Effects of Coleus amboinicus L. Essential Oil and Ethanolic Extracts on Planktonic Cells and Biofilm Formation of Microsporum canis Isolated from Feline Dermatophytosis

Microsporum canis is an important zoonotic fungus that causes dermatophytosis in domestic animals and their owners. Domestic cats are the primary reservoir for M. canis. Antifungal drugs frequently produce adverse effects on the host animal, increasing the demand for novel alternative treatments derived from nature. We evaluated the antifungal activity of Coleus amboinicus essential oil (CEO) and ethanolic extracts (CEE) against M. canis in planktonic and biofilm growth. Twelve clinical isolates of M. canis were identified in feline dermatophyte samples. Using GC-MS, 18 compounds were identified in CEO, with carvacrol being the major constituent. HPLC analysis of CEE revealed that it contained rosmarinic acid, apigenin, and caffeic acid. The planktonic growth of all M. canis isolates was inhibited by C. amboinicus extracts. The minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 128 µg/mL (32–256 µg/mL) for both CEO and CEE. The MIC90 values of CEO and CEE were 128 and 256 µg/mL, respectively. CEO at MIC (128 µg/mL) and 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of weak, moderate, and strong biofilm-producing M. canis. CEE at 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of all isolates. Overall, C. amboinicus extracts inhibited planktonic growth and exhibited a significant antibiofilm effect against M. canis. Thus, C. amboinicus is a potential source of natural antifungal compounds.


Introduction
Microsporum canis is an important zoophilic dermatophyte in domestic cats and dogs; however, it also causes dermatophytosis in humans. Human infections are acquired from domestic animals through direct contact with clinically or subclinically infected animals [1]. M. canis is a major species of keratinophilic and keratinolytic filamentous fungi that cause superficial fungal infections worldwide [2], particularly in Europe, the eastern Mediterranean, and South America [3][4][5]. M. canis infections in cats, dogs, and other domestic animals such as rabbits generally manifest as multifocal alopecia, scaling, and circular lesions [6,7]. Both stray and domesticated cats are considered common reservoirs for M. canis [8][9][10] and anyone in direct contact with these animals, including owners and veterinarians, is at risk of becoming infected [9]. Treatments include oral and topical preparations of antifungal agents such as amphotericin B, griseofulvin, terbinafine, itraconazole,

Chemical Composition of CEO and CEE
The yield of essential oil obtained from fresh C. amboinicus leaves was 0.08% (v/w), and the oil was clear and light yellow. The refractive index, density (g/cm 3 ), and specific gravity of the oil at 20 • C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively. gravity of the oil at 20 °C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.    gravity of the oil at 20 °C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  gravity of the oil at 20 °C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  gravity of the oil at 20 °C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  gravity of the oil at 20 °C were 1.505, 0.935, and 0.937, respectively. Figure 1 presents the chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  chromatogram of the main components of CEO. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 18 compounds, representing 99.84% of the total composition of CEO (Table 1). The mass spectrum of each compound is presented in Supplementary Figure S1. The concentrations of carvacrol, β-caryophyllene, and thymol in CEO were determined and found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively.  The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( The yield of CEE was 2.2% w/w, appearing as a dark green and highly viscous solid. The total phenolic content was 666.0 ± 9.1 mg gallic acid equivalent (GAE)/g sample and the total flavonoid content was 462.3 ± 3.1 mg quercetin equivalent (QE)/g sample. The three compound standards, namely rosmarinic acid, apigenin, and caffeic acid, were qualified via high-performance liquid chromatography ([HPLC]; Figure 2). The concentrations of rosmarinic acid, apigenin, and caffeic acid used for quantification were 1.251, 1.175, and 0.732 mg/g samples, respectively ( Table 2).

Fungal Isolation and Biofilm Formation
We identified 12 M. canis isolates based on morphology, polymerase chain reaction (PCR), and gene sequencing. A total of 720 base pair (bp) amplicons were obtained from positive M. canis samples using targeted ITS1-5.8S-ITS2 PCR. Bidirectional DNA sequencing was performed to demonstrate that all tested feline specimens were M. canis ITS1-5.8S-ITS2 sequences, which shared 100% sequence identity with those isolated from Thai and Belgium cats (sampled from hair and skin) and are deposited in relevant databases (MT487850 and OW988573). Additionally, the sequences identified in this study were perfectly matched with M. canis ITS1-5.8S-ITS2 sequences isolated from dog (ON527777 and KT155637), horse (LC623726 and OW984765), rabbit (OW987260 and OW987262), and human (OW988577) specimens (Supplementary Figure S2).
All 12 M. canis isolates were classified as weak (25%), moderate (50%), or strong biofilm producers (25%) based on their biofilm production. C. albicans and T. rubrum were classified as strong and moderate biofilm producers, respectively ( Table 3). The biofilm production of M. canis is shown in Supplementary Figure S3.

Fungal Isolation and Biofilm Formation
We identified 12 M. canis isolates based on morphology, polymerase chain reaction (PCR), and gene sequencing. A total of 720 base pair (bp) amplicons were obtained from positive M. canis samples using targeted ITS1-5.8S-ITS2 PCR. Bidirectional DNA sequencing was performed to demonstrate that all tested feline specimens were M. canis ITS1-5.8S-ITS2 sequences, which shared 100% sequence identity with those isolated from Thai and Belgium cats (sampled from hair and skin) and are deposited in relevant databases (MT487850 and OW988573). Additionally, the sequences identified in this study were perfectly matched with M. canis ITS1-5.8S-ITS2 sequences isolated from dog (ON527777 and KT155637), horse (LC623726 and OW984765), rabbit (OW987260 and OW987262), and human (OW988577) specimens (Supplementary Figure S2).
All 12 M. canis isolates were classified as weak (25%), moderate (50%), or strong biofilm producers (25%) based on their biofilm production. C. albicans and T. rubrum were classified as strong and moderate biofilm producers, respectively ( Table 3). The biofilm production of M. canis is shown in Supplementary Figure S3.

Effect of CEO and CEE on Planktonic Cells
CEO and CEE inhibited the planktonic cell growth of all M. canis isolates. The results of the minimum inhibitory concentration (MIC) analysis of CEO and CEE are presented in Tables 3 and 4. The minimal fungicidal concentration (MFC) and MIC values of each treatment were comparable. CEO exhibited antifungal activity comparable to that of CEE, with an MIC 50 of 128 µg/mL (32-256 µg/mL). The MIC 90 of CEE was twice that of CEO. Fluconazole had an MIC 50 and MIC 90 of 8 µg/mL (4-16 µg/mL) and 16 µg/mL, respectively (Table 4).  50 , the minimum inhibitory concentration at which ≥50% of the isolates were inhibited; MIC 90 , the minimum inhibitory concentration at which ≥90% of the isolates were inhibited; GM: geometric mean.

Effect of CEO and CEE on Planktonic Cells
CEO and CEE inhibited the planktonic cell growth of all M. canis isolates. The results of the minimum inhibitory concentration (MIC) analysis of CEO and CEE are presented in Tables 3 and 4. The minimal fungicidal concentration (MFC) and MIC values of each treatment were comparable. CEO exhibited antifungal activity comparable to that of CEE, with an MIC50 of 128 µg/mL (32-256 µg/mL). The MIC90 of CEE was twice that of CEO. Fluconazole had an MIC50 and MIC90 of 8 µg/mL (4-16 µg/mL) and 16 µg/mL, respectively (Table 4).

Discussion
To the best of our knowledge, this is the first report of the antifungal activity of C. amboinicus extracts against planktonic cells and the biofilm formation of M. canis. M. canis biofilms are composed of a multidirectionally expanded network of hyphae linked together by a polysaccharide extracellular matrix [16]. Biofilm reduces the penetrability of antifungal agents, thus contributing to treatment failure and recurrent infection [39,40]. The inhibitory effect of antifungal agents on biofilm formation was observed at concentrations higher than those required to inhibit the growth of planktonic cells [41]. Fungal biofilm formation is a key factor in fungal virulence, persistence, and invasion as well as recurrent fungal infections and conventional antifungal resistance [40,42]. The time-dependent adherence of arthroconidia was observed, starting at 2 h and up to 6 h after inoculation. M. canis produced keratinolytic enzymes and secreted endo and exoproteases during adhesion; this process was likely inhibited by chymostatin, a serine protease inhibitor [43]. After biofilm formation for 72 h, a polysaccharide extracellular matrix that links fungal hyphae was observed [16,44]. The extracellular matrices of poor, moderate, and strong biofilm-producing M. canis appear to be related to mechanisms of antifungal resistance; however, further investigations are needed to confirm this. Flucytosine or fluconazole treatment at every 6-24 h could not completely destroy the biofilms of Candida spp. Poor drug penetration might not be a major mechanism of antifungal resistance for Candida biofilms [45]. During the early stage of C. albicans biofilm formation, genes encoding efflux pumps are upregulated, thereby mediating antifungal resistance [46]. Developing new compounds or alternative inhibitors to treat biofilm-related drug resistant fungal infections is essential to veterinary and human medicine [40,42,47]. In this study, fluconazole (4-16 µg/mL) had no effect on the mature biofilms of M. canis isolates. This result was similar to those reported by Bila et al., who found that fluconazole only inhibited the metabolic activity of early-stage biofilms of T. mentagrophytes at 32 mg/L but did not exhibit antibiofilm activity on mature biofilms, even at the highest concentration (512 mg/mL) [48]

Discussion
To the best of our knowledge, this is the first report of the antifungal activity of C. amboinicus extracts against planktonic cells and the biofilm formation of M. canis. M. canis biofilms are composed of a multidirectionally expanded network of hyphae linked together by a polysaccharide extracellular matrix [16]. Biofilm reduces the penetrability of antifungal agents, thus contributing to treatment failure and recurrent infection [39,40]. The inhibitory effect of antifungal agents on biofilm formation was observed at concentrations higher than those required to inhibit the growth of planktonic cells [41]. Fungal biofilm formation is a key factor in fungal virulence, persistence, and invasion as well as recurrent fungal infections and conventional antifungal resistance [40,42]. The time-dependent adherence of arthroconidia was observed, starting at 2 h and up to 6 h after inoculation. M. canis produced keratinolytic enzymes and secreted endo and exoproteases during adhesion; this process was likely inhibited by chymostatin, a serine protease inhibitor [43]. After biofilm formation for 72 h, a polysaccharide extracellular matrix that links fungal hyphae was observed [16,44]. The extracellular matrices of poor, moderate, and strong biofilmproducing M. canis appear to be related to mechanisms of antifungal resistance; however, further investigations are needed to confirm this. Flucytosine or fluconazole treatment at every 6-24 h could not completely destroy the biofilms of Candida spp. Poor drug penetration might not be a major mechanism of antifungal resistance for Candida biofilms [45]. During the early stage of C. albicans biofilm formation, genes encoding efflux pumps are upregulated, thereby mediating antifungal resistance [46]. Developing new compounds or alternative inhibitors to treat biofilm-related drug resistant fungal infections is essential to veterinary and human medicine [40,42,47]. In this study, fluconazole (4-16 µg/mL) had no effect on the mature biofilms of M. canis isolates. This result was similar to those reported by Bila et al., who found that fluconazole only inhibited the metabolic activity of early-stage biofilms of T. mentagrophytes at 32 mg/L but did not exhibit antibiofilm activity on mature biofilms, even at the highest concentration (512 mg/mL) [48].
Our study also demonstrated that C. amboinicus can inhibit planktonic cell growth and biofilm formation of feline zoonotic M. canis. CEO and CEE significantly inhibited the planktonic cell growth of M. canis at 128 µg/mL (32-256 µg/mL). Considering the MIC 90 values, CEO was found to have a higher potency more potent than CEE against all M. canis isolates (MIC 90 of 128 µg/mL vs. 256 µg/mL). C. amboinicus has been reported to exhibit antifungal activity against several fungi, including Aspergillus clavatus, Aspergillus niger, Cladosporium cladosporioides, Chaetomium globosum, Myrothecium verrucaria, Penicillium citrinum, Trichoderma viride, and Mucor sp. [38,49]. It also inhibits the biofilm formation of other pathogenic microorganisms, such as Streptococcus mutans, Streptococcus pyogenes, and S. aureus [50][51][52]. C. amboinicus is rich in monoterpenes, including carvacrol, thymol, eugenol, chavicol, and ethyl salicylate [37,49,53]. The 18 compounds identified in CEO in this study represent 99.84% of the total essential oil and include carvacrol (56.65%), p-cymene (10.89%), and γ-terpinene (9.33%); these three compounds alone comprise 76.87 % of the total essential oil. The concentrations of carvacrol, β-caryophyllene, and thymol were found to be 3.4 ± 0.2, 0.35 ± 0.16, and 0.013 ± 0.08 mg/mL, respectively. This result differs from that reported by da Costa et al., who found thymol to be the major constituent (64.3%), followed by p-cymene (10.3%), γ-terpinene (9.9%), and β-caryophyllene (2.8%) [54]. Previous studies have reported that the phytochemical composition of CEO is significantly influenced by the cultivation location, processes, and methods of essential oil extraction [25,37]; for example, steam distillation produced higher levels of carvacrol in C. amboinicus essential oil than those produced via the hydrodistillation method [37]. CEO at MIC had excellent effects against all clinical isolates. The high potency of CEO may be attributed to the hydrophobic property of essential oil, which adversely affects every step of biofilm formation, including adhesion, growth, maturation, and dissemination. The antibiofilm mechanisms of essential oil include reducing bacterial adhesions, preventing fresh biofilm formation, and destroying existing biofilm [55,56].
In the present study, CEE effectively eradicated the biofilm formation of weak, moderate, and strong biofilm producers at 2× MIC. Total phenolic and flavonoid contents were positively correlated with the antimicrobial activity of the plant extracts [66]. We found higher total phenolic and flavonoid levels in CEE than those reported in previous studies. For example, the ethanolic extract of C. amboinicus leaves obtained from Vietnam had a total phenolic and total flavonoid content of 26.84 ± 0.91 µg GAE/mg sample and 12.14 ± 0.42 µg QE/mg sample, respectively [67]. A methanolic extract of the C. amboinicus stem obtained from India had a total phenolic content of 49.91 mg GAE/g sample and total flavonoid content of 26.6 mg rutin equivalent/g sample [68]. Flavonoids inhibit nucleic acid biosynthesis and spore germination in plant pathogens [69,70]. High phenolic and flavonoid levels may thus be related to the significant antifungal effects of CEE. Importantly, CEE contained remarkable levels of rosmarinic acid (1.251 mg/g sample), apigenin (1.175 mg/g sample), and caffeic acid (0.732 mg/g sample) in this study. Rosmarinic and caffeic acid compounds have significant antifungal effects against Fusarium oxysporum [71]. The antifungal mechanism of rosmarinic acid is poorly understood but is believed to be related to the RTPase enzyme [72]. Apigenin at a concentration of 5 µg/mL exhibited antifungal activity against C. albicans, C. parapsilosis, Malassezia furfur, T. rubrum, and T. beigelii by inhibiting biofilm formation and efflux-mediated pumps of fungi. It also induced cell death by interfering with membrane function and increasing cell permeability [73,74]. Mice infected with T. mentagrophytes recovered after treatment with apigenin ointment administered at concentrations of 2.5 and 5 mg/g on the 12th and 16th days, respectively [75]. Caffeic acid phenethyl ester, a major active component of propolis (Apis trigona), has been shown to exert concentration-dependent effects on planktonic cells and biofilm formation of different Candida species [76] and synergistically enhance the antifungal activity of fluconazole against resistant clinical isolates of C. albicans [77]. Another study reported that the fungicidal activity of caffeic acid against T. rubrum was observed at 86.59 µM; this activity was mediated via plasma membrane damage and reduced ergosterol production, where caffeic acid reduced isocitrate lyase activity and downregulated critical genes (ERG1, ERG6, and ERG11) required for ergosterol synthesis [78].
Although CEO and CEE had different chemical constituents, both exhibited excellent and comparable inhibitory activities against all fungal isolates obtained from feline dermatophyte samples. Our findings suggest that both CEO and CEE act as natural antifungal agents against planktonic cells and biofilm-producing M. canis. Future investigations of the relationship between plant-based compounds, such as carvacrol in CEO and apigenin in CEE, their mechanisms of action, and classification based on biofilm production may contribute to a better understanding and guide the development of safe and effective antifungal agents derived from natural sources.

Plant Preparation and Extractions
C. amboinicus Lour. was harvested from a pesticide-free garden in the Nonthaburi Province, Thailand (13.862162, 100.409385). The plants were identified and housed at the herbarium within the Faculty of Pharmacy, Mahidol University, Thailand. The voucher specimen was PBM-005507-08. The hydrodistillation method was used to process the fresh leaves of C. amboinicus for essential oil extraction. The extraction was performed using a Clevenger-type apparatus operating at atmospheric pressure. The collected CEO was dried with anhydrous sodium sulfate, transferred to amber glass bottles, and stored at 40 • C. The physical properties of the CEO, including color, density, refractive index, and specific gravity, were evaluated and recorded. The yield of CEO was determined based on the weight of the fresh plant material before processing and was expressed in % (v/w) [37].
During the ethanolic extraction process, C. amboinicus leaves were dried in a hot air oven at 60 • C for 72 h and ground into small pieces. The leaf fragments were macerated in 95% ethanol at room temperature (RT) for 5 days. The extract solution was filtered through sterile gauze and a vaporized solvent using a rotary evaporator at 40 • C (BÜCHI, Flawil, Switzerland). The CEE was lyophilized in Labconco FreeZone 4.5 L Freeze Dryer equipped with Lyo-Works™ Operating System (Labconco, Kansas City, MO, USA) and stored at −20 • C. The yield of CEE was determined based on dry weight, weight after lyophilization, and weight of the leaf fragments before processing, and expressed in % (w/w).

GC-MS
The chemical composition of CEO was analyzed via GC-MS. The samples were diluted with methanol and injected in the split mode (1:10 split ratio) into the GC-MS model Agilent 7890A/5977B GC/MSD system equipped with a DB-5HT capillary column (0.1 µm film thickness × 0.25 mm diameter × 30 m length; Agilent Tech., Santa Clara, CA, USA) at a flow rate of 1 mL·min −1 in helium (carrier gas) and an injector temperature of 250 • C. The initial oven temperature was 40 • C (5 min), which was then increased to 250 • C at a rate of 10 • C/min and maintained there for 5 min. The following MS settings were used: ion source temperature, 230 • C; ionization energy, 70 eV; and mass scan range, 35-550 m/z. Compounds were identified by matching their mass spectra against those specified in Wiley Registry 7th Edition MS libraries. The concentrations of the major components were calculated by comparing the peak area of samples with the peak area of standard compounds.

Determination of Total Phenolic Content
The total phenolic content of CEE was determined using Folin-Ciocalteu's colorimetric assay, with slight modifications. The stock extract solution (1000 µg/mL) was mixed with 125 µL of Folin-Ciocalteu reagent (Merck, Darmstadt, Germany) in a 1:1 ratio for 5 min. Subsequently, 400 µL of 7.5% sodium carbonate was added to the mixture, followed by incubation at RT for 30 min. The absorbance of the final mixture was measured at 760 nm using the Synergy H1 Hybrid Multi-Mode Microplate Reader (BIOTEK, Winooski, VT, USA). Gallic acid was used to prepare the standard curve (with a 40-240 µg/mL calibration range). The gallic acid solutions and the results are expressed in GAE/g of the crude extracts [79].

Determination of Flavonoid Content
The modified aluminum chloride colorimetric method was used to determine the flavonoid content of the plant extracts [80]. A 250-µL aliquot of the extract solution (1000 µg/mL) was mixed with 1.25 mL of deionized water, after which a 5% sodium nitrite solution (75 µL) (Sigma Aldrich, St. Louis, MO, USA) was added and the mixture was allowed to stand for 5 min. Subsequently, 150 µL of 10% aluminum chloride (Sigma Aldrich, St. Louis, MO, USA) was added to the extract solution, followed by 500 µL of 1 M sodium hydroxide. The solution was further diluted with 275 µL of deionized water and allowed to stand for 6 min. Finally, the absorbance was measured at a wavelength of 510 nm using the Synergy H1 Hybrid Multi-Mode Microplate Reader (BIOTEK, Winooski, VT, USA). A quercetin solution (30-300 µg/mL) was used to prepare the standard calibration curves.

HPLC
CEE was analyzed using HPLC. The HPLC 1290 Infinity II system: Zorbrax Eclipse Plus C18 column (2.1 × 50 mm, 1.8-Micron; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with an ultraviolet (UV) detector (280 nm) was used at a gradient flow of 0.5 mL/min. The mobile phase composition was 3% acetic acid in water: 1% acetic acid in water acetonitrile. The injection volume was 2 µL. The column temperature was maintained at 30 • C. The stock extract solution (10 mg/mL) was dissolved in methanol and filtered through 0.45-µm nylon membrane filters before performing HPLC. The compounds present in the extracts were characterized according to their UV-vis spectra and identified in terms of their retention time relative to that of known standards: rosmarinic acid, apigenin, and caffeic acid (Sigma Aldrich, St. Louis, MO, USA) [25]. A standard graph was generated using standard solutions of 5-500 µg/mL. The regression equation correlating to the area under the peak (Y) and standard (X) was as follows: Y = 5.04 (X: rosmarinic acid) + 4.37, Y = 8.98(X: apigenin) + 0.31 and Y = 13.04 (X: caffeic acid) + 1.31.

Sample Collection and Fungal Identification
The study protocol was approved by the Faculty of Veterinary Science, Animal Care and Use Committee (MUVS-2019-09-45) and the Faculty of Veterinary Science-Institutional Biosafety Committee (IBC/MUVS-B-005-2562). Skin, nail, and hair specimens were randomly collected from cat patients with feline dermatophytosis during 2019-2020 at Prasu-Arthorn Animal Hospital, Faculty of Veterinary Science, Mahidol University, Thailand. The samples were placed on Difco™ Potato Dextrose Agar (PDA) (Becton, Dickinson and Company, NJ, USA) plates supplemented with 0.1% chloramphenicol. The M. canis isolates were screened based on the morphology of the colonies, including their size, texture, and color. The characteristics, size, and arrangement of microconidia and macroconidia were evaluated by lactophenol cotton blue staining and observed under a light microscope at 10× and 40× magnification [81].

Confirmation of M. canis Using Molecular Techniques
PCR was used to confirm the species of M. canis. Twelve fungal samples obtained from feline patients were grown on PDA at 27 • C for 7 days before DNA extraction using the Veterinary Diagnosis, Faculty of Veterinary Science, Mahidol University, Thailand) were used as the experimental controls.
Each biofilm biomass was quantified using crystal violet assay. The planktonic cells were discarded, and the attached cells were gently washed twice with PBS. After drying the plates at RT for 10 min, the cells were fixed with 200 µL of absolute methanol for 10 min and subsequently dried for 10 min. After 10 min of drying, 100 µL of aqueous 0.3% crystal violet solution was added to each well and the plates were incubated at RT for 20 min. The remaining dye was removed, and the biofilms were washed with PBS to remove any excess dye. After drying for another 10 min, the crystal violet that had accumulated in the biofilm cells was decolorized using 150 µL of 33% acetic acid for 30 s. Finally, each solution was transferred to a new plate, and the optical density was measured immediately at 590 nm using the BIOTEK ELx808 spectrophotometer (BIOTEK, Winooski, VT, USA) [87].

Effects of CEO and CEE on Planktonic Cells
The microdilution method (based on standard Clinical and Laboratory Standards Institute Guidelines, 2008) [88], was used to determine the MIC. CEO and CEE were mixed with 100 mg/mL dimethyl sulfoxide in a RPMI 1640 medium supplemented with L-glutamine, which was buffered to pH 7.0 with 3-[N-morpholino] propane sulfonic acid (Sigma Aldrich, MO, USA) [41,48], and then subjected to two-fold serial dilution to obtain concentrations from 1024 µg/mL to 2 µg/mL. Fluconazole (Sigma Aldrich, St. Louis, MO, USA), an antifungal agent used as the positive control, was prepared at the same concentration as CEO and CEE. Subsequently, 100 µL of each concentration was added into the wells, followed by inoculation with 100 µL of each fungal suspension to obtain a final concentration of 2 × 10 3 CFU/mL. All procedures were performed in triplicate. Culture media was used as the negative control. C. albicans and T. rubrum were used for internal quality control. The microplates were incubated at 35 • C for 96 h. The MIC was defined as the lowest concentration of extract at which no visible growth was observed under an inverted microscope.
MFCs were established by streaking the subcultures taken from the MIC wells without visible growth on an SDA plate. After incubation at 37 • C for 96 h in aerobic conditions, viable fungal growth was evaluated. The MFC was defined as the lowest concentration of extract at which no fungal growth was observed under an inverted microscope.

Effects of CEO and CEE on the Biofilm Formation of M. canis
The susceptibility of mature M. canis biofilms to CEO and CEE was evaluated by exposing the biofilms to extracts at MIC and 2× MIC. The extracts were diluted in RPMI 1640 and added to each well of a 96-well plate containing a mature biofilm, following which the plates were incubated at 37 • C for 96 h. Each treatment was performed in triplicate. After incubation, the biofilm metabolic activity of the fungi was determined via XTT (2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay [89]. XTT (Sigma Aldrich, St. Louis, MO, USA) was prepared according to the manufacturer's instructions, and 100 µL of XTT-menadione solution was added to each well. The plates were then incubated in the dark for 1-2 h at 37 • C, after which absorbance at 490 nm was measured using the Synergy H1 Hybrid Multi-Mode Microplate Reader (BIOTEK, Winooski, VT, USA). The results are expressed as the average of absorbance values.

Statistical Analysis
Data were evaluated for normal distribution using the Shapiro-Wilk test prior to one-way analysis of variance. All statistical analyses were performed using IBM SPSS Statistics version 21.0. A p value of < 0.05 was considered statistically significant.

Conclusions
The increasing resistance in zoonotic fungi and adverse reactions to antifungal agents are major challenges for the development of natural-based agents. We found CEO and CEE to be extremely effective against the planktonic cell growth of clinical M. canis isolates. C. amboinicus also had significant antibiofilm effects on weak, moderate, and strong fungal biofilm producers. Thus, C. amboinicus may emerge as a novel source of natural antifungals. The antifungal mechanisms of C. amboinicus and drug formulations warrant further investigation for developing safe and effective treatments for zoonotic M. canis infections.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/antibiotics11121734/s1, Figure S1: Mass spectrum of the main components of Coleus amboinicus essential oil determined via gas chromatography-mass spectrometry; Figure S2: Phylogenetic tree analysis of Microsporum canis based on nucleotide sequences from a 720 bp fragment of ITS1-5.8S-ITS2 using the neighbor-joining method. Sequences from this study are marked with stars; Figure   Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available within the article.