Overcoming resistance in MDR Klebsiella pneumoniae: a dual approach using thymol and carvacrol to enhance antibacterial activity and inhibit biofilm-mediated virulence

Background The escalating prevalence of resistant bacterial strains, including MDR, XDR, and PDR, poses a major threat to public health. Klebsiella pneumoniae is of primary concern, identified by WHO as a critical priority pathogen in hospital-acquired infections. Therapeutic options were relegated to last-resort antibiotics like colistin, limited by its nephrotoxicity, and tigecycline, which now faces emerging bacterial resistance, raising the need for different treatment options. Natural products offer a compelling alternative, with different bioactive compounds that may exhibit antibacterial activity. Accordingly, this study evaluated the antibacterial and antibiofilm activities of thyme and ajwain alcoholic extracts and their pure active constituents (thymol and carvacrol), as single agents or in combination with tigecycline (as a last-resort antibiotic), against MDR K.pneumoniae isolates. Methods Antibacterial tests, including MIC, time-kill, and bacterial membrane integrity assays, were performed, along with biofilm inhibition assays using crystal violet. At the molecular level, the effects of these agents on various biofilm- and virulence-related genes were investigated using RT-qPCR. Results Thymol and carvacrol, as well as plant extracts of thyme and ajwain, showed potent antibacterial properties against MDR, XDR, and PDR K.pneumoniae isolates(n=47), with thymol being the most potent, with MIC 50 of 250µg/ml and MIC 90 of 500µg/ml. Additionally, Monoterpenes showed a significant effect on disrupting the bacterial membrane. All tested natural products, as well as tigecycline, showed potent biofilm-inhibitory activity against strong and moderate K.pneumoniae biofilm-forming isolates, reaching up to 100%inhibition with Carvacrol at 0.5MIC. Carvacrol and thymol were the most potent, exhibiting significant inhibition even at 0.125MIC concentrations. Furthermore, Carvacrol demonstrated a clear adjuvant effect when combined with tigecycline at 0.25MIC, significantly enhancing tigecycline’s antibiofilm activity. Mechanistically, carvacrol and tigecycline significantly downregulated the expression of different biofilm-related genes( mrkA, lsrK, ecpA , and fimH ) in almost all tested clinical isolates. Conclusion Monoterpenes(thymol/carvacrol) exert a statistically significant disruptive effect on bacterial membrane integrity compared to tigecycline. Moreover, they exhibited potent antibiofilm activity on K.pneumoniae clinical isolates, either alone or in combination with tigecycline. Carvacrol acted as an antibiofilm agent even at very low concentrations, making it a highly promising candidate for clinical use, offering a novel standalone or adjuvant antimicrobial therapy.