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Article

In Vitro Antimicrobial Activity of Piper betle Leaf Extract and Some Topical Agents against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus Strains from Canine Pyoderma

by
Patcharaporn Phensri
1,
Kokaew Thummasema
1,
Udomlak Sukatta
2,
Serge Morand
3,4 and
Chantima Pruksakorn
1,*
1
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, 50 Ngamwongwan Road, Bangkok 10900, Thailand
2
Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok 10900, Thailand
3
MIVEGEC, CNRS-IRD-Montpellier University, 911 Avenue Agropolis, 34394 Montpellier, France
4
Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand
*
Author to whom correspondence should be addressed.
Animals 2022, 12(22), 3203; https://doi.org/10.3390/ani12223203
Submission received: 25 September 2022 / Revised: 1 November 2022 / Accepted: 12 November 2022 / Published: 18 November 2022
(This article belongs to the Section Companion Animals)
Browse Figures
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Abstract

:

Simple Summary

Pyoderma is one of the most common diseases in dogs. The primary pathogen isolated from canine pyoderma is Staphylococcus pseudintermedius, followed by Staphylococcus schleiferi subsp. coagulans. With the emergence of a multidrug-resistant strain, namely, methicillin-resistant staphylococci (MRS), topical antimicrobial therapy is encouraged. In this study, the in vitro antimicrobial activity of crude Piper betle leaf extract and some topical antimicrobials against clinical S. pseudintermedius and S. schleiferi subsp. coagulans, including their MRS strains, were evaluated. It was found that betel leaf extract demonstrated good antimicrobial activity with a higher proficiency than azelaic acid and benzoyl peroxide. Accordingly, betel leaf extract could provide a novel antimicrobial treatment, which may help reduce the need for systemic antibiotics, for canine pyoderma.

Abstract

As multidrug-resistant methicillin-resistant staphylococci (MRS) is becoming more prevalent in canine pyoderma, the discovery of new therapeutic options is required. This study aimed to test the antimicrobial activity of crude Piper betle leaf extract and some topical antimicrobial agents against canine Staphylococcus clinical strains by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The results showed that the mean MICs of chlorhexidine, miconazole, crude P. betle leaf extract, azelaic acid, and benzoyl peroxide against Staphylococcus strains were 1.41, 1.62, 252.78, 963.49, and 1342.70 mg/L, respectively. Therefore, betel leaf extract demonstrated a superior efficacy to azelaic acid and benzoyl peroxide. Furthermore, the ratio of MBC/MIC of betel leaf extract was 1.75, indicating its bactericidal action. When applied to methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP), betel leaf extract was equally efficient towards both groups. S. pseudintermedius strains were more susceptible to betel leaf extract than S. schleiferi subsp. coagulans. In gas chromatography–mass spectrometry analysis, eugenol and hydroxychavicol appeared to be the major components of betel leaf extract. Given its efficacy, dogs with pyoderma could benefit from the use of betel leaf extract as a topical antimicrobial alternative.

1. Introduction

Pyoderma is a common dermatologic disease in dogs [1]. According to previous studies in the United Kingdom and Canada, pyoderma was found in 10.8% and 25.3% of canine dermatological disorders, respectively, which represents an estimated one fifth of the total dog caseloads [1,2]. In another UK survey, a rate of 1.3% pyoderma was reported among the total number of dogs presented to veterinary practitioners [3]. The pathogenesis of pyoderma is described as a bacterial skin infection, typically secondary to underlying primary disorders, including allergies, ectoparasites, and endocrinopathy, that weaken the skin defense mechanisms [4]. Staphylococcus pseudintermedius is the most common bacteria associated with canine pyoderma, along with, to a lesser extent, Staphylococcus schleiferi subsp. coagulans [5,6,7,8,9]. Antibacterial treatment usually relies on systemic antimicrobial therapy [3,10]. Among dogs with pyoderma, 91.9% were prescribed systemic antimicrobials, either alone or in combination with a topical product [3]. However, the growing number of methicillin-resistant staphylococci (MRS) cases has limited the use of many conventional antimicrobial agents [8,10,11,12]. MRS strains are resistant to all beta-lactam antibiotics, with the exception of the newest generation of cephalosporin beta-lactams [13,14]. An additional resistance to other drug classes (macrolides, aminoglycosides, fluoroquinolones, and sulfonamides) is frequently found among MRS isolates and is defined as multidrug resistance [8,15]. Some MRS strains are associated with zoonotic problems and pose a threat to public health [16]. The alarming antimicrobial resistance problem highlights the role of topical therapy in an antimicrobial stewardship strategy in treating canine pyoderma [5,17,18]. Furthermore, the global veterinary dermatology drug market is expected to expand by 9.5% and be worth nearly USD 10 billion by 2031 [19]. With the trend of pet adoption increasing, this is expected to be a key factor to bolster the market. New topological inventions could not only obtain more market value from other sectors but also drive market growth.
Topical therapy is recommended as the sole treatment or as an adjunctive to systemic antimicrobial agents for superficial pyoderma [5,18]. Chlorhexidine alone, chlorhexidine with miconazole, and benzoyl peroxide, in formulations such as shampoos, are preferred for generalized lesions [5]. With Malassezia infections, a combination of chlorhexidine and miconazole has been found to be clinically effective [20]. Topical compounds, including chlorhexidine, miconazole, benzoyl peroxide, fusidic acid, and mupirocin, have demonstrated anti-staphylococcal activity against canine isolates [21,22,23,24,25]. Chlorhexidine and miconazole’s MICs towards canine staphylococcal isolates have been found to be low, with their MIC50 values ranging from 0.5 to 8 mg/L, and differences in susceptibility related to geography could be observed [22,23,24]. A benzoyl peroxide-containing shampoo has been tested for its in vitro efficacy in canine S. pseudintermedius, with an MBC of 1:2 to 1:8 dilutions [25]. However, data on benzoyl peroxide’s MIC and MBC, which can be used to evaluate its efficacy in a more direct way, are lacking. For localized pyoderma lesions, a variety of hydroxyl acids represent a treatment option [5]. In human medicine, azelaic acid, a dicarboxylic acid, is regarded as a topical acne treatment [26]. Particularly, it is recommended as a second-line therapy [26]. The antimicrobial activity of azelaic acid has been documented in many bacteria, including pathogenic Staphylococcus species of human skin [27]. It has not been evaluated in terms of its anti-staphylococcal efficacy for veterinary purposes.
In addition to the in vitro antimicrobial evidence, the clinical efficacy of chlorhexidine shampoo in canine superficial pyoderma has been evaluated to be the most effective, compared with benzoyl peroxide and ethyl lactate shampoos [17,28]. Due to the superior effectiveness of chlorhexidine, it is considered to be one of the most widely used topical agents [29]. Although the insensitivity of bacteria to chlorhexidine has been uncommon in veterinary dermatology, there is evidence suggesting that the use of chlorhexidine and the overall exposure of bacteria to chlorhexidine increases the risk of resistance not only to chlorhexidine, but also to some antimicrobials [30]. There is correspondingly a concern regarding the use of and resistance to fusidic acid and mupirocin in animals, which has led some European countries to reserve these drugs for human medicine [31]. Hence, there is an urgent need for additional treatment options.
Natural remedies have become increasingly interesting in veterinary dermatology, with a focus on plants and their derivatives, which may provide alternative antimicrobial compounds [32,33]. Mangosteen extract, manuka honey, and many essential oils have been reported to have antimicrobial activity against S. pseudintermedius clinical isolates [34,35,36,37]. Among the essential oils is betel vine (Piper betle Linn.) oil [38]. As an alternative to its essential oil, P. betle can also be used as a crude extract, which has been reported to possess greater antibacterial activity [39]. Regarding its background, P. betle is a member of the Piperaceae family, which is widely cultivated in Thailand, Malaysia, Taiwan, India, and Sri Lanka [40]. In traditional medicine, the leaf is used to treat a wide range of health conditions and skin infections [39]. Recent research has shown that P. betle leaves possess antibacterial activity against a variety of Gram-negative and Gram-positive bacteria [41]. Based on studies of its essential oil, P. betle consists of a variety of phytochemically active compounds, classified as alkaloids/amides, propenylphenols, terpenes/sesquiterpenes, steroids, and prenylated hydroxybenzoic acids [42]. Several compounds from the propenylphenol group, such as hydroxychavicol and eugenol, have been detected in P. betle that likely contribute to its antibacterial properties [42,43]. Crude P. betle leaf extracts could be effective against Staphylococcus species of canine pathogens and provide a therapeutic option for dogs suffering from pyoderma.
As benzoyl peroxide, miconazole, and chlorhexidine are standard topical treatments, they were included in this study. In addition, azelaic acid is not a dermatology primary option but another candidate for the treatment of canine pyoderma. This study aimed to assess the in vitro antimicrobial activity of crude P. betle leaf extract against canine Staphylococcus clinical isolates, including methicillin-resistant S. pseudintermedius (MRSP), methicillin-susceptible S. pseudintermedius (MSSP), and methicillin-resistant S. schleiferi subsp. coagulans (MRSS) and to compare it with that of azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine.

2. Materials and Methods

2.1. Bacterial Strains

Seventy-five Staphylococcus clinical isolates, including 31 methicillin-resistant S. pseudintermedius (MRSP), 21 methicillin-susceptible S. pseudintermedius (MSSP), 19 methicillin-resistant S. schleiferi subsp. coagulans (MRSS), 3 methicillin-susceptible S. schleiferi subsp. coagulans (MSSS), and one methicillin-susceptible Staphylococcus simulans, were obtained from a strain collection at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University. These strains had been isolated from cases of canine pyoderma in the dermatology unit at Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand as part of the standard care of the patients and were not related to the present study. Brief details of the identification and characterization of these clinical strains are as follows. The species identification and methicillin resistance were confirmed by a polymerase chain reaction (PCR) based on nuc [44] and mecA [45] genes, respectively. Control strains for PCR included S. aureus ATCC 29213 (DMST 4745, Department of Medical Sciences, Thailand), the mecA-positive strain S. pseudintermedius Thai 36 [15], and S. schleiferi subsp. coagulans VETKU-SSC-5972. The S. schleiferi control was identified with PCR and sequencing. S. simulans were classified via MALDI-TOF-MS (Vitek® MS, bioMérieux SA, Marcy-l’Étoile, France). The strains were stored in Luria–Bertani broth containing 20% glycerol (MilliporeSigma, Burlington, MA, USA) at −80 °C until use, and a culture at 37 °C for 18–24 h was performed on trypticase soy agar (Becton Dickinson, Franklin Lakes, NJ, USA).

2.2. Preparation of Piper betle Leaf Extract and Analysis of Its Chemical Composition

The betel leaf extract was prepared as a crude ethanolic extract. Briefly, betel leaves were purchased from a retail market in Nakhon Pathom Province, Thailand. After cleaning and air drying, the betel leaves were ground into a powder and then extracted via a maceration method using 95% ethanol as a solvent. The extract was condensed by using a rotary evaporator (Buchi Rotavapor R-124, Flawil, Switzerland). The gluey betel crude extract was kept in a desiccator at 4 °C.
The chemical constitution of the betel extract was characterized by gas chromatography and mass spectrometry (GC-MS model GC-2030 mass selective detector, Shimazu, Japan) at the Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (Kasetsart University, Bangkok, Thailand). The GC part, with a column of DB-5MS 30 m × 0.25 mm ID × 0.25 mm, was injected with a sample volume of 1.0 µL (split ratio 1:20). Helium gas flowed into the column at a rate of 1.0 mL/min. The column temperature was programmed using an initial temperature of 60 °C for 3 min and then increasing at a speed of 10 °C/min to a temperature of 280 °C for 1 min. As for the MS operation, the ion source temperature was 250 °C in the electron impact ionization (EI) system. The extraction of the crude betel composition was obtained as a total ion chromatogram (TIC) in the scan mode using a range of mass of 35 to 500 atomic mass units. The identification of the query GC-MS mass spectrum was carried out by matching with the reference mass spectra of the Wiley Version 5 NIST 5 Library, together with a consideration of the Kovats retention index in the Adams table reference. The relative percentage was calculated by comparing its average peak area to the total area.

2.3. Determination of Minimum Inhibitory Concentration

The determination of the MIC was performed via a broth microdilution, according to the CLSI guidelines [46]. A stock solution was prepared at a 10× final concentration and adjusted for the drug’s potency or purity. P. betle leaf extract, fusidic acid (MilliporeSigma, Burlington, MA, USA), azelaic acid (MilliporeSigma, Burlington, MA, USA), benzoyl peroxide (MilliporeSigma, Burlington, MA, USA), miconazole nitrate (MilliporeSigma, Burlington, MA, USA), and chlorhexidine (MilliporeSigma, Burlington, MA, USA) were weighed and dissolved in different solvents at different concentrations (Table 1). The prepared solutions were clarified by filtration through a 0.2 µm syringe filter (Minisart, Darmstadt, Germany). The solvent controls were tested and it was shown that there was no interference with the bacteria growth. A working solution was serially diluted two-fold in cation-adjusted Mueller–Hinton broth (MHB, Becton Dickinson, Franklin Lakes, NJ, USA), and 100 µL of each dilution was added to a well of a 96-well polypropylene microplate (Corning, NY, USA). A bacterial suspension was prepared in a 0.85% sodium chloride (Vivantis, Shah Alam, Malaysia) solution, equivalent to the turbidity of the 0.5 McFarland standard, and further diluted to give a cell density approximately of 106 colony forming units (CFU/mL). The aforementioned wells were then filled with a 10 µL volume of the prepared inoculum. The inoculum quantity control was performed by colony plating with the acceptable range of 2 × 105 to 8 × 105 CFU/mL [46]. Positive and negative growth control wells with MHB were included. Control wells without an inoculation to the serially diluted working reagent were also run in parallel. The plates were incubated at 37 °C for 18–24 h. The S. pseudintermedius Thai 36 was used as a quality control strain between independent assays. The MICs were determined as the lowest concentration of the bacterial test substance showing no visible growth. The reading of the microbial growth for MICs of betel extract, benzoyl peroxide, miconazole, and chlorhexidine was aided by the addition of 30 µL of resazurin (MilliporeSigma, Burlington, MA, USA) at a 0.02% concentration. The plates were incubated for a further 2 to 4 h [47]. Visible growth changed the blue dye (no growth) to a purple or pink color. The MIC values obtained by the colorimetric assay and visual observation were identical, with a difference of no more than one dilution. Due to azelaic acid’s reactivity with resazurin dye, the MIC was read by observing the broth’s clearness (no growth) and turbidity. Each isolate was assessed in triplicate and the discrepancy was accepted at only one dilution. The MIC test was performed concurrently for fusidic acid with the CLSI quality control strain, S. aureus ATCC 29213. According to the colorimetric and visual observations, the MIC values of fusidic acid against S. aureus ATCC 29213 were within the CLSI acceptable range [48].

2.4. Determination of Minimum Bactericidal Concentration

The determination of the MBC was carried out with the broth microdilution method [49]. Briefly, an aliquot of 10 µL from each well of the MIC-tested plates was spotted on the surface of TSA (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 37 °C for 24 h. The culture plates were then counted for the bacterial colonies, and the lowest concentration showing a 99.9% killing of the final inoculum was recorded as the MBC.

2.5. Statistical Analysis

Analyses were performed using NCSS software version 2021 (NCSS, LLC, East Kaysville, UT, USA). The MIC and MBC data are presented as the means and standard deviations. The differences between the betel leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine towards Staphylococcus isolates, and differences between Staphylococcus groups (MRSP, MSSP, and MRSS), were compared using non-parametric Kruskal–Wallis one-way ANOVA tests. The chosen level of significance for the differences between the testing reagents was p < 0.005 and that for the differences between Staphylococcus groups was p < 0.017, according to the Bonferroni correction.

3. Results

3.1. Phytochemicals of Crude Ethanolic Piper betle Extract

A phytochemical assay on P. betle ethanolic extract was performed using GC-MS analysis, as shown in Table 2. Eugenol was the predominant constituent, with a 44.17% peak area, followed by hydroxychavicol (26.34%). Chavicol, acetate (7.02%), γ-muurolene (5.27%), hexadecanoic acid, ethyl ester (3.60%), δ-cadinene (2.11%), 2,4 di-tert-butyl-phenol (1.84%), n-hexadecanoic acid (1.55%), and cis-calamenene (1.33%) were present in the lower percentages. Other components representing 6.78% of the peak area were 3-ethyl-3-methyl heptane, 5-methyl-5-propylnonane, trans-caryophyllene, α-selinene, α-calacorene, spathulenol, hexadecane, n-pentadecanal, tau-cadinol acetate, neophytadiene, and globulol. Since eugenol and hydroxychavicol are among the reported antibacterial compounds [43], the P. betel extract is considered to have an antibacterial potency in the majority proportion. The anti-staphylococcal activity of the betel leaf extract was then demonstrated.

3.2. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) of Piper betle Leaf Extract, Azelaic Acid, Benzoyl Peroxide, Miconazole, and Chlorhexidine against Staphylococcus Clinical Isolates of Canine Pyoderma

The antibacterial activity of betel leaf extract, azelaic acid, benzoyl peroxide, and miconazole against 75 canine Staphylococcus clinical isolates at the minimum inhibitory and bactericidal concentrations is shown in Figure 1. Chlorhexidine’s MIC towards 39 Staphylococcus isolates, including 16 MRSP, 11 MSSP, 10 MRSS, and 2 MSSS, is also included in the figure. As compared to azelaic acid (mean MIC 963.49 mg/L) and benzoyl peroxide (mean MIC 1342.70 mg/L), the betel leaf extract had a significantly lower MIC (mean 252.78 mg/L) (p < 0.0001). Miconazole and chlorhexidine demonstrated the lowest MICs (mean 1.62 and 1.41 mg/L, respectively) (p < 0.0001). The MICs of the betel leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine ranged from 125 to 500, 500 to 1000, 250 to 2000, 0.39 to 3.12, and 0.5 to 2 mg/L, respectively. The MIC50 and MIC90 of the betel leaf extract were 250 mg/L, while azelaic acid and chlorhexidine displayed an MIC50 and MIC90 of 1000 and 1 mg/L, respectively. Miconazole and benzoyl peroxide exhibited only a two-fold difference in the values of the MIC50 (1.56 and 1000 mg/L, respectively) and MIC90 (3.12 and 2000 mg/L, respectively). The mean MBCs of the betel leaf extract, azelaic acid, benzoyl peroxide, and miconazole, as shown in Figure 1, were 443.06, 1348.68, 2070.32, and 6.55 mg/L, respectively, consistent with the MIC results. When the MBC/MIC ratio was considered, all the compounds except miconazole displayed an MBC/MIC ≤ 4, indicating bactericidal action. In addition, the MICs of the betel leaf extract, azelaic acid, benzoyl peroxide, and miconazole towards S. aureus ATCC 29213 were 500, 2000, 4000, and 1.56 mg/L, respectively (data not shown).

3.3. Minimum Inhibitory Concentrations (MICs) of Piper betle Leaf Extract, Azelaic Acid, Benzoyl Peroxide, Miconazole, and Chlorhexidine against Methicillin-Resistant Staphylococcus pseudintermedius (MRSP), Methicillin-Susceptible Staphylococcus pseudintermedius (MSSP), and Methicillin-Resistant Staphylococcus schleiferi subsp. coagulans (MRSS)

The mean MICs and standard deviations of the betel leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine were characterized according to the MRSP, MSSP, and MRSS groups (Figure 2). There was a statistically significant difference in the betel MICs, in which MRSS (mean 346.49 mg/L) showed a lower susceptibility than MRSP (mean 212.37 mg/L) and MSSP (mean 216.27 mg/L) (p < 0.0001). The mean MICs of azelaic acid towards MRSP, MSSP, and MRSS were 976.19, 920.63, and 982.46 mg/L, respectively. Benzoyl peroxide showed mean MICs against MRSP, MSSP, and MRSS of 1589.86, 1099.21, and 1175.44 mg/L, respectively. In addition, the MRSP group had a significantly higher benzoyl peroxide MIC than the MSSP and MRSS groups (p < 0.0001). Miconazole gave mean MICs for MRSP, MSSP, and MRSS of 1.55, 1.74, and 1.59 mg/L, respectively. The mean MICs of chlorhexidine towards MRSP, MSSP, and MRSS were 1.31, 1.23, and 1.75 mg/L, respectively. MRSP, MSSP, and MRSS were susceptible to azelaic acid, miconazole, and chlorhexidine, with no significant difference between these strains.

3.4. Minimum Bactericidal Concentrations (MBC) of Piper betle Leaf Extract, Azelaic Acid, Benzoyl Peroxide, and Miconazole against Methicillin-Resistant Staphylococcus pseudintermedius (MRSP), Methicillin-Susceptible Staphylococcus pseudintermedius (MSSP), and Methicillin-Resistant Staphylococcus schleiferi subsp. coagulans (MRSS)

The MBCs of the betel leaf extract, azelaic acid, benzoyl peroxide, and miconazole against MRSP, MSSP, and MRSS are shown in Table 3. The MBCs of the betel leaf extract for the MRSP and MSSP groups were 312.50 and 295.63 mg/L, respectively, while the MBC of 710.53 mg/L was obtained for the MRSS group. Azelaic acid showed MBCs in the MRSP, MSSP, and MRSS groups of 1250.38, 1185.19, and 1605.26 mg/L, respectively. The benzoyl peroxide MBCs in the MRSP, MSSP, and MRSS groups were 1898.62, 1908.73, and 2491.23 mg/L, respectively. Miconazole exhibited MBCs in the MRSP, MSSP, and MRSS groups of 4.80, 5.37, and 8.69 mg/L, respectively. There was no significant difference in the MBCs of all compounds in the MRSP and MSSP groups, while MRSS gave significantly higher MBCs for all compounds compared to the other groups (p < 0.0001 for betel leaf extract, azelaic acid, and miconazole and p < 0.017 for benzoyl peroxide). Regarding the MBC/MIC ratio for the MRSP, MSSP, and MRSS groups, the ratios of betel leaf extract, azelaic acid, and benzoyl peroxide were less than four. However, the miconazole ratio in the MRSS group was the highest, at 5.47.

4. Discussion

The increased prevalence of multidrug-resistant (MDR) strains, the emergence of methicillin-resistant staphylococci (MRS) in canine pyoderma, and the limited choices of antimicrobial drugs have led to the necessity of the discovery of new therapeutic alternatives [8,10,11,12]. The use of a topical antimicrobial treatment is likewise recommended to improve the treatment efficiency and reduce the systemic antimicrobial use [5,18]. Meanwhile, certain topical antimicrobials pose concerns regarding their use and resistance in animals [30,31]. Plants and their antimicrobial derivatives could be studied to respond to the need for additional treatment options in veterinary dermatology.
In this study, based on the testing of the MIC and MBC, betel leaf extract appeared to be a promising candidate for an antimicrobial treatment in dogs with pyoderma. Overall, the relevant Staphylococcus isolates were susceptible to betel leaf extract, with an MIC of 252.78 mg/L, which is considered to have a significant antimicrobial activity cut-off of 1000 mg/L [50]. It was also found that betel leaf extract had a superior efficacy to azelaic acid and benzoyl peroxide. For MRSP and MSSP, betel leaf extract showed an inhibitory effect with no measurable difference in the susceptibility. While the inhibition of MRSS required a higher MIC than the other two groups, this concentration is still considered noteworthy. Based on an MBC/MIC ratio of less than four, the betel leaf extract is bactericidal towards MRSP, MSSP, and MRSS, consistent with previous studies of methicillin-resistant Staphylococcus aureus (MRSA) [51,52]. Although there has been no confirmation that cidal agents are better than static agents, bactericidal agents are preferred in immunocompromised patients.
Several studies have demonstrated the anti-staphylococcal activity of P. betle leaf extract against S. aureus [41,51,52]. The current study reported the remarkable antimicrobial activity of ethanolic betel leaf extract against clinical MRSP, MSSP, and MRSS isolates in the MIC range of 125 to 500 mg/L. In a previous study on MRSA, betel MICs of 0.31 to 2.5 mg/mL (310 to 2500 mg/L) were reported [52]. While there is an overlap in these MIC values with those obtained in the current study, the susceptibility to betel leaf extract among MRSP, MRSS, and MRSA requires future work. In the GC-MS-based analysis, eugenol and hydroxychavicol appeared to be the most abundant components in the betel leaf extract. These phenolic compounds have been identified as major antibacterial compounds of betel leaves [42,43]. In previous studies on S. aureus, the MICs of eugenol and hydroxychavicol pure compounds have been reported to be 100 to 400 mg/L, and 200 mg/L, respectively [53,54]. This corresponds with the current study that found a betel MIC of 252.78 mg/L and the eugenol and hydroxychavicol peak areas of 44.17% and 26.24%. Accordingly, the activity of the betel leaf extract against the canine Staphylococcus clinical isolates could be mainly attributed to the eugenol and hydroxychavicol constituents. The mechanism of action of eugenol against S. aureus by targeting the bacterial cell membrane has been observed [53]. In addition, a study of the microarray analysis has revealed that MRSA responded to eugenol by an upregulated gene expression in unique metabolic pathways for a bacterial survival [55]. However, differences in species susceptibility can be expected, which could lead to differences in the mechanism of inhibition. Research into the mechanism of action of eugenol, as well as hydroxychavicol on canine Staphylococcus species, could contribute to the discovery of new targets for drug development.
The relatively narrow MIC50 and MIC90 values suggested that a resistance to betel leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine is unlikely. However, the statistically significant difference between MRSP, MSSP, and MRSS in response to the activity of benzoyl peroxide was interesting. Whether this represents the impact of the species or methicillin resistance on benzoyl peroxide’s susceptibility is unclear. The lowest MICs and highest efficacy among the tested reagents were found for miconazole and chlorhexidine. Miconazole’s MICs of 1.55 and 1.74 mg/L were demonstrated for MRSP and MSSP, respectively, which is consistent with previous reports [23,56]. Chlorhexidine’s MICs towards MRSP (1.31 mg/L) and MSSP (1.23 mg/L) in the current and previous reports are consistent [23,24]. Low MIC values of miconazole and chlorhexidine were also revealed against MRSS, whose MICs were not significantly different from those of MRSP and MSSP. Hence, the anti-staphylococcal activity of miconazole and chlorhexidine was further supported by this study. Regarding the bactericidal action, there was evidence for the betel leaf extract, azelaic acid, and benzoyl peroxide, but not for miconazole. Benzoyl peroxide and azelaic acid are known for their bactericidal activity, which corresponds with the present study [27]. However, the lack of bactericidal action of miconazole needs further elucidation.
In addition, the results of this study indicate that azelaic acid could be another acid candidate for the treatment of canine pyoderma. Although its efficacy was lower than that of chlorhexidine, miconazole, and betel leaf extract, the azelaic acid MICs of 500 to 1000 mg/L are well below the concentrations achievable with 20% azelaic acid applications. Furthermore, MRSP, MSSP, and MRSS were susceptible to azelaic acid with no significant difference.

5. Conclusions

To the best of the authors’ knowledge, this is the first study on the in vitro antibacterial activity of crude P. betle leaf extract against S. pseudintermedius and S. schleiferi subsp. coagulans of canine pyoderma isolates, including their methicillin-resistant strains. The antibacterial efficacy of betel leaf extract was superior to that of azelaic acid and benzoyl peroxide and was lower than that of chlorhexidine and miconazole. Information on MICs and MBCs regarding the use of betel leaf extract as a topical antibacterial agent was also provided. Further studies on the microbial spectrum, toxicity, product formulation, stability, and clinical efficacy should be undertaken prior to the application of betel leaf extract in canine pyoderma.

Author Contributions

Conceptualization, P.P. and C.P.; methodology, P.P., K.T., U.S. and C.P.; software, P.P.; validation, U.S., S.M. and C.P.; formal analysis, C.P.; investigation, P.P., K.T., U.S. and C.P.; resources, C.P.; data curation, P.P. and C.P.; writing—original draft preparation, P.P. and C.P.; writing—review and editing, P.P., S.M. and C.P. All authors have read and agreed to the published version of the manuscript.

Funding

National Research Council of Thailand (NRCT), grant number N41D640003 and Faculty of Veterinary Medicine, Kasetsart University (Bangkok, Thailand), grant numbers 62-2/04 and 60/SRF11.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

This research and innovation activity is funded by National Research Council of Thailand (NRCT). Financial support from the Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand. The authors would like to thank Chalermpol Lekcharoensuk for valuable advice on statistical analysis, and Walaiporn Tonpitak for giving S. pseudintermedius Thai 36. The authors also thank Jatuporn Rattanasrisomporn for commenting on the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hill, P.B.; Lo, A.; Eden, C.A.N.; Huntley, S.; Morey, V.; Ramsey, S.; Richardson, C.; Smith, D.J.; Sutton, C.; Taylor, M.D.; et al. Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice. Vet. Rec. 2006, 158, 533–539. [Google Scholar] [CrossRef] [PubMed]
  2. Scott, D.W.; Paradis, M. A survey of canine and feline skin disorders seen in a university practice: Small Animal Clinic, University of Montréal, Saint-Hyacinthe, Québec (1987–1988). Can. Vet. 1990, 31, 830–835. [Google Scholar]
  3. Summers, J.F.; Hendricks, A.; Brodbelt, D.C. Prescribing practices of primary-care veterinary practitioners in dogs diagnosed with bacterial pyoderma. BMC Vet. Res. 2014, 10, 240. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Sykes, J.E.; Terry, M.N.; Stephen, D.W. Chapter 84—Pyoderma, otitis externa, and otitis media. In Canine and Feline Infectious Diseases; Sykes, J.E., Ed.; Saunders: Saint Louis, MO, USA, 2014; pp. 800–813. [Google Scholar]
  5. Hillier, A.; Lloyd, D.H.; Weese, J.S.; Blondeau, J.M.; Boothe, D.; Breitschwerdt, E.; Guardabassi, L.; Papich, M.G.; Rankin, S.; Turnidge, J.D.; et al. Guidelines for the diagnosis and antimicrobial therapy of canine superficial bacterial folliculitis (Antimicrobial guidelines working group of the international society for companion animal infectious diseases). Vet. Dermatol. 2014, 25, 163–175. [Google Scholar] [CrossRef]
  6. Eckholm, N.G.; Outerbridge, C.A.; White, S.D.; Sykes, J.E. Prevalence of and risk factors for isolation of meticillin-resistant Staphylococcus spp. from dogs with pyoderma in northern California, USA. Vet. Dermatol. 2013, 24, 154-e34. [Google Scholar] [CrossRef]
  7. Hariharan, H.; Gibson, K.; Peterson, R.; Frankie, M.; Matthew, V.; Daniels, J.; Martin, N.A.; Andrews, L.; Paterson, T.; Sharma, R.N. Staphylococcus pseudintermedius and Staphylococcus schleiferi subspecies coagulans from canine pyoderma cases in Grenada, West Indies, and their susceptibility to beta-lactam drugs. Vet. Med. Int. 2014, 2014, 850126. [Google Scholar] [CrossRef] [Green Version]
  8. Kawakami, T.; Shibata, S.; Murayama, N.; Nagata, M.; Nishifuji, K.; Iwasaki, T.; Fukata, T. Antimicrobial susceptibility and methicillin resistance in Staphylococcus pseudintermedius and Staphylococcus schleiferi subsp. coagulans isolated from dogs with pyoderma in Japan. J. Vet. Med. Sci. 2010, 72, 1615–1619. [Google Scholar]
  9. Sasaki, T.; Kikuchi, K.; Tanaka, Y.; Takahashi, N.; Kamata, S.; Hiramatsu, K. Reclassification of phenotypically identified Staphylococcus intermedius strains. J. Clin. Microbiol. 2007, 45, 2770–2778. [Google Scholar] [CrossRef] [Green Version]
  10. Loeffler, A.; Lloyd, D.H. What has changed in canine pyoderma? A narrative review. Vet. J. 2018, 235, 73–82. [Google Scholar] [CrossRef] [Green Version]
  11. Morris, D.O.; Rook, K.A.; Shofer, F.S.; Rankin, S.C. Screening of Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus schleiferi isolates obtained from small companion animals for antimicrobial resistance: A retrospective review of 749 isolates (2003–04). Vet. Dermatol. 2006, 17, 332–337. [Google Scholar] [CrossRef]
  12. Yoo, J.H.; Yoon, J.W.; Lee, S.Y.; Park, H.M. High prevalence of fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates from canine pyoderma and otitis externa in veterinary teaching hospital. J. Microbiol. Biotechnol. 2010, 20, 798–802. [Google Scholar] [PubMed]
  13. Hartman, B.J.; Tomasz, A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J. Bacteriol. 1984, 158, 513–516. [Google Scholar] [CrossRef] [PubMed]
  14. Peacock, S.J.; Paterson, G.K. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu. Rev. Biochem. 2015, 84, 577–601. [Google Scholar] [CrossRef]
  15. Kadlec, K.; Weiß, S.; Wendlandt, S.; Schwarz, S.; Tonpitak, W. Characterization of canine and feline methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand. Vet. Microbiol. 2016, 194, 93–97. [Google Scholar] [CrossRef] [PubMed]
  16. Swe, T.; Naing, A.T.; Baqui, A.; Khillan, R. Methicillin-Resistant Staphylococcus schleiferi subspecies coagulans infection in a patient with hepatocellular carcinoma. J. Investig. Med. High Impact. Case Rep. 2016, 26, 1–3. [Google Scholar] [CrossRef] [Green Version]
  17. Loeffler, A.; Cobb, M.A.; Bond, R. Comparison of a chlorhexidine and a benzoyl peroxide shampoo as sole treatment in canine superficial pyoderma. Vet. Rec. 2011, 169, 249. [Google Scholar] [CrossRef]
  18. Valentine, B. Treating pyoderma without the use of systemic antibiotics. Can. Vet. J. 2019, 60, 1361–1363. [Google Scholar] [PubMed]
  19. Transparency Market Research. 2021. Veterinary Dermatology Drugs Market. Available online: https://www.transparencymarketresearch.com/veterinary-dermatology-drugs-market.html (accessed on 24 September 2022).
  20. Bond, R.; Rose, J.F.; Ellis, J.W.; Lloyd, D.H. Comparison of two shampoos for treatment of Malassezia pachydermatis-associated seborrhoeic dermatitis in basset hounds. J. Small Anim. Pract. 1995, 36, 99–104. [Google Scholar] [CrossRef]
  21. Loeffler, A.; Baines, S.J.; Toleman, M.S.; Felmingham, D.; Milsom, S.K.; Edwards, E.A.; Lloyd, D.H. In vitro activity of fusidic acid and mupirocin against coagulase-positive staphylococci from pets. J. Antimicrob. Chemother. 2008, 62, 1301–1304. [Google Scholar] [CrossRef] [Green Version]
  22. Valentine, B.K.; Dew, W.; Yu, A.; Weese, J.S. In vitro evaluation of topical biocide and antimicrobial susceptibility of Staphylococcus pseudintermedius from dogs. Vet. Dermatol. 2012, 23, 493–495. [Google Scholar] [CrossRef]
  23. Clark, S.M.; Loeffler, A.; Bond, R. Susceptibility in vitro of canine methicillin-resistant and-susceptible staphylococcal isolates to fusidic acid, chlorhexidine and miconazole: Opportunities for topical therapy of canine superficial pyoderma. J. Antimicrob. Chemother. 2015, 70, 2048–2052. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Clark, S.M.; Loeffler, A.; Schmidt, V.M.; Chang, Y.M.; Wilson, A.; Timofte, D.; Bond, R. Interaction of chlorhexidine with trisEDTA or miconazole in vitro against canine meticillin-resistant and -susceptible Staphylococcus pseudintermedius isolates from two UK regions. Vet. Dermatol. 2016, 27, 340-e84. [Google Scholar] [CrossRef] [PubMed]
  25. Young, R.; Buckley, L.; McEwan, N.; Nuttall, T. Comparative in vitro efficacy of antimicrobial shampoos: A pilot study. Vet. Dermatol. 2012, 23, 36–40. [Google Scholar] [CrossRef] [PubMed]
  26. Thiboutot, D.M.; Dréno, B.; Abanmi, A.; Alexis, A.F.; Araviiskaia, E.; Cabal, M.I.B.; Bettoli, V.; Casintahan, F.; Chow, S.; Costa, A.; et al. Practical management of acne for clinicians: An international consensus from the global alliance to improve outcomes in acne. J. Am. Acad. Dermatol. 2018, 78, S1–S23. [Google Scholar] [CrossRef] [PubMed]
  27. Blaskovich, M.A.T.; Elliott, A.G.; Kavanagh, A.M.; Ramu, S.; Cooper, M.A. In vitro antimicrobial activity of acne drugs against skin-associated bacteria. Sci. Rep. 2019, 9, 14658. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Mueller, R.S.; Bergvall, K.; Bensignor, E.; Bond, R. A review of topical therapy for skin infections with bacteria and yeast. Vet. Dermatol. 2012, 23, 330–341. [Google Scholar] [CrossRef] [PubMed]
  29. Jeffers, J.G. Topical therapy for drug-resistant pyoderma in small animals. Vet. Clin. N. Am. Small Anim. Pract. 2013, 43, 41–50. [Google Scholar] [CrossRef]
  30. Kampf, G. Acquired resistance to chlorhexidine—Is it time to establish an ‘antiseptic stewardship’ initiative? J. Hosp. Infect. 2016, 94, 213–227. [Google Scholar] [CrossRef]
  31. Cobb, M.A.; Edwards, H.J.; Jagger, T.D.; Marshall, J.; Bowker, K.E. Topical fusidic acid/betamethasone-containing gel compared to systemic therapy in the treatment of canine acute moist dermatitis. Vet. J. 2005, 169, 276–280. [Google Scholar] [CrossRef]
  32. Budgin, J.B.; Flaherty, M.J. Alternative therapies in veterinary dermatology. Vet. Clin. N. Am. Small Anim. Pract. 2013, 43, 189–204. [Google Scholar] [CrossRef]
  33. Tresch, M.; Mevissen, M.; Ayrle, H.; Melzig, M.; Roosje, P.; Walkenhorst, M. Medicinal plants as therapeutic options for topical treatment in canine dermatology? A systematic review. BMC Vet. Res. 2019, 15, 174. [Google Scholar] [CrossRef] [PubMed]
  34. Lima, C.O.; Barreto, H.M.; Lima, E.O.; Souza, E.L.; Squeira, J.P. Antimicrobial effect of the essential oil from Rosmarinus officinalis L. against Staphylococcus pseudintermedius isolated from dogs. Braz. J. Biol. Sci. 2013, 11, 280–283. [Google Scholar]
  35. Larsuprom, L.; Rungroj, N.; Lekcharoensuk, C.; Pruksakorn, C.; Kongkiatpaiboon, S.; Chen, C.; Sukatta, U. In vitro antibacterial activity of mangosteen (Garcinia mangostana Linn.) crude extract against Staphylococcus pseudintermedius isolates from canine pyoderma. Vet. Dermatol. 2019, 30, 487-e145. [Google Scholar] [CrossRef] [PubMed]
  36. Brown, H.; Metters, G.; Hitchings, M.D.; Wilkinson, T.S.; Sousa, L.; Cooper, J.; Dance, H.; Atterbury, R.J.; Jenkinsa, R. Antibacterial and anti-virulence activity of manuka honey against genetically diverse Staphylococcus pseudintermedius. Appl. Environ. Microbiol. 2020, 86, e01768-20. [Google Scholar] [CrossRef] [PubMed]
  37. Nocera, F.P.; Mancini, S.; Najar, B.; Bertelloni, F.; Pistelli, L.; Filippis, A.; Fiorito, F.; Martino, D.L.; Fratini, F. Antimicrobial activity of some essential oils against methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius-associated pyoderma in dogs. Animals 2020, 10, 1782. [Google Scholar] [CrossRef]
  38. Aiemsaard, J.; Kamollert, C.; Singh, R.; Suwannathada, P.; Suwannathada, P.; Thongkham, E. Anti-biofilm activity of Thai herbal essential oils against Staphylococcus pseudintermedius causing canine pyoderma. Sci. Asia 2020, 46, 514–519. [Google Scholar] [CrossRef]
  39. Nayaka, N.M.D.M.W.; Sasadara, M.M.V.; Sanjaya, D.A.; Yuda, P.E.S.K.; Dewi, N.L.K.A.A.; Cahyaningsih, E.; Hartati, R. Piper betle (L): Recent review of antibacterial and antifungal properties, safety profiles, and commercial applications. Molecules 2021, 26, 2321. [Google Scholar] [CrossRef]
  40. Nagori, K.; Singh, M.; Alexander, A.; Kumar, T.; Dewangan, D.; Badwaik, H.; Tripathi, D.K. Piper betle L.: A review on its ethnobotany, phytochemistry, pharmacological profile and profiling by new hyphenated technique DART-MS (Direct Analysis in Real Time Mass Spectrometry). J. Pharm. Res. 2011, 4, 2991–2997. [Google Scholar]
  41. Sureshkumar, M.; Babu, S. Antibacterial effect of crude aqueous extract of Piper betle L. against pathogenic bacteria. Int. J. Res. Pharm. Biomed. Sci. 2013, 4, 42–46. [Google Scholar]
  42. Singtongratana, N.; Vadhanasin, S.; Singkhonrat, J. Hydroxychavicol and eugenol profiling of betel leaves from Piper betle L. obtained by liquid-liquid extraction and supercritical fluid extraction. Witthayasan Kasetsat. 2013, 47, 614–623. [Google Scholar]
  43. Syahidah, A.; Saad, C.R.; Hassan, M.D.; Rukayadi, Y.; Norazian, M.H.; Kamarudin, M.S. Phytochemical analysis, identification and quantification of antibacterial active compounds in betel leaves, Piper betle methanolic extract. Pak. J. Biol. Sci. 2017, 20, 70–81. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Sasaki, T.; Tsubakishita, S.; Tanaka, Y.; Sakusabe, A.; Ohtsuka, M.; Hirotaki, S.; Hirotaki, S.; Kawakami, T.; Fukata, T.; Hiramatsu, K. Multiplex-PCR method for species identification of coagulase-positive staphylococci. J. Clin. Microbiol. 2010, 48, 765–769. [Google Scholar] [CrossRef] [Green Version]
  45. Kondo, Y.; Ito, T.; Ma, X.X.; Watanabe, S.; Kreiswirth, B.N.; Etienne, J.; Hiramatsu, K. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob. Agents Chemother. 2007, 51, 264–274. [Google Scholar] [CrossRef] [Green Version]
  46. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, CLIS Document M07-A8; Clinical Laboratory and Standards Institute: Wayne, PA, USA, 2009. [Google Scholar]
  47. Elshikh, M.; Ahmed, S.; Funston, S.; Dunlop, P.; McGaw, M.; Marchant, R.; Banat, I.M. Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants. Biotechnol. Lett. 2016, 38, 1015–1019. [Google Scholar] [CrossRef] [Green Version]
  48. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement; Clinical Laboratory and Standards Institute: Wayne, PA, USA, 2015. [Google Scholar]
  49. CLSI. Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Guideline; National Committee for Clinical Laboratory Standards Institute: Wayne, PA, USA, 1999. [Google Scholar]
  50. Orchard, A.; Vuuren, S.V. Commercial essential oils as potential antimicrobials to treat skin diseases. Evid. Based. Complement. Alternat. Med. 2017, 2017, 4517971. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Valle, D.L.; Andrade, J.; Puzon, J.J.M.; Cabrera, E.C.; Rivera, W.L. Antibacterial activities of ethanol extracts of Philippine medicinal plants against multidrug-resistant bacteria. Asian Pac. J. Trop. Biomed. 2015, 5, 532–540. [Google Scholar] [CrossRef]
  52. Saeloh, D.; Visutthi, M. Efficacy of thai plant extracts for antibacterial and anti-biofilm activities against pathogenic bacteria. Antibiotics 2021, 10, 1470. [Google Scholar] [CrossRef]
  53. Yadav, M.K.; Chae, S.W.; Im, G.J.; Chung, J.W.; Song, J.J. Eugenol: A phyto-compound effective against methicillin-resistant and methicillin-sensitive Staphylococcus aureus clinical strain biofilms. PLoS ONE 2015, 10, e0119564. [Google Scholar] [CrossRef] [Green Version]
  54. Jesonbabu, J.; Spandana, N.; Lakshmi, A.K. The potential activity of hydroxychavicol against pathogenic bacteria. J. Bacteriol. Parasitol. 2011, 2, 2–5. [Google Scholar] [CrossRef]
  55. Buru, A.S.; Neela, V.K.; Mohandas, K.; Pichika, M.R. Microarray analysis of the genomic effect of eugenol on methicillin-resistant Staphylococcus aureus. Molecules 2022, 27, 3249. [Google Scholar] [CrossRef]
  56. Weese, J.S.; Walker, M.; Lowe, T. In vitro miconazole susceptibility of methicillin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus. Vet. Dermatol. 2012, 23, 400-e74. [Google Scholar] [CrossRef] [PubMed]
Animals 12 03203 g001 550
Figure 1. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine against 75 Staphylococcus clinical isolates of canine pyoderma. Only chlorhexidine’s MIC was determined in 39 Staphylococcus isolates, and its MBC was not determined. Data were expressed as means and standard deviations. a, b, c, d Above mean MIC bars and a*, b*, c*, d* above mean MBC bars carrying different superscripts are significantly different by Kruskal–Wallis one-way ANOVA (p < 0.0001).
Figure 1. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine against 75 Staphylococcus clinical isolates of canine pyoderma. Only chlorhexidine’s MIC was determined in 39 Staphylococcus isolates, and its MBC was not determined. Data were expressed as means and standard deviations. a, b, c, d Above mean MIC bars and a*, b*, c*, d* above mean MBC bars carrying different superscripts are significantly different by Kruskal–Wallis one-way ANOVA (p < 0.0001).
Animals 12 03203 g001
Animals 12 03203 g002 550
Figure 2. Minimum inhibitory concentrations (MICs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine against methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-susceptible Staphylococcus pseudintermedius (MSSP), and methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSS). Only chlorhexidine’s MIC was determined in 16 MRSP, 11 MSSP, and 10 MRSS. Data were expressed as means and standard deviations. a, b above mean MIC bars carrying different superscripts is significantly different by Kruskal–Wallis one-way ANOVA (p < 0.0001).
Figure 2. Minimum inhibitory concentrations (MICs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, miconazole, and chlorhexidine against methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-susceptible Staphylococcus pseudintermedius (MSSP), and methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSS). Only chlorhexidine’s MIC was determined in 16 MRSP, 11 MSSP, and 10 MRSS. Data were expressed as means and standard deviations. a, b above mean MIC bars carrying different superscripts is significantly different by Kruskal–Wallis one-way ANOVA (p < 0.0001).
Animals 12 03203 g002
Table
Table 1. Stock solutions prepared.
Table 1. Stock solutions prepared.
Stock Solution Concentration (mg/L)Solvents
P. betle leaf extract320,000100% DMSO
Fusidic acid20,480Water
Azelaic acid640,0005% Tween 80 and 5% ethanol
Benzoyl peroxide320,000100% DMSO
Miconazole4000100% DMSO
Chlorhexidine1280Water
Table
Table 2. Phytochemical composition of Piper betle ethanolic leaf extract.
Table 2. Phytochemical composition of Piper betle ethanolic leaf extract.
CompoundsRetention TimeKovats Retention IndexPeak Area
(%)
Chavicol, acetate9.87511957.02
Eugenol11.675139244.17
Hydroxychavicol12.970142426.34
γ-Muurolene13.22014785.27
2,4 di-tert-butyl-phenol13.475-1.84
δ-Cadinene13.74015232.11
cis-calamenene13.84015281.33
n-hexadecanoic acid18.60519681.55
Hexadecanoic acid, ethyl ester18.96519783.60
Others--6.78
Table
Table 3. Minimum bactericidal concentrations (MBCs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, and miconazole against methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-susceptible Staphylococcus pseudintermedius (MSSP), and methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSS).
Table 3. Minimum bactericidal concentrations (MBCs) of Piper betle leaf extract, azelaic acid, benzoyl peroxide, and miconazole against methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-susceptible Staphylococcus pseudintermedius (MSSP), and methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSS).
ReagentsMRSP (n = 31)MSSP (n = 21)MRSS (n = 19)
MBC (mg/L)
Mean ± SD		MBC/MIC
Ratio		MBC (mg/L)
Mean ± SD		MBC/MIC RatioMBC (mg/L)
Mean ± SD		MBC/MIC Ratio
Betel leaf extract312.50 ± 119.68 a1.47295.63 ± 100.26 a1.37710.53 ± 295.76 b2.05
Azelaic acid1250.38 ± 382.93 a1.281185.19 ± 357.17 a1.291605.26 ± 462.18 b1.63
Benzoyl peroxide1898.62 ± 485.89 a1.191908.73 ± 881.09 a1.742491.23 ± 772.75 b2.12
Miconazole4.80 ± 3.23 a3.105.37 ± 2.99 a3.098.69 ± 4.99 b5.47
a, b Mean values within the same row carrying different superscripts are significantly different by Kruskal–Wallis one-way ANOVA (p < 0.0001 for betel leaf extract, azelaic acid, and miconazole; p < 0.017 for benzoyl peroxide).
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Phensri, P.; Thummasema, K.; Sukatta, U.; Morand, S.; Pruksakorn, C. In Vitro Antimicrobial Activity of Piper betle Leaf Extract and Some Topical Agents against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus Strains from Canine Pyoderma. Animals 2022, 12, 3203. https://doi.org/10.3390/ani12223203

AMA Style

Phensri P, Thummasema K, Sukatta U, Morand S, Pruksakorn C. In Vitro Antimicrobial Activity of Piper betle Leaf Extract and Some Topical Agents against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus Strains from Canine Pyoderma. Animals. 2022; 12(22):3203. https://doi.org/10.3390/ani12223203

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Phensri, Patcharaporn, Kokaew Thummasema, Udomlak Sukatta, Serge Morand, and Chantima Pruksakorn. 2022. "In Vitro Antimicrobial Activity of Piper betle Leaf Extract and Some Topical Agents against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus Strains from Canine Pyoderma" Animals 12, no. 22: 3203. https://doi.org/10.3390/ani12223203

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