Antimicrobial and Biofilm-Preventing Activity of l-Borneol Possessing 2(5H)-Furanone Derivative F131 against S. aureus—C. albicans Mixed Cultures

Candida albicans and Staphylococcus aureus are human pathogens that are able to form mixed biofilms on the surface of mucous membranes, implants and catheters. In biofilms, these pathogens have increased resistance to antimicrobials, leading to extreme difficulties in the treatment of mixed infections. The growing frequency of mixed infections caused by S. aureus and C. albicans requires either the development of new antimicrobials or the proposal of alternative approaches to increase the efficiency of conventional ones. Here, we show the antimicrobial, biofilm-preventing and biofilm-eradicating activity of 2(5H)-furanone derivative F131, containing an l-borneol fragment against S. aureus–C. albicans mixed biofilms. Furanone F131 is also capable of inhibiting the formation of monospecies and mixed biofilms by S. aureus and C. albicans. The minimal biofilm-prevention concentration (MBPC) of this compound was 8–16 μg/mL for S. aureus and C. albicans mono- and two-species biofilms. While the compound demonstrates slightly lower activity compared to conventional antimicrobials (gentamicin, amikacin, fluconazole, terbinafine and benzalkonium chloride), F131 also increases the antimicrobial activity of fluconazole–gentamicin and benzalkonium chloride against mixed biofilms of S. aureus–C. albicans, thus reducing MBPC of fluconazole–gentamicin by 4–16 times and benzalkonium chloride twofold. F131 does not affect the transcription of the MDR1, CDR1 and CDR2 genes, thus suggesting a low risk of micromycete resistance to this compound. Altogether, combined use of antibiotics with a F131 could be a promising option to reduce the concentration of fluconazole used in antiseptic compositions and reduce the toxic effect of benzalkonium chloride and gentamicin. This makes them an attractive starting point for the development of alternative antimicrobials for the treatment of skin infections caused by S. aureus–C. albicans mixed biofilms.


Introduction
Staphylococcus aureus is a common opportunistic microorganism causing various infectious diseases in human and animals. Together with various virulence factors allowing host colonization, tissue invasion, and host immune system evasion, this bacterium also possesses different tools for overcoming the treatment with antimicrobials [1,2], that makes S. aureus one of the most dangerous nosocomial pathogens [3,4]. Thus, based on its susceptibility to antibiotics, S. aureus is classified into methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) [5,6]. In the last decade, MRSA quickly became the most common drug-resistant pathogen identified in many parts of the world [7][8][9].
The biofilm formation by S. aureus on various immunogenic and non-immunogenic surfaces of the human body represents an additional challenge in medicine [10]. While tion. All solvents were purified and distilled by standard procedures. IR spectra were recorded on a Bruker Tensor-27 spectrometer fitted with a Pike MIRacle ATR accessory (diamond/ZnSe crystal plate). IR spectra were recorded of solids with characteristic absorption wavenumbers (ν max ) reported in cm −1 . NMR spectra were measured on a Bruker Avance III 400 spectrometer at 400.17 MHz ( 1 H) and 100.62 MHz ( 13 C) at 20 • C in the deuterated chloroform. The chemical shifts (δ) are expressed in parts per million (ppm) and are calibrated using residual undeuterated solvent peak as the internal reference (CDCl 3 : δ H 7.26, δ C 77. 16). All coupling constants (J) are reported in Hertz (Hz) and multiplicities are indicated as: s (singlet), m (multiplet). High-resolution mass spectra (HRMS) were obtained by electrospray ionisation (ESI) with positive (+) ion detection on a Bruker micrOTOF-III quadrupole time-of-flight mass spectrometer. Analytical thin layer chromatography (TLC) was carried out on Sorbfil PTLC-AF-A-UF plates using dichloromethane as the eluent and UV light as the visualizing agent. The melting points were measured on an OptiMelt Stanford Research Systems MPA100 automated melting point apparatus and were not corrected. Optical rotations were measured on a Perkin-Elmer model 341 polarimeter at λ 589 nm and at 20 • C in chloroform (concentration c is given as g/100 mL).

Antimicrobials
Amikacin, gentamicin, benzalkonium chloride, terbinafine and fluconazole were purchased from Sigma, (St. Louis, MO, USA). Solutions were prepared at a concentration of 20 mg/mL in deionized water, or in dimethyl sulfoxide (DMSO) for fluconazole. Solutions of F131 were prepared at a concentration of 10 mg/mL in DMSO.

Strains and Growth Conditions
The antimicrobial activity of the compounds was assessed on methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 29213 and 9 clinical isolates of S. aureus (3 MSSA and 6 MRSA) provided by the Pharmaceuticals Research Center of Kazan Federal University. Antimycotic activity was assessed on 12 clinical isolates of Candida albicans (6 fluconazoleresistant strains, and 6 fluconazole-susceptible strains) obtained from the Kazan Research Institute of Epidemiology and Microbiology. The bacterial strains were stored in 10% (v/v) glycerol stocks, fungal strains in 50% glycerol stock at −80 • C, and freshly streaked on Luria Agar (LA) plates following by their overnight growth at 37 • C before use. To obtain a mature biofilm, bacterial and fungal cells were grown in basal medium (BM broth) [54] in tissue-culture-treated (TC-treated) plates under static conditions for 48 h at 37 • C. A mannitol salt agar (peptone 10 g, meat extract 1 g, NaCl 75 g, D-mannitol 10 g, agar-agar 12 g in 1.0 L tap water, Oxoid) and Sabouraud's agar (Difco) were used for differential count of S. aureus and C. albicans, respectively, from S. aureus-C. albicans mixed cultures.

Determination of the Minimal Inhibitory (MIC) and the Minimal Bactericidal/Fungicidal Concentrations (MBC/MFC) of F131 and Reference Antimicrobials
The minimum inhibitory concentration (MIC) was determined by serial 2-fold microdilution in 96-well plates according to the EUCAST rules for antimicrobial susceptibility testing [61] with some modifications. The maximal final concentration of each tested compound was 512 µg/mL. The next well contained two-fold decreasing concentration of the antimicrobial in the range of 0.5-512 µg/mL. The wells were seeded with microbial culture with a final density of 10 6 CFU/mL in a BM broth [54] as universal medium for all further experiments. The plates were incubated for 24 hours at 37 • C without shaking. The minimal inhibitory concentration of compounds was defined as the concentration that ensures complete suppression of the visible growth of the strains. The minimal bactericidal/fungicidal concentration (MBC/MFC) was determined by inoculation in 3 mL of nutrient broth with 3 µL of culture fluid from wells in which no visible growth was observed. MBC/MFC was considered as the minimum concentration of the compound, which ensures the complete absence of microorganisms growth.

Analysis of the Biofilm-Prevention Concentration (BPC)
To determine the minimal biofilm-prevention concentration, bacterial and fungal cells were grown in 96-well adhesive plastic plates for 48 hours without shaking at 37 • C in 200 µL BM broth with an initial density 3 × 10 7 CFU/mL in the presence of the test compounds with concentration in the range of 0.5-512 µg/mL. Next, wells were washed with water and subjected to a staining with crystal violet as described in [62]. The minimal biofilm inhibitory concentration was defined as the lowest concentration at which the residual biofilm was two-fold less compared to untreated wells.

Assessment of Synergy between F131 and Conventional Antimicrobials
To assess a synergy between F131 and antimicrobials, a checkerboard assay was performed as described previously [41]. Each plate contained serial dilutions of a F131 and various antimicrobials in a checkerboard pattern. One of the antimicrobial substances [A] was diluted horizontally, and the F131 [B] vertically on a 96-well plate. The extreme lines and columns contained only one of the considered compounds to determine their MICs in each experiment. The initial concentration of each of the studied antimicrobial agents was 2 × MIC. The plates were incubated at 37 • C for 24 h. The experiments were performed in a triplicate, a growth control without the addition of any antimicrobial agent was included in each plate. The fractional inhibitory concentration index (FICI) for each double combination was calculated as follows: Interpretation of the obtained FICI values was carried out according to Odds [63]; FICI ≤ 0.5 corresponded to synergy, 0.5 < FICI ≤ 4 an additive effect, while FICI > 4 corresponded to antagonism.

Quantification of Viable Cells
To assess the viability of detached cells and cells in the biofilm in the presence of antimicrobials, a drop plate assay [64] with modifications was used. Briefly, a series of ten-fold dilutions of cell suspension from each well were prepared in 3 technical repeats and 5 µL from each dilution were dropped onto both mannitol salt agar and Sabouraud agar with ciprofloxacin (10 µg/mL) to differentiate S. aureus and C. albicans cells, respectively. CFUs were counted from the two last drops where 5-15 colonies were grown and averaged. In the case of the biofilm-embedded cells, the wells were pre-washed twice with sterile 0.9% NaCl and biofilms were suspended in 0.9% NaCl by scratching the well bottoms. Additionally, the wells were sonicated for 2 min to disintegrate the remained cell clumps [65]. The dilutions of obtained suspension were prepared and CFUs were counted as described above.

Confocal laser Scanning Microscopy (CLSM)
To assess the localization of F131 in bacterial and fungal cells, confocal laser scanning microscopy was performed using an Olympus IX83 inverted microscope supplemented with a STEDYCON ultrawide extension platform. Both monospecies and bacterial-fungal cultures of S. aureus and C. albicans were grown on cell imaging cover slips (Eppendorf) for 48 h in BM broth. The fluorescent derivative of 2(5H)-furanone F145 [53] was added to the cells at a final concentration of 20 µg/mL and biofilms were analyzed at blue (405/410-508 nm) channel.

RNA Isolation and Real-Time One-Step qRT-PCR
To analyze the transcription of the efflux system genes MDR1, CDR1, and CDR2, the PCR was performed using PCR amplifier "BioRad CFX96" (BioRad, Singapore) using Extra Mix for reverse transcription and quantitative real-time PCR in a one-step method (BioLabMix, Novosibirsk, Russia) with SYBR Blue under conditions recommended by the manufacturer. The oligonucleotides used for the qRT-PCR are shown in Table 1 [66][67][68]. A total RNA was extracted from planktonic C. albicans 688 cells subjected to 48-h treatment with either fluconazole or F131. Reaction mixture (50 µL) contained 1× RT-qPCR SYBR Blue buffer, 0.1 µM of each primers, 0.1 µM of each dNTPs, 2.5% DMSO, 5% RT-qPCR extra-mix and nuclease-free water (DEPC). The RT-PCR program included reverse transcription at 45 • C for 30 min, followed by 37 cycles of primer annealing-elongationmelting. Primer annealing temperatures were calculated using the Tm Calculator service (https://tmcalculator.neb.com, accessed on 6 January 2022). The elongation time was calculated based on the size of the synthesized fragment and the speed of the DNA polymerase. Real-time PCR monitoring was carried out at the stage of final synthesis at 72 • C. The 18s rRNA and GAPDH genes were used as references; the transcription level of qCDR1, qCDR1 and MDR1 genes was normalized by the 18s rRNA transcription level.

Data Analysis
All experiments were performed in three biological replicates with three technical replicates. The data was analyzed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA, USA). The significance of difference has been checked by using the Kruskal-Wallis test. Significant differences from control were considered at p < 0.05.

Synthesis of Furanone F131
Synthesis of novel 2(5H)-furanone derivative F131 possessing sulfonyl group and l-borneol moiety was achieved via three steps from commercially available mucochloric acid 1 (Figure 1). Following a previously published procedure, mucochloric acid 1 was first converted to the corresponding 5-(l-bornyloxy) derivative, obtained as a mixture of two diastereomers 2a + 2b [52,60]. The pure stereoisomer 2a with S-configuration of the carbon atom C 5 of the γ-lactone cycle was isolated after two recrystallizations from hexane. 72 °С. The 18s rRNA and GAPDH genes were used as references; the transcription level of qCDR1, qCDR1 and MDR1 genes was normalized by the 18s rRNA transcription level.

Data Analysis
All experiments were performed in three biological replicates with three technical replicates. The data was analyzed using GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). The significance of difference has been checked by using the Kruskal-Wallis test. Significant differences from control were considered at p < 0.05.

Synthesis of Furanone F131
Synthesis of novel 2(5H)-furanone derivative F131 possessing sulfonyl group and l-borneol moiety was achieved via three steps from commercially available mucochloric acid 1 (Figure 1). Following a previously published procedure, mucochloric acid 1 was first converted to the corresponding 5-(l-bornyloxy) derivative, obtained as a mixture of two diastereomers 2a + 2b [52,60]. The pure stereoisomer 2a with S-configuration of the carbon atom C 5 of the γ-lactone cycle was isolated after two recrystallizations from hexane.   Next, regioselective introduction of 4-chlorothiophenol fragment into the molecule of the isolated stereoisomer 2a was carried out. It is well-known that interaction of 3,4-dihaloderivatives of 2(5H)-furanone with different thiols in the presence of basic compounds goes with the substitution of halogen atom in the fourth position of the lactone cycle [69,70]. The thiilation reaction was performed in dichloromethane at room temperature using the equimolar ratio of furanone 2a, thiol and triethylamine. Chiral thioether 3 was obtained with 71% yield and subjected to a further oxidation reaction. We used a simple and convenient method of the synthesis of furanone sulfonyl derivatives, based on the treatment of thioethers with excess of hydrogen peroxide in acetic acid at room temperature [70,71]. Thus, the desired novel sulfone F131 in a stereomerically pure form was isolated with 70% yield as colorless crystals ( Figure 1). The structure of borneol-containing furanones 3 and F131 has been characterized by IR and NMR spectroscopy ( Figures A1-A4).

Antimicrobial and Biofilm Preventing Activity of F131
The antimicrobial and antifungal properties of F131 were evaluated using a range of S. aureus clinical isolates (n = 9) and methicillin-sensitive S. aureus ATCC 29213 (Table 2), as well as twelve C. albicans clinical isolates with different susceptibility to fluconazole ( Table 3). All clinical isolates of S. aureus were resistant to gentamicin. MRSA strains were also resistant to amikacin, while MSSA strains remained sensitive to the antibiotic. Six of the twelve clinical isolates of C. albicans were resistant to fluconazole. MIC of F131 ranged within 8-16 µg/mL for S. aureus isolates, and 32-128 µg/mL for C. albicans isolates (Tables 2 and 3) suggesting that susceptibility of isolates to F131 does not correlate with resistance to conventional antimicrobials. Of note, the l-borneol itself was ineffective against both S. aureus and C. albicans (MIC > 1024 for all isolates). The minimal bactericidal concentration (MBC) and the minimal fungicidal concentration (MFC) values of F131 ranged within 32-128 µg/mL and 128-1024 µg/mL respectively (Tables 2 and 3). These data indicate a considerable bactericidal and fungicidal activity of F131, as the MIC/MBC ratio lies within 2-4, with a few exceptions. * BAC-benzalkonium chloride, Gen-gentamicin, Ami-amikacin; ** Ecoff-epidemiological cut-off value; the MIC value is above the ECOFF defines that the bacterium is likely to be resistant [61]. Further, since the biofilm-inhibiting properties for many 2(5H)-furanone derivatives have been reported, the ability of F131 to repress the biofilm formation by S. aureus and C. albicans was assessed. For that S. aureus and C. albicans were grown for 48h under static conditions in the presence of F131. Then, the biofilm was quantified by both crystal violet staining and direct count of viable cells. The compound led to two-fold reduction of the S. aureus biofilm biomass at the concentration of 8 µg/mL, whereas the three-log decrease of viable cells in the biofilm was observed at 16 µg/mL (Figure 2A). For fluconazoleresistant C. albicans 688, the biofilm biomass was reduced twice at 128 µg/mL, and 64 µg/mL led to a 1000-fold decrease of viable cell in the biofilm ( Figure 2B). Of note, the dose-dependent dynamics of viable cells decrease was similar for both planktonic and adherent (biofilm-forming) ones, suggesting that the observed biofilm prevention activity is driven, apparently, rather by the reduction of viable cells than the true inhibition of the biofilm formation. This hypothesis is also supported by fact; that the conventional antimicrobials such as gentamicin, amikacin, fluconazole, terbinafine and benzalkonium chloride also reduced the amount of viable cells in the biofilm at their respective two-fold MICs (Figures 3 and 4).

Antibiofilm Activity of F131 on S. aureus and C. albicans Mixed Biofilms
S. aureus ATCC 29213 and C. albicans 688 fluconazole-resistant isolates were chosen to apply tests on mixed bacterial-fungal biofilms. The S. aureus-C. albicans mixed culture was grown under static conditions in BM broth in the presence of F131 in concentrations varying from 2 to 256 µg/mL. After 48h, the biofilms were quantified by the crystal violet staining and CFUs count by plating on mannitol salt agar, and Sabouraud agar with ciprofloxacin (10 µg/mL) to differentiate S. aureus and C. albicans cells, respectively. The F131 at 128 µg/mL completely inhibited the biofilms formation in mixed cultures (Figure 5a). The growth of S. aureus and C. albicans in mixed culture was repressed at 16 µg/mL and 64 µg/mL, respectively (Figure 5b), similarly to monocultures. Comparison of antibiofilm activity of antimycotics against C. albicans isolate 688 FR . C. albicans isolate was grown for 48 h under static conditions in a 96-well flat-bottom plate in the presence of sub-MICs of fluconazole, terbinafine, benzalkonium and F131. The concentrations are given in relative units as X-fold MIC, which were as follows: 1024 mg/L for fluconazole, 128 mg/L for terbinafine, 4 mg/L for benzalkonium, and 64 mg/L for F131. The asterisks (*) denote a statistically significant difference in cells viability in the untreated wells and treated ones in Kruskal-Wallis test (p < 0.05).

Antibiofilm Activity of F131 on S. aureus and C. albicans Mixed Biofilms
S. aureus ATCC 29213 and C. albicans 688 fluconazole-resistant isolates were chosen to apply tests on mixed bacterial-fungal biofilms. The S. aureus-C. albicans mixed culture was grown under static conditions in BM broth in the presence of F131 in concentrations varying from 2 to 256 μg/mL. After 48h, the biofilms were quantified by the crystal violet staining and CFUs count by plating on mannitol salt agar, and Sabouraud agar with ciprofloxacin (10 μg/mL) to differentiate S. aureus and C. albicans cells, respectively. The F131 at 128 μg/mL completely inhibited the biofilms formation in mixed cultures ( Figure  5a). The growth of S. aureus and C. albicans in mixed culture was repressed at 16 μg/mL and 64 μg/mL, respectively (Figure 5b), similarly to monocultures. To further analyze whether F131 not only prevents biofilm formation but also eradicates already established ones, different concentrations (5-320 μg/mL) of F131 or reference antimicrobials were added to the established 24 h old mixed biofilms. After 24 h, biofilms and detached cells clumps in culture liquid were quantified by differential CFUs counting. For the detached S. aureus cell clumps, the efficiency of F131 was comparable with those of both aminoglycosides. Further, both F131 and aminoglycosides reduced the CFUs number of S. aureus in biofilm by three orders of magnitude at a concentration of 80 μg/mL (2.5-5×MBC), whereas 10 μg/mL (5×MBC) of benzalkonium chloride was required to achieve the same effect (Figure 6b). On C. albicans, the efficiency of F131 was comparable with those of terbinafine. Of note, neither terbinafine nor F131 led to three-order reduction in the CFUs count even at concentrations up to 640 μg/mL (Figure 6d). To further analyze whether F131 not only prevents biofilm formation but also eradicates already established ones, different concentrations (5-320 µg/mL) of F131 or reference antimicrobials were added to the established 24 h old mixed biofilms. After 24 h, biofilms and detached cells clumps in culture liquid were quantified by differential CFUs counting. For the detached S. aureus cell clumps, the efficiency of F131 was comparable with those of both aminoglycosides. Further, both F131 and aminoglycosides reduced the CFUs number of S. aureus in biofilm by three orders of magnitude at a concentration of 80 µg/mL (2.5-5 × MBC), whereas 10 µg/mL (5 × MBC) of benzalkonium chloride was required to achieve the same effect ( Figure 6B). On C. albicans, the efficiency of F131 was comparable with those of terbinafine. Of note, neither terbinafine nor F131 led to three-order reduction in the CFUs count even at concentrations up to 640 µg/mL ( Figure 6D).
These findings raised questions about the penetration possibility of furanone derivatives into the biofilm matrix formed by S. aureus-C. albicans. To confirm these suggestions, the fluorescent analogue of 2(5H)-furanone named F145, possessing the fluorescent moiety instead of l-borneol and described earlier [72], was added to established 24-hours-old mono-and mixed-biofilms of S. aureus-C. albicans, and after 20 min, incubation samples were analyzed by confocal laser scanning microscopy. The fluorescence of F145 was observed throughout the whole biofilm (Figure 7), confirming its fast penetration through the S. aureus-C. albicans biofilm matrix. These findings raised questions about the penetration possibility of furanone derivatives into the biofilm matrix formed by S. aureus-C. albicans. To confirm these suggestions, the fluorescent analogue of 2(5H)-furanone named F145, possessing the fluorescent moiety instead of l-borneol and described earlier [72], was added to established 24-hours-old mono-and mixed-biofilms of S. aureus-C. albicans, and after 20 min, incubation samples were analyzed by confocal laser scanning microscopy. The fluorescence of F145 was observed throughout the whole biofilm (Figure 7), confirming its fast penetration through the S. aureus-C. albicans biofilm matrix.

Synergistic Effects of F131 with Other Antimicrobials on S. aureus -C. albicans Mixed Biofilms
Previously, it has been shown that the 2(5H)-furanone derivative F105 containing l-menthol and sulfonyl moieties, exhibits a synergy with aminoglycosides and benzalkonium сhloride against S. aureus [41] and potentiates the antifungal activity of fluconazole and terbinafine against C. albicans cells [53]. Taking into account the similar structures of compounds F105 and F131, which are both chiral furanone sulfones, pos-

Synergistic Effects of F131 with Other Antimicrobials on S. aureus-C. albicans Mixed Biofilms
Previously, it has been shown that the 2(5H)-furanone derivative F105 containing l-menthol and sulfonyl moieties, exhibits a synergy with aminoglycosides and benzalkonium chloride against S. aureus [41] and potentiates the antifungal activity of fluconazole and terbinafine against C. albicans cells [53]. Taking into account the similar structures of compounds F105 and F131, which are both chiral furanone sulfones, possessing a fragment of terpene alcohol in the fifth position of the lactone cycle, we assumed similar properties for the latter.
Therefore, the synergism of F131 in combination with conventional antimicrobials was analyzed by the checkerboard assay on S. aureus ATCC 29213 and C. albicans  (Table 5). In turn, FIC values of F131 decreased up to eight-fold when combining with gentamicin, fluconazole, and benzalkonium chloride.  Further, we questioned whether the synergistic effect of 2(5H)-furanone derivatives could be used to eradicate mixed S. aureus-C. albicans infection. Therefore, we tested the effect of combination of F131 with conventional antimicrobials on mixed S. aureus-C. albicans biofilms formation and eradication, since the biofilm-embedded consortium became extremely resistant to treatment compared to planktonic cells. Firstly, the S. aureus-C. albicans mixed cultures were inoculated in BM broth in the presence of various concentrations of antimicrobials as indicated.  For convenience, we plotted a graph representing the antifungal and antibacterial effect of fluconazole-gentamicin mix and benzalkonium chloride alone and in combination with fixed concentration of F131 (8 µg/mL) against S. aureus-C. albicans mixed biofilms ( Figure 9). As could be seen from the Figure 9, in the presence of 8 µg/mL F131, the concentrations of fluconazole, gentamicin and benzalkonium chloride, required to decreases CFUs number by three orders of magnitude are four-fold lower when compared with solely antimicrobials. Taken together, these data clearly indicate that F131 at a concentration of 8 µg/mL (1/2 BPC for S. aureus, and 1/8 BPC for C. albicans) is able to significantly reduce the number of viable cells when combining with sub-BPCs of conventional antifungals and antibacterial drugs.

The Combination of F131 with Fluconazole Significantly Decreased the Expression Level of CDR1 and CDR2 Genes
The mRNA levels of the efflux system genes MDR1, CDR1, and CDR2 were analyzed by quantitative real-time RT-PCR in C. albicans 688 isolate (resistant isolate). Cells were grown in the presence of 32 μg/mL fluconazole and/or 8 μg/mL F131 for 24 hours. The same volume of solvents (DMSO and ethanol) was added to the untreated control group of cells. The results showed that F131 solely did not affect the expression of the mentioned genes, since the coefficient of relative expression of MDR1, CDR1 and CDR2 were 1.12, 1.29 and 1.31, respectively. When treating with fluconazole, the expression level was suppressed of the MDR1, CDR1 and CDR2 genes, although underestimated (relative expression 0.71, 0.62 and 0.69, respectively). It is noteworthy that the expression of CDR1 and CDR2 genes decreased significantly after the combined treatment with fluconazole and F131 (relative expression 0.22 and 0.31, respectively) ( Figure 10).

The Combination of F131 with Fluconazole Significantly Decreased the Expression Level of CDR1 and CDR2 Genes
The mRNA levels of the efflux system genes MDR1, CDR1, and CDR2 were analyzed by quantitative real-time RT-PCR in C. albicans 688 isolate (resistant isolate). Cells were grown in the presence of 32 µg/mL fluconazole and/or 8 µg/mL F131 for 24 hours. The same volume of solvents (DMSO and ethanol) was added to the untreated control group of cells. The results showed that F131 solely did not affect the expression of the mentioned genes, since the coefficient of relative expression of MDR1, CDR1 and CDR2 were 1.12, 1.29 and 1.31, respectively. When treating with fluconazole, the expression level was suppressed of the MDR1, CDR1 and CDR2 genes, although underestimated (relative expression 0.71, 0.62 and 0.69, respectively). It is noteworthy that the expression of CDR1 and CDR2 genes decreased significantly after the combined treatment with fluconazole and F131 (relative expression 0.22 and 0.31, respectively) ( Figure 10).

Discussion
Taking into account the high mortality and morbidity rates associated with S. aureus -C. albicans mixed infections, there is an always pursuit to find new potential alternatives that ensure better efficiency and lower toxicity during the management of candida mixed infections, especially in the course of strains that are resistant to fluconazole. Here, we show that the novel 2(5H)-furanone derivative F131, consisting of two pharmacophores (2(5H)-furanone сycle and l-borneol moiety), exhibits an antimicrobial and biofilm-preventing activity as well as potentiates conventional antibiotics and antifungal agents against S. aureus and C. albicans and their mixed culture. Earlier, we showed that the structural analogue of F131 with l-menthol fragment (3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfon yl]-2(5H)-furanone F105) exhibited comparable antibacterial and antifungal activity against S. aureus [41] and C. albicans [53]. F131 possesses the l-borneol instead of l-menthol fragment and demonstrates higher antimicrobial activity. Interestingly, many previous studies reported the anti-inflammatory and antimicrobial activity of borneol derivatives [73][74][75]. Some aromatic borneol compounds were significantly active with MIC50 = 125 μg/mL for both S. aureus and C. albicans [76]. In our previous study we examined the relationship between the chemotype and antimicrobial activity of 2(5H) derivatives. We found that the l-borneol moiety is an essential part of the molecule of 3,4-dichloro-5(S)-[(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy]-2(5H)-furanone (F123), with respect to demonstrating its antibacterial activity against gram-positive bacterium B. cereus [52]. This data raised a question whether the borneol fragment is responsible for the observed antimicrobial activity of F131. For that, we tested the activity of terpene alcohol l-borneol against both S. aureus and C. albicans in mono and mixed cultures. However, our data showed that l-borneol itself is not able to inhibit the growth of either S. aureus or C. albicans, suggesting that this terpene alcohol apparently facilitates the penetration of the multipharmacophore drug into both the cells, probably thereby also providing the synergy with other antimicrobials (Tables 2,3), and into biofilms (Figure 7) thus exhibiting biofilm-eradicating activity ( Figure 6).
Our data show that F131 demonstrates bactericidal and fungicidal activity, as the MIC/MBC ratio lies within 2-4, with a few exceptions (Tables 2,3). Moreover, the sus-

Discussion
Taking into account the high mortality and morbidity rates associated with S. aureus-C. albicans mixed infections, there is an always pursuit to find new potential alternatives that ensure better efficiency and lower toxicity during the management of candida mixed infections, especially in the course of strains that are resistant to fluconazole. Here, we show that the novel 2(5H)-furanone derivative F131, consisting of two pharmacophores (2(5H)-furanone cycle and l-borneol moiety), exhibits an antimicrobial and biofilm-preventing activity as well as potentiates conventional antibiotics and antifungal agents against S. aureus and C. albicans and their mixed culture. Earlier, we showed that the structural analogue of F131 with l-menthol fragment (3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone F105) exhibited comparable antibacterial and antifungal activity against S. aureus [41] and C. albicans [53]. F131 possesses the l-borneol instead of l-menthol fragment and demonstrates higher antimicrobial activity. Interestingly, many previous studies reported the anti-inflammatory and antimicrobial activity of borneol derivatives [73][74][75]. Some aromatic borneol compounds were significantly active with MIC 50 = 125 µg/mL for both S. aureus and C. albicans [76]. In our previous study we examined the relationship between the chemotype and antimicrobial activity of 2(5H) derivatives. We found that the l-borneol moiety is an essential part of the molecule of 3,4-dichloro-5(S)-[(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy]-2(5H)-furanone (F123), with respect to demonstrating its antibacterial activity against gram-positive bacterium B. cereus [52]. This data raised a question whether the borneol fragment is responsible for the observed antimicrobial activity of F131. For that, we tested the activity of terpene alcohol l-borneol against both S. aureus and C. albicans in mono and mixed cultures. However, our data showed that l-borneol itself is not able to inhibit the growth of either S. aureus or C. albicans, suggesting that this terpene alcohol apparently facilitates the penetration of the multipharmacophore drug into both the cells, probably thereby also providing the synergy with other antimicrobials (Tables 2 and 3), and into biofilms (Figure 7) thus exhibiting biofilm-eradicating activity ( Figure 6).
Our data show that F131 demonstrates bactericidal and fungicidal activity, as the MIC/MBC ratio lies within 2-4, with a few exceptions (Tables 2 and 3). Moreover, the susceptibility of isolates to F131 does not correlate with resistance to conventional antimicrobials, making this compound suitable to the treatment of infections caused by resistant strains, including mixed C. albicans-S. aureus infections. In addition, F131 was able to prevent the biofilm formation by both pathogens, either separately or in the mixed culture (Figures 2 and 5). Generally, BPCs of F131 were found to approximately correspond to MICs, suggesting indirect biofilm-preventing activity of F131, apparently because of the reduction of viable cells rather than the true inhibition of the biofilm formation. Additionally, this assumption is supported by noting that the amount of viable planktonic cells also decreased by three orders of magnitude at the corresponding BPCs of F131 against both S. aureus and C. albicans (Figure 2). Thus, it could be suggested that the biofilm suppression was rather the consequence of cell growth repression. Interestingly, for C. albicans the BPCs were a little higher than MICs. This phenomenon might be attributed to the induction of biofilm formation by the fungi under sub-lethal concentrations of furanone derivatives [53]. Having a closer look at the F131 chemotype, F131 is a halogenated furanone containing a chlorine atom in the third position of a five-membered cycle, but our findings are not in agreement with previous studies that reported a direct biofilm-inhibiting activity of some halogenated furanones against gram-positive bacteria such as Bacilli and Staphylococci [48,54].

Conclusions
Taken together, our in vitro data allows the assumption of the F131 chemotype as a biofilm-preventing agent, especially in the course of skin infections caused by Candida-Staphylococcus mixed biofilms. In addition, due to its synergistic effect with conventional antimicrobials, the combination of the latter with F131 or F131-like compounds would give rise to the minimization of the required therapeutic doses and thus reduction of side effects, toxicity and tolerance development by pathogens. Nevertheless, further in vitro assays for the biosafety of F131 and in vivo studies are required to understand further perspectives to managing Candida-Staphylococcus mixed infections. Funding: This work was funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities, project №FZSM-2022-0017. The synthetic part of this work was funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (no. 0671-2020-0063).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: All data are included in the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.