In vitro Assessment of Brazilian Red Propolis against Mycobacteria: Antibacterial Potency, Synergy, Biofilm Disruption, and Intramacrophage Effects

S B Martins¹Samuel Cota Teixeira¹G De Souza¹Bader Alhatlani^2*Emad M Abdallah^2*Sergio Ricardo Ambrosio³T. S. Silva³Jairo Kenupp Bastos⁴M H Tanimoto⁴Bellisa Freitas Barbosa¹Eloisa Amália Vieira Ferro¹Carlos Henrique Gomes Martins¹

• ¹Federal University of Uberlândia, Minas Gerais, Brazil
• ²Qassim University, Buraidah, Saudi Arabia
• ³University of Franca, São Paulo, Brazil
• ⁴University of São Paulo, São Paulo, Brazil

Tuberculosis persists as a major global health threat and remains the leading cause of death from infectious disease. Efforts to control the disease are increasingly hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. At the same time, non-tuberculous mycobacteria are an expanding clinical concern, with few effective therapies available. Brazilian red propolis (BRP) has shown broad-spectrum antibacterial activity, yet its efficacy against mycobacteria is poorly characterized. In this study, we evaluated the in vitro antimycobacterial potential of a crude hydroalcoholic extract of BRP (CHEBRP). We determined minimum inhibitory concentrations against drug-susceptible and rifampicin-resistant M. tuberculosis strains (M. tuberculosis H37Rv – ATCC 27294, clinical isolate, and rifampicin-resistant clinical isolate; M. kansasii ATCC 12478 and clinical isolate) and M. avium ATCC 25291 and clinical isolate). We also calculated fractional inhibitory concentration indices to assess interactions with isoniazid and rifampicin, measured biofilm inhibition, and examined cytotoxicity in RAW 264.7 macrophages. Intracellular activity was quantified using infected macrophage cultures. CHEBRP exhibited potent activity across most M. tuberculosis strains tested, including those resistant to rifampicin. Its combination with isoniazid or rifampicin yielded an indifferent interaction, supporting the feasibility of co-administration; moreover, BRP’s known immunomodulatory effects may provide additional therapeutic benefit. CHEBRP significantly inhibited biofilm formation, demonstrated minimal cytotoxicity toward macrophages, and achieved substantial clearance of intracellular bacilli. These in vitro findings highlight CHEBRP as a promising candidate for adjunctive antimycobacterial therapy. Future studies should explore its in vivo efficacy, pharmacokinetics, and activity against a broader spectrum of mycobacterial species.