Pyoderma is a common infectious skin disease in dogs that produce distressing symptoms such as itching and pain [1]. Epidemiological estimates indicate that it affects 1.25%–2.67% of the general canine population and up to 11% of dogs presenting with dermatological conditions [2–5]. The disorder is typically bacterial and secondary in nature, often triggered by predisposing factors including allergies, hypothyroidism, or fungal infections [1]. Staphylococcus pseudintermedius is the predominant bacterial species isolated, accounting for up to 92% of cases, and occurs in both methicillin-resistant S. pseudintermedius methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) forms [6]. Other pathogens, such as Staphylococcus schleiferi and certain Gram-negative bacteria, are less frequently involved [1, 7, 8].

The growing threat of antimicrobial resistance (AMR) poses a significant challenge under the One Health framework, which links human, animal, and environmental health [9]. Multidrug-resistant strains, particularly MRSP and methicillin-resistant Staphylococcus schleiferi (MRSS) subsp. coagulans, have emerged as major concerns in veterinary medicine, severely limiting therapeutic options [10, 11]. Prevalence study by Beck et al. [8] reports MRSP in 40.5% of canine pyoderma cases and MRSS in 2.9%. The increasing resistance of S. pseudintermedius is alarming, and some methicillin-resistant strains also pose zoonotic risks [12]. These findings highlight the urgent need for new antiseptic and disinfection strategies aligned with the One Health concept [1, 6, 13, 14].

Topical antibacterial therapy remains a cornerstone in the management of canine pyoderma. It may be applied as monotherapy for localized infections or combined with systemic antimicrobials in more severe cases [15]. Long-term topical therapy is also recommended to prevent relapse and slow the development of AMR [15, 16]. Conventional antiseptics include chlorhexidine, benzoyl peroxide, ethyl lactate, and acetic acid [16]. Chlorhexidine is widely used due to its well-documented in vivo antibacterial efficacy [17]; however, its prolonged or excessive application can induce side effects such as pruritus and erythema [18]. Moreover, reports of reduced susceptibility to chlorhexidine reinforce the demand for novel antimicrobial agents, especially those derived from natural sources [17].

Medicinal plants are increasingly recognized as promising alternatives for infection control in veterinary medicine [19]. Their diverse phytochemical compositions provide broad-spectrum antimicrobial activity, and they are generally considered to have a lower potential for inducing resistance [19, 20].

Piper betel L. (locally known as “Phlu” in Thailand) exemplifies such potential. Conventionally used across India and Southeast Asia, it is rich in flavonoids and phenols with established antimicrobial properties [21, 22]. Previous research by Phensri et al. [23] demonstrated that betel leaf extract exhibits potent in vitro antibacterial activity against canine staphylococcal isolates, including methicillin-resistant strains. Notably, its minimum inhibitory concentration (MIC) was significantly lower than that of benzoyl peroxide, suggesting strong potential for development as a topical treatment for canine pyoderma.

Cannabis (Cannabis sativa L.) derivatives, particularly cannabidiol (CBD), are also attracting growing interest in veterinary therapeutics [24]. Among the more than 120 phytocannabinoids identified, CBD is preferred for medical applications due to its lack of psychoactive effects, unlike tetrahydrocannabinol [25]. CBD has demonstrated antibacterial efficacy against methicillin-resistant Staphylococcus aureus [26] and antibacterial as well as anti-biofilm activity against Streptococcus mutans [27]. However, its effectiveness against clinically relevant canine isolates such as MRSP and MRSS remains underexplored, warranting further investigation.

Despite their potential, approximately 90% of natural products face limitations, including poor water solubility, restricted bioavailability, and low stability, which hinder their clinical efficacy [28]. For instance, both betel leaf extract and CBD suffer from limited solubility, reducing their therapeutic potential unless optimized with advanced delivery systems [29, 30]. Furthermore, penetration of natural compounds through the stratum corneum remains a barrier in in vivo applications [31, 32]. Nanoemulsions, with their submicron droplet size and high colloidal stability, represent a promising strategy to overcome these limitations. They enhance solubility, improve dermal absorption, and increase antimicrobial potency by facilitating better delivery of lipid-soluble compounds [33, 34]. For example, nanoemulsions of bay leaf extract and clove essential oil demonstrated superior antibacterial activity against S. aureus and Escherichia coli compared with unformulated extracts [35, 36]. These findings underscore the value of nanoemulsions in enhancing the therapeutic potential of natural products.

Taken together, the shortcomings of current topical therapies and the limitations of natural agents underscore a critical research gap. Although the antibacterial potential of Piper betel and CBD has been individually documented, no natural formulation has yet proven clinically effective against MRSP or MRSS in veterinary medicine. Challenges such as achieving effective therapeutic concentrations without toxicity and the lack of standardized, controlled veterinary trials demonstrating clinical efficacy and safety continue to impede progress [37].

Despite the growing recognition of AMR in veterinary dermatology, particularly in canine pyoderma caused by MRSP and MRSS subsp. coagulans, treatment options remain limited. Conventional topical antiseptics such as chlorhexidine, benzoyl peroxide, and acetic acid are increasingly associated with adverse effects, reduced susceptibility, and patient compliance issues. Medicinal plants, such as P. betel and bioactive compounds such as CBD have demonstrated promising antibacterial properties in vitro, yet their clinical translation has been hindered by poor solubility, low bioavailability, and insufficient dermal penetration. Although nanoemulsion-based delivery systems have been shown to enhance antimicrobial activity of natural compounds, no previous study has systematically compared betel leaf extract nanoemulsion with different CBD formulations against clinically relevant canine staphylococcal isolates. Moreover, their combined potential as natural, topical alternatives to synthetic antiseptics remains unexplored. This lack of comparative evaluation and formulation optimization constitutes a critical knowledge gap in the search for safe, effective, and sustainable topical therapies for canine pyoderma.

The present study aimed to evaluate and compare the in vitro antibacterial activity of betel leaf extract (in solvent-based and nanoemulsion forms) and three CBD formulations (ethanol-dissolved CBD, water-soluble CBD, and CBD nanoemulsion) against clinical isolates of MRSP, MSSP, and MRSS from dogs with pyoderma. In addition, the antibacterial potential of betel leaf extract nanoemulsion was tested against Pseudomonas aeruginosa, an important Gram-negative pathogen commonly associated with canine skin infections. By determining MIC and minimum bactericidal concentration (MBC) values, this study sought to identify the most effective formulations and establish a scientific foundation for developing plant-based antiseptic alternatives. Ultimately, the goal was to provide evidence for the potential application of P. betel and CBD nanoformulations as novel, natural topical agents that may reduce reliance on conventional antiseptics and support antimicrobial stewardship in veterinary medicine.