Evaluation of Resistance Modulation in MDR Pseudomonas aeruginosa and Klebsiella pneumoniae Using Peppermint Oil Nanoemulsion: Integrating Antibacterial Assays and Molecular Modelling

Sardar Ali^1*Firasat Hussain^2*Tehmeena Nousheen²Kashif Rahim²Hamid Majeed²Kamal Niaz²Muhammad Nadeem Khan^3,4*

• ¹Shandong Xiehe University, Jinan, China
• ²Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan
• ³Department of Cell Biology and Genetics, Shantou Universtiy Medical College, Shantou, China
• ⁴Shantou University Medical College, Shantou, China

Multidrug-resistant urinary tract pathogens, mainly Klebsiella pneumoniae and Pseudomonas aeruginosa, pose a growing challenge to public health. The overuse of antibiotics has accelerated resistance development, necessitating alternative antimicrobial strategies. This study evaluated the antibacterial efficacy of peppermint oil nanoemulsion (PEONE) against clinical multi drug resistant (MDR) K. pneumoniae and P. aeruginosa, with a focus on resistance modulation and mechanism of action. Clinical isolates were obtained from UTI patients and tested for antibiotic susceptibility. PEONE was prepared via ultrasonic emulsification and characterized for droplet size of 190.21±0.5 and 0.15±0.021 PDI. Antibacterial activity was assessed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), DNA/protein leakage assays, and time-kill kinetics. Molecular docking and dynamic simulations (100 ns) were performed against β-lactamase enzymes (PDB: 4EXY from K. pneumoniae, 6R73 from P. aeruginosa) to identify key bioactive components. The bacterial cocktail mixture was resistant to Levofloxacin, Penicillin G, Ceftazidime, and amoxicillin-clavulanic acid (AMC).Augmentin. PEONE exhibited potent antibacterial activity with an MIC of 0.1% v/v and MBC of 0.14% v/v, corresponding to a droplet size of 1.277 nm. DNA and protein leakage increased significantly (p < 0.05) with higher PEONE concentrations, indicating membrane disruption. Time-kill assays demonstrated a sustained reduction in bacterial growth over 72 hours, with significant differences emerging after 12 hours. Docking revealed that caryophyllene, a major component of PEO, had the highest binding affinity against both targets (-9.2 kcal/mol for 4EXY; -7.1 kcal/mol for 6R73), and molecular dynamics confirmed stable binding. PEONE effectively inhibited and killed MDR K. pneumoniae and P. aeruginosa, likely through membrane disruption leading to leakage of intracellular contents as supported by computational data. These findings suggest PEONE as a promising, plant-based alternative to combat antibiotic-resistant UTIs. Further in vivo studies are warranted to explore clinical applicability and safety.