Inhibition of Mixed Biofilms of Candida albicans and Staphylococcus aureus by β-Caryophyllene-Gold Nanoparticles

Polymicrobial biofilms, consisting of fungal and bacterial pathogens, often contribute to the failure of antimicrobial treatment. The growing resistance of pathogenic polymicrobial biofilms to antibiotics has led to the development of alternative strategies to combat polymicrobial diseases. To this end, nanoparticles synthesized using natural molecules have received significant attention for disease treatment. Here, gold nanoparticles (AuNPs) were synthesized using β-caryophyllene, a bioactive compound isolated from various plant species. The shape, size, and zeta potential of the synthesized β-c-AuNPs were found to be non-spherical, 17.6 ± 1.2 nm, and -31.76 ± 0.73 mV, respectively. A mixed biofilm of Candida albicans and Staphylococcus aureus was used to test the efficacy of the synthesized β-c-AuNPs. The results revealed a concentration-dependent inhibition of the initial stages of formation of single-species as well as mixed biofilms. Furthermore, β-c-AuNPs also eliminated mature biofilms. Therefore, using β-c-AuNPs to inhibit biofilm and eradicate bacterial-fungal mixed biofilms represents a promising therapeutic approach for controlling polymicrobial infections.


Introduction
Polymicrobial interactions at the interspecies or cross-kingdom level significantly influence pathogenic behaviors, including virulence, growth, host immune system tolerance, and antimicrobial resistance [1][2][3]. The interactions between Staphylococcus aureus and Candida albicans are major contributors to skin, mucosal, and bloodstream infections [4][5][6]. S. aureus is the third most common bacterial pathogen that is often responsible for various infections such as blood, burn wound, and catheter infections, as well as denture stomatitis and peri-implantitis [5,7,8]. The effect of injecting sublethal doses of S. aureus and C. albicans into animal models on mortality rates have been previously investigated [9,10]. Pathogenicity due to the synergistic effect of S. aureus and C. albicans also increases antimicrobial resistance. Physical adherence and chemical signaling have been identified to be responsible for the synergistic interactions between S. aureus and C. albicans. S. aureus physically adheres onto the surface of C. albicans, which is coated with an extracellular matrix [11,12]. The involvement of secretory molecules produced by S. aureus and C. albicans in interspecies interactions further demonstrated the presence of chemical interactions. Thus, biofilm formation is enhanced, and drug resistance is increased [12]. The synthesized β-c-AuNPs were fully characterized using several instrumental techniques. Moreover, their antimicrobial activities against S. aureus and C. albicans were investigated. Since the majority of previously developed nanoparticles have been evaluated against single-species biofilms [39][40][41], the inhibitory activity of β-c-AuNPs on polymicrobial biofilms is a novel contribution to efforts in combatting the emergence of antimicrobial resistance. Figure 1 shows the procedure of the synthesis of β-c-AuNPs followed in this study. A color change from yellow to deep wine-red was used to confirm the formation of AuNPs. The UV-vis absorption spectra showed a continuous increase, with a maximum peak at 534 nm ( Figure 2A). An FTIR analysis revealed that β-c-AuNPs exhibit vibration band characteristics at 3400, 2970, 1568, 1510, 1397, and 1054 cm −1 , respectively ( Figure 2B). The broadband at 3400 cm −1 and a shoulder at 2970 cm −1 represent the hydroxyl group of the compound, which is involved in metal ion reduction [28]. The peak at 1397 cm −1 corresponds to the O-H bending of phenol, whereas the peak at 1054 cm −1 corresponds to the CO-O-CO stretching of anhydride. The average size of β-c-AuNPs was determined to be 17.6 ± 1.2 nm ( Figure 2C) using dynamic light scattering. The zeta potential of β-c-AuNPs was measured as −31.76 ± 0.73 mV ( Figure 2D). The UV-vis absorption spectra showed a continuous increase, with a maximum peak at 534 nm ( Figure 2A). An FTIR analysis revealed that β-c-AuNPs exhibit vibration band characteristics at 3400, 2970, 1568, 1510, 1397, and 1054 cm −1 , respectively ( Figure 2B). The broadband at 3400 cm −1 and a shoulder at 2970 cm −1 represent the hydroxyl group of the compound, which is involved in metal ion reduction [28]. The peak at 1397 cm −1 corresponds to the O-H bending of phenol, whereas the peak at 1054 cm −1 corresponds to the CO-O-CO stretching of anhydride. The average size of β-c-AuNPs was determined to be 17.6 ± 1.2 nm ( Figure 2C) using dynamic light scattering. The zeta potential of β-c-AuNPs was measured as −31.76 ± 0.73 mV ( Figure 2D). The field emission transmission electron microscope (FE-TEM) analysis of β-c-AuNPs morphology revealed a non-uniform shape ( Figure 3A-C). The scanning electron microscopy (SEM) analysis further confirmed the non-uniform shape of β-c-AuNPs as well ( Figure 4B). The presence of Debye-Scherrer rings in selected area diffraction pattern (SAED) confirmed the crystalline nature of β-c-AuNPs ( Figure 3D). Furthermore, an Xray diffractometer (XRD) analysis revealed distinctive peaks at 2ϴ values of 38.2°, 45.5°, 55.4°, 66.2°, 75.3°, and 77.5°, which are similar to the characteristic peaks observed in other AuNPs obtained using green synthesis methods ( Figure 4A). The presence of Au in β-c-AuNPs was also confirmed by elemental mapping ( Figure 4C). The presence of a gold signal in β-c-AuNPs was confirmed by energy dispersive X-ray (EDX) analysis ( Figure  4D). Thermal gravimetric analysis (TGA) of the synthesized AuNPs confirmed a steady weight loss from 20 to 600 °C, with a total loss of up to 900 °C. ( Figure 4E). The field emission transmission electron microscope (FE-TEM) analysis of β-c-AuNPs morphology revealed a non-uniform shape ( Figure 3A-C). The scanning electron microscopy (SEM) analysis further confirmed the non-uniform shape of β-c-AuNPs as well ( Figure 4B). The presence of Debye-Scherrer rings in selected area diffraction pattern (SAED) confirmed the crystalline nature of β-c-AuNPs ( Figure 3D). Furthermore, an X-ray diffractometer (XRD) analysis revealed distinctive peaks at 2θ values of 38.2 • , 45.5 • , 55.4 • , 66.2 • , 75.3 • , and 77.5 • , which are similar to the characteristic peaks observed in other AuNPs obtained using green synthesis methods ( Figure 4A). The presence of Au in β-c-AuNPs was also confirmed by elemental mapping ( Figure 4C). The presence of a gold signal in β-c-AuNPs was confirmed by energy dispersive X-ray (EDX) analysis ( Figure 4D). Thermal gravimetric analysis (TGA) of the synthesized AuNPs confirmed a steady weight loss from 20 to 600 • C, with a total loss of up to 900 • C. ( Figure 4E).

Determination of the Minimum Inhibitory Concentration of β-c-AuNPs
The antimicrobial activities of β-c-AuNPs were investigated using the microbroth dilution method. The minimum inhibitory concentration (MIC) of β-c-AuNPs against S. aureus and C. albicans ( Figure 5) was found to be 512 μg/mL. We also determined the MIC values of β-caryophyllene for these pathogens, and found that the values were different from those of β-c-AuNPs. The MIC of β-caryophyllene against S. aureus and C. albicans was found to be >2048 μg/mL, which is four-fold higher than the MIC of β-c-AuNPs. The MIC values of tetracycline and fluconazole as control drugs against S. aureus and C. albicans were found to be >0.5 μg/mL and >32 μg/mL, respectively. These values are consistent with previously published values [42].

Determination of the Minimum Inhibitory Concentration of β-c-AuNPs
The antimicrobial activities of β-c-AuNPs were investigated using the microbroth dilution method. The minimum inhibitory concentration (MIC) of β-c-AuNPs against S. aureus and C. albicans ( Figure 5) was found to be 512 µg/mL. We also determined the MIC values of β-caryophyllene for these pathogens, and found that the values were different from those of β-c-AuNPs. The MIC of β-caryophyllene against S. aureus and C. albicans was found to be >2048 µg/mL, which is four-fold higher than the MIC of β-c-AuNPs. The MIC values of tetracycline and fluconazole as control drugs against S. aureus and C. albicans were found to be >0.5 µg/mL and >32 µg/mL, respectively. These values are consistent with previously published values [42].

Figure 5.
Growth study for determining the MIC values of β-c-AuNPs against S. aureus and C. albicans. *** p < 0.0001 and ** p < 0.01 are statistically significant compared to the control, whereas ns is non-significant.

Inhibitory Activity of β-c-AuNPs on Biofilms
The counting of microbial colony forming units (CFU) was used to examine the effects of sub-MIC β-c-AuNPs on the initial-stage biofilms of S. aureus and C. albicans, as well as the mixed biofilm. The biofilm inhibitory effects of β-c-AuNPs on S. aureus were found to be concentration-dependent ( Figure 6A). The maximum level of inhibition on the S. aureus biofilm was observed at a concentration of 256 μg/mL, with a reduction of 3.34 log CFU compared to the control. β-c-AuNPs were found to inhibit C. albicans biofilms in a concentration-dependent manner as well. Here, the maximum level of inhibition was found to be a 2.45 log CFU reduction of cells at a concentration of 256 μg/mL of β-c-AuNPs ( Figure 6B). The inhibition of the initial stage of the mixed biofilm of S. aureus and C. albicans was also found to be concentration-dependent, as measured by the CFU values of S. aureus and C. albicans on a selective agar plate containing an antibiotic or antifungal agent ( Figure 6C). The reduction in the log CFU value of S. aureus and C. albicans from the mixed biofilm was found to be 4.4 and 3.0, respectively, at 256 μg/mL of β-c-AuNPs. The inhibitory effect of β-c-AuNPs towards single-species biofilms of S. aureus and C. albicans was found to be higher compared to the mixed biofilms. albicans. *** p < 0.0001 and ** p < 0.01 are statistically significant compared to the control, whereas ns is non-significant.

Inhibitory Activity of β-c-AuNPs on Biofilms
The counting of microbial colony forming units (CFU) was used to examine the effects of sub-MIC β-c-AuNPs on the initial-stage biofilms of S. aureus and C. albicans, as well as the mixed biofilm. The biofilm inhibitory effects of β-c-AuNPs on S. aureus were found to be concentration-dependent ( Figure 6A). The maximum level of inhibition on the S. aureus biofilm was observed at a concentration of 256 µg/mL, with a reduction of 3.34 log CFU compared to the control. β-c-AuNPs were found to inhibit C. albicans biofilms in a concentration-dependent manner as well. Here, the maximum level of inhibition was found to be a 2.45 log CFU reduction of cells at a concentration of 256 µg/mL of β-c-AuNPs ( Figure 6B). The inhibition of the initial stage of the mixed biofilm of S. aureus and C. albicans was also found to be concentration-dependent, as measured by the CFU values of S. aureus and C. albicans on a selective agar plate containing an antibiotic or antifungal agent ( Figure 6C). The reduction in the log CFU value of S. aureus and C. albicans from the mixed biofilm was found to be 4.4 and 3.0, respectively, at 256 µg/mL of β-c-AuNPs. The inhibitory effect of β-c-AuNPs towards single-species biofilms of S. aureus and C. albicans was found to be higher compared to the mixed biofilms.

Microscopic Examination of Biofilms Treated with β-c-AuNPs
The SEM analysis further confirmed the inhibitory effects of β-c-AuNPs on singlespecies as well as mixed biofilms. Our findings here revealed biofilms densely populated with C. albicans cells in the control sample ( Figure 7A), yet only a few cells on the membrane in the β-c-AuNPs-treated cells ( Figure 7B). Similarly, the untreated S. aureus biofilms were found to be thicker ( Figure 7C) than those treated with β-c-AuNPs ( Figure 7D). The surface adherence of both cells was very low in the mixed biofilm of S. aureus + C. albicans when incubated with β-c-AuNPs, compared to the control mixed biofilms ( Figure  7E,F). Based on the SEM analysis, we concluded that the sub-MIC levels of β-c-AuNPs have biofilm inhibitory effects on single-and mixed-species biofilms of bacterial and fungal pathogens.

Microscopic Examination of Biofilms Treated with β-c-AuNPs
The SEM analysis further confirmed the inhibitory effects of β-c-AuNPs on singlespecies as well as mixed biofilms. Our findings here revealed biofilms densely populated with C. albicans cells in the control sample ( Figure 7A), yet only a few cells on the membrane in the β-c-AuNPs-treated cells ( Figure 7B). Similarly, the untreated S. aureus biofilms were found to be thicker ( Figure 7C) than those treated with β-c-AuNPs ( Figure 7D). The surface adherence of both cells was very low in the mixed biofilm of S. aureus + C. albicans when incubated with β-c-AuNPs, compared to the control mixed biofilms ( Figure 7E,F). Based on the SEM analysis, we concluded that the sub-MIC levels of β-c-AuNPs have biofilm inhibitory effects on single-and mixed-species biofilms of bacterial and fungal pathogens.

Effect of β-c-AuNPs towards Mature Biofilms
In addition to the inhibition of initial-stage biofilms, the dispersal of mature singleand mixed-species biofilms was also evaluated using the colony counting method. Different concentrations of β-c-AuNPs were used to test the inhibition of mature biofilms compared to those used in initial-stage biofilm inhibition experiments. The inhibition of mature biofilms was tested using β-c-AuNPs at sub-MIC, MIC, and above MIC values. The inhibition of mature biofilms of S. aureus and C. albicans was found to be significantly higher at the MIC and above the MIC value of β-c-AuNPs (Figure 8). Accordingly, the reduction in CFU counts of the S. aureus biofilm cells at 1024 µg/mL (>MIC) and 512 µg/mL (MIC) were found to be 3.2 log CFU and 1.9 log CFU, respectively ( Figure 8A). Similarly, the level of eradication of C. albicans mature biofilms at 1024 µg/mL (>MIC) and 512 µg/mL (MIC) was found to be a 3.98 log CFU and 2.11 log CFU reduction of cells, respectively, as compared to the control ( Figure 8B). Individual mature biofilms of S. aureus and C. albicans were eradicated in both cases in a concentration-dependent manner. The CFU counts of the mature mixed biofilm were also significantly reduced at MIC values and above the MIC value of β-c-AuNPs ( Figure 8C). The level of reduction of S. aureus and C. albicans cells in the mixed mature biofilm at the above MIC value (1024 µg/mL) of β-c-AuNPs was found to be 1.43 log CFU and 2.11 log CFU, respectively. Similarly, the reduction of S. aureus and C. albicans at the MIC value (512 µg/mL) of β-c-AuNPs in the mixed mature biofilm was found to be 0.97 log CFU and 1.12 log CFU, respectively.

Effect of β-c-AuNPs towards Mature Biofilms
In addition to the inhibition of initial-stage biofilms, the dispersal of mature singleand mixed-species biofilms was also evaluated using the colony counting method. Different concentrations of β-c-AuNPs were used to test the inhibition of mature biofilms compared to those used in initial-stage biofilm inhibition experiments. The inhibition of mature biofilms was tested using β-c-AuNPs at sub-MIC, MIC, and above MIC values. The inhibition of mature biofilms of S. aureus and C. albicans was found to be significantly higher at the MIC and above the MIC value of β-c-AuNPs (Figure 8). Accordingly, the reduction in CFU counts of the S. aureus biofilm cells at 1024 μg/mL (>MIC) and 512 μg/mL (MIC) were found to be 3.2 log CFU and 1.9 log CFU, respectively ( Figure 8A). Similarly, the level of eradication of C. albicans mature biofilms at 1024 μg/mL (>MIC) and 512 μg/mL (MIC) was found to be a 3.98 log CFU and 2.11 log CFU reduction of cells, respectively, as compared to the control ( Figure 8B). Individual mature biofilms of S. aureus and C. albicans were eradicated in both cases in a concentration-dependent manner. The CFU counts of the mature mixed biofilm were also significantly reduced at MIC values and above the MIC value of β-c-AuNPs ( Figure 8C). The level of reduction of S. aureus and C. albicans cells in the mixed mature biofilm at the above MIC value (1024 μg/mL) of β-c-AuNPs was found to be 1.43 log CFU and 2.11 log CFU, respectively. Similarly, the reduction of S. aureus and C. albicans at the MIC value (512 μg/mL) of β-c-AuNPs in the mixed mature biofilm was found to be 0.97 log CFU and 1.12 log CFU, respectively.

Discussion
The rising number of antimicrobial therapy failures in polymicrobial infections has become a major concern worldwide [43]. The cross-kingdom interaction between pathogenic bacteria and fungal pathogens is a well-studied example of polymicrobial infection [11,44]. In vivo and in vitro studies have shown the existence of synergistic physical and chemical interactions between S. aureus and C. albicans [10,45,46]. Here, we developed a strategy to control biofilm formation using nanotechnology approaches by using S. aureus and C. albicans as examples of polymicrobial pathogens. For this purpose, AuNPs were synthesized using a plant-derived compound called β-caryophyllene. Several instrumental techniques were used to characterize the synthesized β-c-AuNPs. The sizes of the β-c-AuNPs were discovered to be relatively small (17.6 ± 1.2 nm), rendering them highly effective in treating microbial pathogens due to their large surface area [47]. Previously, AuNPs synthesized by utilizing a hexane fraction of O. sanctum comprising methyl eugenol and β-caryophyllene were likewise found to have a modest size in the range of 1 to 50 nm [28]. Similarly, AgNPs synthesized using β-caryophyllene showed a similar size range of 5 to 100 nm [48].
The synthesized β-c-AuNPs were found to have an irregular shape, similar to those previously reported for AuNPs synthesized using natural products [49]. However, spherical AuNPs synthesized with a hexane fraction containing β-caryophyllene have previously been reported [28]. The high zeta potential of β-c-AuNPs confirms that β-caryophyllene acts as a stabilizing agent, as previously described [28]. Previously, various plant products containing β-caryophyllene have been used to produce a variety of polymeric and metallic nanomaterials, each with a unique size and shape (Table 1). These nanoparticles have been shown to have antimicrobial activities against bacterial and fungal pathogens ( Table 1). The β-c-AuNPs synthesized in this study were found to have antimicrobial activity against S. aureus and C. albicans. Our investigation showed an identical MIC value of β-c-AuNPs against S. aureus and C. albicans, despite the fact that the MIC values of different AuNPs synthesized using naturally derived chemicals show varying activities against various microbial pathogens [50]. Here, nanoparticles were synthesized using a natural pure compound called β-caryophyllene, which enables the pinpointing of key molecules involved in antimicrobial activity [15]. However, most studies have synthesized nanoparticles using extracts containing β-caryophyllene with a wide spectrum of physiologically active ingredients, making it difficult to specify the components related to nanoparticle formation and biological activity (Table 1).
Polymicrobial biofilms are a major cause of antimicrobial treatment failure, immune system evasion, the development of antimicrobial resistance, and the progression of chronic or persistent infections [51,52]. The mixed biofilms of S. aureus and C. albicans were exposed to the sub-MIC of β-c-AuNPs to control the initial-stage biofilm formation. The S. aureus and C. albicans biofilms that developed individually or in mixed forms were inhibited in a concentration-dependent manner. Previous studies have shown similar inhibitory effects of AuNPs synthesized using natural compounds against polymicrobial biofilms of S. aureus and C. albicans in a concentration-dependent manner [50]. However, drug resistance has become a major challenge in eradicating established mature biofilms [53]. Here, the eradication of mature mixed biofilms of S. aureus and C. albicans was found to be significant, particularly at and above the MIC of β-c-AuNPs. The eradication effects of β-c-AuNPs on the single-species biofilms of S. aureus and C. albicans were also higher at and above MIC. The recalcitrant nature of biofilm polymeric substances results in high drug concentrations required for the inhibition of mature biofilms [50,54]. Previous studies have shown that AuNPs synthesized using natural compounds at and above MIC can destroy mature singleor mixed-species biofilms [50,55]. However, because of the enhanced biofilm formation in a synergistic manner in mixed-species biofilms, inhibiting them at the initial stage or eradicating mature biofilms requires a high concentration of drugs [50].

Microbes, Culture Media, and Reagents
The bacterial pathogen S. aureus (KCTC 1916) and fungal pathogen C. albicans (KCCM 11282) were used as reference strains [42]. The growth media for S. aureus and C. albicans were tryptic soy broth (TSB) (Difco Laboratory Inc., Detroit, MI, USA) and potato dextrose broth (PDB) with glucose (5%). Growth media containing 50% TSB and 50% PDB with glucose (5%) were used to grow the mixed S. aureus and C. albicans cell cultures. The culture shock was maintained in 2% glycerol, and culturing was carried out on agar plates (TSA/PDA). Both pathogens were grown at 37 • C. β-caryophyllene (≥98.5%) and gold(III) chloride trihydrate were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

Synthesis and Characterization of β-c-AuNPs
The synthesis of β-c-AuNPs was carried out exactly as described previously [50]. In brief, the reaction was carried out in a volume of 200 mL of deionized water by first dissolving 1 mM of HAuCl 4 .3H 2 O (pH 9.0) and stirring at 60 • C. A drop-wise solution of β-caryophyllene (1 mM) was added to the reaction mixture and stirred continuously at 60 • C. During the reaction, the color of the liquid changed from yellow to a deep winered. Furthermore, the nanoparticle synthesis was confirmed by measuring the UV-visible absorption spectra using a microplate reader (BioTek, Winooski, VT, USA) with the scanned absorption spectra ranging from 200 to 700 nm. A Fourier transform infrared spectrometer (FTIR, JASCO (FT-4100), Tokyo, Japan) was used to measure the ionic interaction in β-c-AuNPs. The Litesizer 500 was used to determine the size and zeta potential of β-c-AuNPs (Anton Paar, GmbH, Graz, Austria). An XRD (X-Ray Diffractometer, Rigaku (Japan), Ultima IV) was used to determine the crystalline nature of β-c-AuNPs.

Microbroth Dilution for MIC Determination
The antimicrobial activities of β-c-AuNPs were assessed by determining the minimal inhibitory concentration (MIC) against S. aureus and C. albicans, as recommended by the Clinical and Laboratory Standards Institute (CLSI) [56]. S. aureus and C. albicans cell cultures (OD 600 = 0.05) were individually incubated with β-c-AuNPs or β-caryophyllene. The concentration of β-c-AuNPs or β-caryophyllene ranged from 128 to 2048 µg/mL. The treated and untreated cell culture (for the control group) was incubated at 37 • C for 24 h before measuring the OD 600 . As previously reported, tetracycline and fluconazole were used as control drugs to determine the MIC value [42]. The MIC value was calculated based on the absence of visible growth as well as a growth inhibition greater than 90% as measured by the OD 600 . The experiment was carried out in triplicate.

Biofilm Inhibition Assays
The S. aureus (OD 600 = 0.05 in TSB) and C. albicans (OD 600 = 0.05 in PDB with 5 % glucose) cell cultures were incubated separately in a 96-well polystyrene microtiter plate (SPL Life Sciences Co., Ltd. Republic of Korea) containing sub-MIC β-c-AuNPs (ranging from 64 to 256 µg/mL). In the mixed biofilm experiment, 50% S. aureus (prepared in TSB) and 50% C. albicans cell cultures (prepared in PDB containing 5 % glucose) were added to a microplate containing sub-MIC β-c-AuNPs and incubated for 24 h at 37 • C. The cells that were not treated with β-c-AuNPs were considered the control group. The surface-attached biofilm was counted using the plate counting method described previously [42,50]. After 24 h of incubation, the planktonic cells were removed, and the biofilm cells were rinsed thrice with sterile TSB. The washed cells were scraped off using a sterile pipette tip and resuspended in sterile TSB. The cell culture was serially diluted and plated on an agar plate (TSA for S. aureus and PDA for C. albicans). The colonies on the agar plates were counted, and the CFU value was computed.

Inhibition of Established Mature Biofilms
The mature biofilm was eradicated using β-c-AuNPs in the same manner as previously described [50]. As mentioned in the biofilm inhibition experiments, the S. aureus and C. albicans cell cultures were initially allowed to produce mature biofilms independently and in combination. After 24 h of incubation at 37 • C, the planktonic cells were removed and the surface-adhered cells were treated with varying doses of β-c-AuNPs (ranging from 128 to 1024 µg/mL). A control group of untreated cell cultures was used. The microplate was incubated for a further 24 h. The surface-adhered cells were counted using the colony counting method described in the biofilm inhibition assays.

SEM Analysis of Biofilm Architecture
SEM was used to visualize the biofilm cells of S. aureus and C. albicans treated with β-c-AuNPs, either in mono-species or mixed form [42]. In brief, the cell cultures of single microbial species with an OD 600 of 0.05 and mixed cultures were placed in a 24-well microplate with a nylon membrane. These cultures were treated with sub-MIC β-c-AuNPs (256 µg/mL) and incubated at 37 • C for 24 h. Formaldehyde (2%) and glutaraldehyde (2.5%) were used to fix the biofilm cells. The fixed cells were washed with phosphate-buffered saline (pH 7.4) and dehydrated in increasing concentrations of ethyl alcohol. The nylon membrane was attached to the SEM stub and visualized using TESCAN (Vega II LSU, Brno, Czech Republic) microscopy at 10 kV voltage and with a magnification of 3.5 kx (20 µm).

Statistical Analysis
GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA) was used to make all the graphs. One-way ANOVA was used for the statistical analysis carried out by one-way ANOVA with Dunnett's multiple comparisons tests. p < 0.0001, p < 0.01 and p < 0.05 were considered as significant.

Conclusions
Here, we synthesized β-c-AuNPs to inhibit the polymicrobial biofilms of S. aureus and C. albicans. The β-c-AuNPs were synthesized using a pure plant-derived compound, β-caryophyllene, with multiple biological roles. Table 1 summarizes the application of several plant materials containing β-caryophyllene in nanoformulations for treating microbiological infections. The synthesized β-c-AuNPs were found to be highly stable with irregular shapes and an average size of 17.6 ± 1.2 nm. β-c-AuNPs were also found to show antimicrobial activities against S. aureus and C. albicans. The sub-MIC level of β-c-AuNPs inhibited the initial-stage biofilms of S. aureus and C. albicans as well as the mixed biofilm. These inhibitory effects were concentration-dependent. Furthermore, the mature biofilm eradication of S. aureus and C. albicans was found to be effective at the MIC and above MIC value of β-c-AuNPs in individual cell cultures and mixed cultures.
Although the synthesized β-c-AuNPs may be biocompatible due to the use of natural compounds, further investigation is still required to examine the toxicity of β-c-AuNPs on different types of animal cells. In future, we intend to focus on elucidating the action mechanism of β-c-AuNPs on the polymicrobial biofilms of S. aureus and C. albicans by analyzing the biofilm and virulence-related gene expression. Furthermore, the findings of this in vitro study will also be validated in an in vivo system and in host-mimicking media that resemble the host environment.
Author Contributions: F.K., conceptualization, methodology, investigation, data curation, writingoriginal draft, and writing-review and editing; N.T., methodology, investigation, data curation, writing-original draft, and writing-review and editing; G.-J.J., data curation, and writing; W.-K.J., supervision, funding, and review and editing; Y.-M.K., supervision, funding, and writing-review and editing. All authors have read and agreed to the published version of the manuscript.

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