Planktonic Growth and Biofilm Formation in Pseudomonas aeruginosa under Combined Treatment with Rifampicin and Moringa oleifera Leaf Ethanolic Extract

Biofilm formation by Pseudomonas aeruginosa complicates antimicrobial treatment, particularly in persistent infections, and motivates the assessment of plant-derived antibiotic adjuncts. The influence of Moringa oleifera leaf ethanolic extract on rifampicin responses across planktonic growth and biofilm formation requires concentration-resolved evaluation. Three independent experiments assessed planktonic growth and crystal violet-stained biofilm biomass across ethanolic extract–rifampicin concentration matrices. Conventional PCR screened selected biofilm-associated genes. The ethanolic extract alone yielded 99.33–119.33% planktonic growth relative to controls at 1–64 µg/mL. Rifampicin alone reduced growth to 58.67 ± 1.15% and 6.67 ± 0.58% at 16 and 32 µg/mL, respectively. Adding 16 µg/mL ethanol fraction to 16 µg/mL rifampicin yielded 44.67 ± 30.99% growth, compared with 58.67 ± 1.15% for rifampicin alone. At 32 µg/mL rifampicin, the same fraction addition yielded 16.00 ± 13.00% growth, compared with 6.67 ± 0.58% for rifampicin alone. Biofilm biomass at 16 µg/mL rifampicin was 55.67 ± 67.00%, reaching 70.00 ± 86.88% with ethanol fraction addition. All 49 paired comparisons per assay yielded false-discovery-rate-adjusted q-values above 0.05. PCR detected pelA, pslA, pslD, pqsA, lasR, and ndvB. The concentration matrices revealed distinct planktonic growth and biofilm biomass responses. These findings position complementary planktonic and biofilm measurements as essential criteria for assessing plant–antibiotic combinations, providing concentration-specific evidence to guide formulation screening and antimicrobial candidate prioritization.