Antibacterial and antibiofilm activity of methanolic Indian propolis extract against ESBL Pseudomonas isolated from buffalo mastitis

Increasing antimicrobial resistance poses a major challenge to mastitis treatment in dairy animals, highlighting the need for alternative therapeutics. This study analysed the antibacterial and antibiofilm activity of methanolic propolis extract (MPE) against extended-spectrum β-lactamase (ESBL) positive Pseudomonas spp. recovered from mastitis affected Murrah buffaloes. Of the 175 environmental isolates from mastitis-affected buffalo milk (n= 472 animals), Pseudomonas spp. accounted for 23.4% (n=41), making it the second most prevalent pathogen after Escherichia coli (24.6%). Six clinical ESBL positive Pseudomonas isolates were selected for analysis. MPE exhibited antibacterial activity with minimum inhibitory concentration (MIC) in the range of 7.81 to 15.62 mg/mL. Scanning electron microscopy and fluorescence microscopy revealed membrane disruption in treated cells, and flow cytometry further confirmed a high proportion of apoptotic cells post-treatment. All isolates were strong biofilm producers, and MPE showed significant antibiofilm activity with 72.5± 0.39% and 71.2 ± 0.95% biofilm inhibition at 2MIC and MIC, respectively. The findings indicate that Indian propolis is a promising natural alternative with potent antibacterial and antibiofilm activity against Pseudomonas associated with bovine mastitis.