Human Vaginal Lactobacillus -Derived (-)-Terpinen-4-ol restores antibiotic sensitivity by inhibiting efflux pumps in drug resistant E. coli and K. pneumoniae

Probiotics present a promising preventive strategy for addressing urinary tract infections (UTIs) and serve as a viable alternative to conventional antimicrobial treatments. The integration of Lactobacillus -derived efflux pump inhibitors (EPIs) with conventional antibiotics presents a promising strategy to enhance the efficacy of treatments against antibiotic-resistant superbugs. In this study, we isolated eight vaginal Lactobacillus spp. from 54 healthy Indian women to explore their probiotic properties, specifically the ability of cell-free supernatant (CFS) from Lactobacillus jensenii. to inhibit efflux in MDR E. coli and K. pneumoniae . From the CFS, we obtained seven fractions, among which compound 7 (C7) markedly reduced the MIC of erythromycin against the K. pneumoniae MTCC 432 strain by 32-fold from 64 µg to 2 µg and restored its sensitivity, indicating potent efflux inhibitory activity. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified one major compound from the C7 fraction as Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)-, also known as (-)-Terpinen-4-ol. In-vivo studies using zebrafish infected with clinical strain of K. pneumoniae 1845 followed by treatment with (-)-Terpinen-4-ol and erythromycin caused a significant 7-log reduction in bacterial bioburden.. Additionally, time-kill assay demonstrated that the combination of erythromycin with (-)-Terpinen-4-ol caused a remarkable 7-log reduction in both K. pneumoniae , and E. coli cell counts. Furthermore, (-)-Terpinen-4-ol -erythromycin combination exhibited a six-log reduction in bacterial burden in infected T24 bladder cell lines and was found to be non-toxic. This study underscores the efflux inhibitory potential of (-)-Terpinen-4-ol extracted from the CFS of human vaginal probiotics, paving the way for future pharmacological research and therapeutic applications.