Abstract
Propolis, also known as bee glue, is a natural resinous mixture produced by Western honeybees and has long been recognized for its potential therapeutic properties. Recent research has focused on its diverse bioactivities, particularly its antimicrobial effects against a broad spectrum of microorganisms, including human but also animal pathogens. However, further investigation is required to fully elucidate the pharmacological properties and potential toxicity of propolis to support its broader application. This study investigated the antimicrobial efficacy and safety of an ethanol extract of Portuguese propolis collected from the Gerês region (G23.EE). The antimicrobial activity was assessed in vitro against both Gram-positive and Gram-negative bacteria associated with infections in companion animals, using the agar dilution method. To evaluate potential toxicity, the extract was administered via injection and topical application in an in vivo Galleria mellonella larval model, with health parameters monitored over a 96 hours period. The in vitro results showed that G23.EE was more effective against Gram-positive bacteria, including Staphylococcus spp. (e.g., S. felis, S. hominis, S. simulans, and S. pseudintermedius; MIC = 0.5 mg/mL) and Enterococcus faecium (MIC = 1.5 mg/mL), than against Gram-negative bacteria, such as Escherichia coli and Klebsiella oxytoca (MIC > 8.0 mg/mL). No significant adverse effects were observed in G. mellonella larvae following injection or brushing with propolis extract concentrations up to 8.0 mg/mL. Overall, these findings suggest that Portuguese propolis from Gêres is a promising, safe, and effective natural antimicrobial agent for targeting Gram-positive bacterial infections in companion animals.