grown for 24 h in liquid BHI and RPMI at 37°C with DMSO 2% or betulinic acid 25 µg/mL. Suspensions were washed once with PBS (centrifugation 5000 × g, 5 min) and adjusted at an OD₆₀₀nm of 0.3 in PBS (A₀). A volume of 2 mL was added to 1 mL toluene and vortexed for 2 min in a glass tube. Separated phases were obtained after 15 min at room temperature; the aqueous phase was recovered and its absorbance measured at 600 nm (A₁). The percentage of CSH was calculated using the following equation:

$$CSH\% = \frac{A_0 - A_1}{A_0} \times 100.$$

The ability of betulinic acid to disturb the C. albicans membrane integrity was also assessed using the non-polar membrane-impermeable fluorescent probe 1-N-phenylnaphthylamine (NPN; Sigma-Aldrich). Briefly, a fresh culture of C. albicans at 10⁶ CFU/mL was grown in BHI at 37°C with DMSO 2%, betulinic acid 25 µg/mL, betulin 25 µg/mL or amphotericin B at half its minimum inhibitory concentration (1/2 MIC = 0.35 µg/mL) [31]. Amphotericin B was used as a control due to its known cytotoxic effect, resulting from

loss of membrane integrity and increased membrane permeability following treatment [32].

Anisotropy measurements were performed 2, 5, 12 and 24 h after these treatments. Treated cells were washed twice with PBS (centrifugation 5000 × g, 5 min). Yeast suspensions were adjusted at an OD₆₀₀nm of 1, and 100 µL added to 100 µL NPN solution at 100 µM in PBS (final concentration 50 µM), in wells of a 96-well microplate (F-bottom µCLEAR® black cellstar®, Greiner BioOne, Kremsmünster, Austria). Each condition was repeated in three separate wells; controls were prepared by replacing NPN with PBS, to take into account the inherent fluorescence of the yeast suspension. The overall fluorescence was measured after 10 min of incubation in the dark, with excitation at 355 nm and emission at 410 nm (Infinite M Plex absorbance reader, TECAN, Zürich, Switzerland). Each condition was repeated at least three times. The percentage of extra-fluorescence was calculated by comparing the treated condition (x⁻T) with the control one (x⁻C) using the following formula:

$$fluorescence\ increase\ (%) = \frac{\bar{x}_T - \bar{x}_C}{\bar{x}_C} \times 100$$

## 2.9. Gene expression in mono- and bi-species early-stage biofilms following betulinic acid treatment

In order to increase biofilm biomass, S. aureus and C. albicans cells (10⁶ CFU/mL) were incubated for 5 h at 37°C in 15 mL BHI liquid medium with DMSO 2% (control) or betulinic acid at 25 µg/mL, in 50 mL polypropylene tubes (Falcon™, Fisher Scientific, Hampton, USA) instead of microtitre plates. Then, adhered cells were carefully washed once with PBS (centrifugation 5000 × g, 5 min) and detached from the surface by sonication for 10 min, completed by stirring for 30 s. RNA was extracted from cell pellets using the RNeasy Mini kit with the QIAcube workstation according to the manufacturer's instructions (Qiagen, Hilden, Germany). Contaminating DNA was removed using RNase-free DNase I (Turbo DNA-free kit, ThermoFisher Scientific), according to the manufacturer's instructions. RNA concentration and purity were measured using the NanoDropTM 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The samples were stored at -80°C until analysis. Total RNA was reverse-transcribed using the GoScript Reverse Transcriptase kit (Promega, Madison, WI, USA) according to the manufacturer's recommendation. The first-strand cDNA products were used to carry out quantitative polymerase chain reaction (PCR) to analyse gene expression. Seven genes involved in quorum sensing, oxidative stress responses and microbial surface components recognising adhesive matrix molecules were targeted for each species, including fnbB, sasF, icaC, agrC, RNAIII, sasE, sodA for S. aureus with gyrB and rpoB as reference genes, and als3, rbt1, bcr1, rlm1, dpp3, cdr1, sod1, for C. albicans with act1, pma1 and tef1 as reference genes. The primers originated from literature or were designed using the Primer-BLAST tool (NCBI, Rockville Pike, Bethesda, MD, USA) from reference genomes. All primer sequences are compiled in Supplementary Table S1. The specificity and absence of cross-amplification between S. aureus and C. albicans were investigated prior to the gene expression experiments.

Quantitative PCR was carried out using the Takyon™ No ROX SYBR 2X MasterMIX blue dTTP reactional mix (Eurogentec, Seraing, Belgium) in a LightCycler 480 II (Roche Molecular Systems, Bäle, Switzerland). Reactions were prepared in a total volume of 10 µL containing 5 µL 2 × SYBR mix, 2.4 µL H₂O₂, 2 µL 20-fold diluted cDNA template and 0.3 µL 10 µM primers. PCR cycling was performed as follows: initial denaturation 95°C for 10 min, then 45 cycles at 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. To check the specificity of PCR products, a melting curve analysis was performed ranging from 65 to 95°C.
The relative quantification method ($2^{-\Delta\Delta Ct}$) was used to evaluate gene expression variations following betulinic acid treatment [33]. The DMSO-incubated condition was used as reference for calculations. Quantitative PCR was performed at least five times from independent biological cultures to ensure the accuracy of the results. Decreased mRNA expression was defined as fold-change $\le 0.5$, increased mRNA expression as fold-change $\ge 2.0$ and 'normal expression' in the range of fold-change from 0.5 to 2.

## 2.10. Statistical analyses

All biological experiments were performed at least three times in triplicate for each condition. The Kruskal-Wallis test with Dunn's multiple comparisons test were applied to determine the statistical significance between obtained measures, with treated biofilms and controls, using GraphPad Prism® version 6.01 (Graph-Pad Software Inc, San Diego, CA, USA).

# 3. Results and discussion

## 3.1. Betulinic acid as a promising antibiofilm agent with a non-microbicidal activity

Fluorescence microscopy allowed the comparative observation of *S. aureus*–*C. albicans* biofilms in the absence and presence of treatment with a concentration allowing the solubility of betulinic acid 25 µg/mL, providing an overview of the effect of betulinic acid on biofilm architecture. To ensure the antibiofilm activity resulted specifically from betulinic acid, DMSO was added to the control condition, as this solvent was used for betulinic acid stock solution preparation. All observations showed that in the control condition, both species together produced a reproducible dense biofilm on polystyrene plates. Three-dimensional structured 48 h-old bi-species biofilms displayed a homogeneous thickness with a full coverage of bacterial and fungal microcolonies across the surface (Fig. 1A, B), whatever the prepared sample. In contrast, 24 h-old bi-species biofilms treated with betulinic acid for 24 h appeared heterogeneous (Fig. 1D, E), less structured, free of cells in some areas and showed microbial aggregates irregularly dispersed across the surface (Fig. 1D, E). Based on visual observations, the functional cells appeared strongly impacted by betulinic acid, especially (Fig. 1F compared with Fig. 1C), with a reduced biomass. The cryoscanning electron microscopy approach showed that betulinic acid did not irreversibly compromise the cell surface integrity (Fig. 1G, H) as the cells in the biofilm remained identifiable (bacteria vs yeast and hyphae) with no observable membrane damaged cells. Bacteria and fungi were still present in the biofilms after betulinic acid treatment, suggesting a non-microbicidal activity of betulinic acid under our conditions.

Thus, these first results suggested that betulinic acid would be able to reduce a preformed biofilm without having a microbicidal action. Quantitative analyses were assessed to confirm this hypothesis, especially using flow cytometry.

## 3.2. Betulinic acid damages interkingdom biofilms

The activity of solubilised betulinic acid on interkingdom biofilms was assessed in polystyrene wells using flow cytometry to quantify simultaneously bacteria and yeast populations, based on their sizes.

A significant decrease ($P < 0.05$) of 37% for *S. aureus* and 49% for *C. albicans* biomass was confirmed on 24 h bi-species biofilms treated with betulinic acid for 24 h (Fig. 2).

Interestingly, such decreases were also observed when 24 h mono-species biofilms were treated with betulinic acid for 24 h,

with 48% and 34% biomass reduction for bacteria and yeasts, respectively (Fig. 2). Betulinic acid was therefore effective at reducing mature *S. aureus*–*C. albicans* biofilms, as well as mature *S. aureus* and *C. albicans* mono-species biofilms, which shows a broad spectrum of antibiofilm activity, on to distinct kingdoms. Also, at concentrations ranging from 6.25 to 50 µg/mL, betulinic acid had no activity on planktonic growth of either *C. albicans* or *S. aureus*, indicating that the antibiofilm activity of betulinic acid observed was not due to reduced viability and growth of *S. aureus* and/or *C. albicans* (Supplementary Figure S1A, B). These results suggested that betulinic acid may rather affect the microbial adhesion to abiotic surface and/or the microbial cell-cell interactions of both microbial species. These hypotheses were subsequently investigated.

## 3.3. Betulinic acid inhibits cell adhesion on abiotic surfaces only when used free-form

A series of tests was carried out to assess the influence of betulinic acid on adhesion, the early phase of biofilm formation, and to understand how it acted. The degree of microbial colonisation of betulinic acid-impregnated polystyrene surfaces was assessed, after washing or not washing the impregnated surfaces prior to microbial seeding. The colonisation level was also compared with that obtained when betulinic acid was added simultaneously with bacteria and yeasts, without prior impregnation (Fig. 3). In parallel, surfaces were pretreated with a non-ionic surfactant (Tween®80), leading, as expected, to the inhibition of the biofilm formation ($P < 0.001$), whatever the conditions tested (Fig. 3A, B), thus validating the impregnation approach. The overall biomass adhered to surfaces impregnated with betulinic acid was comparable ($P > 0.05$) to that obtained with the negative control impregnation (DMSO) (Fig. 3A, B). In contrast, when betulinic acid was used as a free soluble form and added simultaneously to planktonic bacteria and yeasts, the resulting sessile biomass decreased up to 40% compared with the DMSO control, which is consistent with the results obtained using Tween®80 impregnation (Fig. 3C). Therefore, betulinic acid impregnation over microplate surfaces was ineffective, betulinic acid needing to be added as a soluble form to affect adhesion.

Our hypothesis was that the anti-adhesion effect of betulinic acid would be caused by a direct contact with cell surfaces and/or after entry into the cell. Based on the microscopy examinations (Fig. 1E, F) and biofilm biomass quantification using flow cytometry (Fig. 2), subsequent experiments were conducted on the species *C. albicans*.

## 3.4. Betulinic acid interacts with C. albicans cell wall and membrane

To understand better how betulinic acid acts, *C. albicans* yeasts were treated with betulinic acid prior to biofilm formation (preculture treatment, PT) and/or during biofilm formation (biofilm formation treatment, BFT). The combination of both resulted in four assay conditions (Fig. 4): PT-/BFT- (control; in grey with line pattern); PT-/BFT+; PT+/BFT-; PT+/BFT+. Betulinic acid significantly ($P < 0.05$) reduced the sessile biomass compared with control, regardless of the treatment modalities. The highest biomass reduction (75%) was observed when betulinic acid was present both prior to and during biofilm formation. However, even if extending the contact time between betulinic acid and microorganisms seemed to strengthen its antibiofilm effect, the impact of PT seemed limited, the additional reduction being only 9% (PT+/BFT+: 75%; PT-/BFT+: 66%, $P < 0.05$) (Fig. 4). Moreover, the pretreatment by betulinic acid carried out alone (PT+/BFT-) significantly reduced the formed biofilm (48% reduction, $P < 0.01$), its persistent interaction with cell wall and/or membrane and/or its transport
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Representative micrographs from independent experiments of 24 h-old bi-species biofilms treated for 24 h with dimethyl sulfoxide (DMSO) 2% as the control condition and betulinic acid (BA) at a final concentration of 25 µg/mL, using fluorescence microscopy (A–F) and scanning electronic microscopy (G, H). Syto9 stained cells in green, whereas calcofluor white stained yeasts in red. (B, C, E, F) Three-dimensional reconstructions of fluorescence micrographs from bi-species biofilms [(C) and (F): *Candida albicans* only, from (B) and (E), respectively]. On (G) and (H): b indicates a *Staphylococcus aureus* cell, y indicated *C. albicans* yeast and h indicates *C. albicans* hyphae.</figcaption></figure>

through the fungal cell wall and membrane allowing its concen-
tration in fungal cytoplasm. Finally, these additional results were
consistent with previous ones and suggested that betulinic acid ac-
tion involves one or more cellular targets.

3.5. *Betulinic acid inhibits the yeast-to-hyphae transition of C. albicans*

The morphology of *C. albicans* can switch from blastospore to
hyphae [34]. The yeast-to-hyphae transition is one of the events
that occur during biofilm maturation, along with matrix produc-
tion. The capacity of betulinic acid to interfere with *C. albicans* fil-

amentation was thus evaluated (Fig. 5A), in comparison with two
control conditions: DMSO as a negative control and EDTA 25 mm
as a positive one, this molecule being known to completely in-
hibit filamentation (Fig. 5B–D). Betulinic acid reduced the yeast-to-
hyphae transition of *C. albicans*, the yeast/hyphae proportion being
around 23% with betulinic acid compared with 10% with the nega-
tive control (Fig. 5A, C). It has already been shown that the yeast-
to-hyphae transition is triggered by environmental signalling [35].
As the hyphae form of *C. albicans* contributes to its virulence, our
results suggest that betulinic acid treatment could attenuate the
pathogenicity of *C. albicans*. Additionally, a decrease in *S. aureus*–
*C. albicans* interactions may be expected, as *S. aureus* adheres pre-
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** *Staphylococcus aureus* and *Candida albicans* cell quantification of 24 h-old mono- and bi-species biofilms treated for an additional 24 h with dimethyl sulfoxide (DMSO) 2% as the control (in white) and betulinic acid (BA) at a final concentration of 25 µg/mL (in grey) using flow cytometry and Syto9 staining. Data represent the mean (± standard error of the mean) of nine independent experiments. \*P < 0.05; \*\*P < 0.01. The percentage cell decrease is reported on the graph.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Quantification of *Candida albicans* mono-species biofilm produced from a yeast preculture treated with dimethyl sulfoxide (DMSO; in grey) and betulinic acid (BA; in blue). The 24 h-old biofilms formed from the treated yeast precultures were treated with DMSO 2% (dashed) and BA 25 µg/mL (transparent) at 37°C for an additional 24 h, using crystal violet staining and optical density (OD<sub>510nm</sub>) measurement. Data represent the mean (± standard deviation) of independent experiments. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001. The percentage cell decrease is reported on the graph. PT, preculture treatment; BFT, biofilm formation treatment.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Cell quantification of bi-species biofilm biomass treated with dimethyl sulfoxide (DMSO), Tween 80 and betulinic acid (BA) according to two treatment modes: from a solution-impregnated plate surface or as a soluble form in medium, using crystal violet staining and optical density (OD<sub>570nm</sub>) measurement. (A) *Staphylococcus aureus* and *Candida albicans* cells were added into DMSO-, Tween 80- and BA-impregnated wells after removing the impregnation solution but without a previous well wash step. (B) The impregnated wells were washed with phosphate-buffered saline (PBS) before being seeded with microorganisms and (C) the plate wells were seeded with microorganisms together with DMSO 2%, Tween 80 at 2 mg/mL or BA at 25 µg/mL, without impregnation of wells. Data represent the mean (± standard deviation) of independent experiments. \*\*P < 0.01; \*\*\*P < 0.001.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Estimation of the filamentation of *Candida albicans* cultured in RMPI with 10% SVF liquid medium treated for 3 h with dimethyl sulfoxide (DMSO) 2%, betulinic acid (BA) 25 µg/mL or ethylenediamine tetraacetic acid (EDTA) 25 mM (yeast form-promoting), enumeration by light microscopy (A). Yeast hyphae form proportion data represent the mean (± standard deviation) of nine independent experiments. \*\*P < 0.01. Representative micrographs from independent experiments of *C. albicans* treated with (B) DMSO, (C) BA and (D) EDTA in the previously described conditions.</figcaption></figure>

dominantly to *C. albicans* hyphae [36,37] while betulinic acid promoted yeast forms.

Additional experiments demonstrated that betulinic acid inhibited the formation of both *C. albicans* (*P* < 0.05) and *S. aureus*-C. albicans (*P* < 0.0001) biofilms in RPMI (Supplementary Figure S2A) and BHI (Fig. 4). Even if the seeded fungi had a yeast morphology, RPMI favoured the yeast-to-hyphal growth transition. The biomasses of single- and bi-species biofilm obtained after a 24 h contact were reduced by up to 51% (Supplementary Figure S2A).

Nevertheless, betulinic acid treatment of a 24 h-old bi-species biofilms preformed in RPMI did not significantly reduce the biofilm biomass (Supplementary Figure S2B). These results suggested that the antibiofilm activity of betulinic acid targeted the yeast forms of *C. albicans* rather than the hyphae.

### 3.6. Betulinic acid affects the chemical properties of C. albicans yeast and hyphae surfaces

The effect of betulinic acid on the CSH of *C. albicans* cells was analysed using the MATH assay, which measures the ability of cells to adhere to a hydrophobic surface [30]. To ensure that the obtained results were only the consequence of the action of betulinic acid, hydrophobicity was previously measured in the different solutions and media used for cell growth and biofilm formation in the absence and presence of betulinic acid (Supplementary Figure S3). Similar drop diameters were measured with or without be-

tulinic acid, indicating no direct influence of these solutions and media on the cell surface of *C. albicans* (Supplementary Figure S3).

Thereafter, we compared two growth conditions, using either a BHI medium promoting yeast forms (Fig. 6A) or an RPMI medium promoting hyphae forms (Fig. 6B) in order to assess the impact on morphology. Betulinic acid treatment caused a slight decrease in the fungal CSH regardless of the medium, thus the morphology. The CSH reduction measured after betulinic acid contact for 24 h was almost two-fold higher in RPMI (around 10% decrease) compared with that obtained in BHI (5%). The CSH of hyphae forms (around 79%, Fig. 6B) was higher than that of yeast forms (32%, Fig. 6A); yeast forms tended to remain in aqueous phase and betulinic acid treatment seemed to encourage it, as previously observed (Fig. 5). According to Ovchinnikova et al. [38], the presence of mannoproteinaceous, hydrophilic surface fibrils, accounts for a higher CSH for hyphae compared with yeast forms. These results supported that betulinic acid would modulate the chemical surface properties of *C. albicans* cells.

### 3.7. Betulinic acid modulates the cell membrane permeability of C. albicans

The membrane permeability of *C. albicans* was monitored in response to betulinic acid and betulin. The enhanced permeability of the cell membrane was determined as the increase in fluorescence of the probe NPN, a hydrophobic molecule that interacts with lipids from the membrane of *C. albicans* [39,40]. *C. albicans* cells
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Cell surface hydrophobicity of *Candida albicans* cultured in (A) brain heart infusion (BHI) medium or (B) RPMI1640 medium, supplemented for 24 h with dimethyl sulfoxide (DMSO) 2% as the control condition and betulinic acid (BA) at 25 µg/mL (± standard deviation). (C) Membrane integrity of *C. albicans* cultured in BHI medium supplemented with BA (25 µg/mL), betulin (BT, 25 µg/mL) and amphotericin B at half minimum inhibitory concentration (0.35 µg/mL) compared with control condition (DMSO 2%) (± standard error of the mean). Data represent the mean of nine independent experiments. \*P < 0.05 significance compared with control.</figcaption></figure>

were grown in BHI with betulinic acid or betulin, both at 25 µg/mL, in order to compare these treatments with the negative control condition (DMSO). Fluorescence anisotropy (which is proportional to membrane disturbing) was measured 2, 5, 12 and 24 h after these treatments (Fig. 6C). Amphotericin B (0.35 µg/mL) was used as a positive control, as its cytotoxic effect should strongly disturb the membrane of *C. albicans*. Our results confirmed that amphotericin B compromised the cell wall and membrane as an increase in fluorescence anisotropy up to 25% was observed at 2 h incubation. Results at 24 h incubation suggested cell disruption over time due to an extreme membrane disturbance under this treatment. For betulin, which is a lupane triterpenoid derivate close to betulinic acid but has no antibiofilm activity observed in our conditions (Supplementary Figure S4A, B), an increase in anisotropy up to 40% was observed after 2 h of contact; however, this fluidising effect was transitory, as the membrane nearly returned to its initial state after 5 h of contact, indicating a maintenance of cell integrity.

An increased anisotropy up to 40% was also observed with betulinic acid after 2 h of contact. This effect was also transitory, betulinic acid treatment resulting in an abrupt anisotropy decrease (8%, i.e. a 32% decrease), indicating complete recovery of the state of the membrane recorded after 5 h of contact. However, after 24 h, an anisotropy reversal up to 20% again suggested a weakened state of the membranes, not found in the case of betulin treatment. However, this effect was not comparable with that of amphotericin B, which argues in favour of a fungistatic activity of betulinic acid in the tested conditions.

The results showed that despite their close structure (Supplementary Figure S4A), betulinic acid and betulin interact differently with fungal cell wall and membrane, suggesting an involvement of the carboxylic acid group only present in betulinic acid [22,41].

The results also indicated that betulinic acid modulated the membrane fluidity of *C. albicans* with a membrane state after 24 h treatment more disturbed than its initial state.

From our experiments, the antibiofilm activity of betulinic acid could be related to a reduction in CSH with a modulation of the cell permeability, thus resulting in changes in cell ability to adhere to the plastic and/or cell surfaces, which especially concerns early stages of biofilm formation.

### 3.8. Betulinic acid may modulate gene expression involved in adhesion between the microbial cells

Finally, the influence of betulinic acid on the expression of candidate genes directly or indirectly involved in biofilm formation of *S. aureus* and *C. albicans* was evaluated using reverse transcriptase-quantitative PCR in mono- and bi-species biofilm conditions. We focused on the first 5 h, corresponding to the early stages of biofilm growth, i.e. to reversible and irreversible attachment to abiotic or biotic surfaces. After 5 h, no change was observed in the relative expression of the bacterial candidate genes (Fig. 7A) involved in quorum sensing (*agrC*, RNAIII and *saeS*), exopolysaccharide synthesis (*icaC*), the expression of a cell surface-bound protein that binds to both fibronectin and fibrinogen (*fnbB*) and oxidative stress response (*sodA*; Fig. 7A). Nevertheless, a significant two-fold downregulation of the *sasF* gene expression was observed in both mono- and bi-species biofilms under betulinic acid treatment. SasF may play an important role as a cell wall-anchored adhesin for the early stages of biofilm formation [42]. Also, an upregulation of SasF was reported in a rat model of orthopaedic implant-associated infection leading to consider this adhesin as an important virulence factor because of its role in biofilm formation [43,44]. Interestingly,
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Relative gene expression in mono- and bi-species early-stage biofilms formed from *Staphylococcus aureus* and *Candida albicans* cultures treated with betulinic acid 25 µg/mL, after 5 h static incubation at 37°C. Expression levels of *fnbB*, *sasF*, *icaC*, *agrC*, *RNAIII*, *saeS*, *sodA* and *als3*, *rbt1*, *bcr1*, *rlm1*, *dpp3*, *cdr1* and *sod1* were normalised against those of *rpoB* and *act1* (considered as reference genes) for *S. aureus* (A) and *C. albicans* (B), respectively. The dimethyl sulfoxide (DMSO)-treated condition was used as the reference for calculations. Results were confirmed using the other selected reference genes (data not shown). Mono- and bi-species early-stage biofilms are represented in transparent colour and with shaded boxes respectively, for *S. aureus* and *C. albicans*.</figcaption></figure>

Schlecht et al. [45] reported that a ΔsasF mutant of *S. aureus* displayed a reduced ability to bind *C. albicans* hyphae, up to 40% [45].

Similarly, after 5 h, no change occurred in the relative expression of the fungal candidate genes involved in quorum sensing (*dpp3*), in efflux pump (*cdr1*), surface components recognising adhesive matrix (*als3*, *rbt1*, *bcr1*, *rlm1*) and oxidative stress response (*sodA*; Fig. 7B).

Although all being reported as biofilm-related, most of the *S. aureus* and *C. albicans* genes selected in our study may not be specifically involved in the early stage of biofilm formation. As reversible and irreversible cell–polystyrene and cell–cell adhesion occur in the early stage, gene expression levels may also considerably vary within the bacterial and fungal populations. The results suggested at least an action of betulinic acid on cell–cell adhesion during biofilm formation as a significant downregulation of *sasF* gene in *S. aureus* was observed 5 h after treatment. The results argue in favour of a reduction in *S. aureus–C. albicans* physical interactions consistent with the data of Schlecht et al. [45], which could partly explain the reduced *S. aureus* biomass in bi-species biofilms treated with betulinic acid (Fig. 1D, E, 2).

Thus, whereas the microbial cell membrane would be the primary target of betulinic acid, our results also suggest that betulinic acid acts on more pathways, independent of surface sensing. Transcriptional analysis with methicillin-resistant *S. aureus* (MRSA) and methicillin-sensible *S. aureus* (MSSA) strains showed that betulinic acid treatment affected ABC transporters, two-component systems, ribosomes and DNA replication in MRSA, whereas the gene expression pattern of MSSA exhibited minimal changes, with only a significant downregulation of *kdpC* in the two-component systems and *murG* in peptidoglycan biosynthesis [21]. Interestingly, different in silico protein homology modelling and molecular docking analyses also suggested interactions between betulinic acid and nuclear and metabolic enzymes in bacterial and fungal cells, such as gyrase, beta-lactamase-like enzymes for *S. aureus* and CYP 51 and dihydrofolate reductase enzymes for *C. albicans* [26,27].

Also, in the literature, results dealing with the efficacy of betulinic acid as an antimicrobial were diverse, its activity depending on many factors. The susceptibility and resistance to betulinic acid vary considerably within and between species; for example, ranging from an MIC of 561 µM for *S. aureus* ATCC 13150, according to Rodrigues et al. [27], to an MIC of 140 µM for *S. aureus* ATCC 43300 and ATCC 29213 but from 8.8 to 1126 µM for clinical *S. aureus*, according to Chung et al. [21]. The variability in experimental parameters (inoculum size, betulinic acid solubilisation and tested concentrations, biofilm growth medium) used to assess biofilm formation or treat preformed biofilms makes it difficult

to compare results between studies. Contrary to *C. albicans* [46], to date, clear results on the antibiofilm activity of betulinic acid against *S. aureus* have been reported in the literature. Gilabert et al. [22] showed stimulation by 88% of *S. aureus* ATCC 6538 biofilm formation under betulinic acid treatment at a final concentration of 110 µM, whereas results described by Silva et al. [25] indicated that betulinic acid displays a 37% inhibition of *S. aureus* ATCC 25904 biofilm formation at 100 µM without any antibiotic activity.

## 4. Conclusions

Betulinic acid seems to be a promising candidate for the prevention and treatment of *S. aureus–C. albicans* biofilms. The associated infections are currently treated with traditional therapies individually targeting the causative agents. Effective treatments are still lacking to cure polymicrobial infections, including those related to interkingdom biofilms, despite the discovery of different natural active structures displaying antibiofilm potential, such as triterpenes. The mechanism of action of betulinic acid against complex biofilms needs to be investigated in-depth: its effect on cytoplasmic membrane and transcriptional responses deserves further experiments to be fully understood.

## Declarations

**Funding:** This research was funded by Region Nouvelle-Aquitaine (AAPR2020-2019-8408110 Research Grant) and University of Poitiers (AAP ARIC 2019 Research Grant) (France). This project was also supported by the French Society of Medical Mycology (SFMM) (AAP SFMM 2022 Research Grant).

**Competing Interests:** The authors declare no conflict of interest and no use of AI tools to analyse and draw insights from data as part of the research process.

**Ethical Approval:** Not required.

**Sequence Information:** Not applicable.

**Author Contributions:** CI, MG and GH were responsible for conceptualisation. GH, WA, AM, JB and FT were responsible for methodology. GH was responsible for validation. GH and MM carried out the formal analysis. GH prepared the original draft. CI, WA, AM, JB, MG, MM and GH reviewed and edited the manuscript. CI and MG were responsible for supervision and project administra-
tion and funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** The authors would like to thank D. Debail for revising the English text.

**Supplementary materials**

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2024.107166.

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