Antibacterial and anti-biofilm activities of thyme oil, oregano oil, and their combination against Klebsiella pneumoniae and Acinetobacter baumannii polymicrobial biofilms.

Biofilm formation is a key virulence factor in Klebsiella pneumoniae and Acinetobacter baumannii, and their coexistence leads to highly drug-resistant polymicrobial biofilms. We tested the antibacterial, antibiofilm, and biofilm-disrupting activities of thyme oil, oregano oil, and their combination against dual species culture of K. pneumoniae and A. baumannii. Chemical composition was characterized by GC-MS. Antibacterial activity was evaluated using agar well diffusion, with MIC and MBC determined by broth dilution. Antibiofilm activities of oils and their combinations were tested using crystal violet staining and live/dead fluorescence imaging. RT-PCR was performed to examine the expression of biofilm-associated genes, and anti-inflammatory activity was assessed using protein denaturation inhibition. Both oils exhibited dose-dependent antibacterial activity, with oregano showing stronger bactericidal effects (MIC 5 µL/mL; MBC 10 µL/mL) and thyme exhibiting superior protein denaturation inhibitory activity. The combination of oils acted synergistically (FIC index = 0.5), achieving > 90% disruption of dual-species biofilms. RT-PCR confirmed significant downregulation of mrkA, fimA, ompA, and bap, with the combination showing the strongest suppression (p < 0.0001). GC-MS analysis confirmed carvacrol as the major constituent of oregano oil and thymol as the dominant component of thyme oil, providing a chemical basis for the observed biological activities. Overall, the results highlight the therapeutic potential of oregano and thyme essential oils, individually and in combination, for managing polymicrobial biofilm-associated infections caused by K. pneumoniae and A. baumannii.