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Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance. This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate (LNA) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). LNA alone exhibited bactericidal activity at 2.5% (V/V), and in combination with meropenem (MPM) at 1.25% (V/V). Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in LNA-treated KPC-KP cells. Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability. Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells, inferring membrane damage. Furthermore, intracellular leakage of nucleic acid and proteins was detected upon LNA treatment. Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular materials. LNA was found to induce oxidative stress by generating reactive oxygen species (ROS) that initiate lipid peroxidation and damage the bacterial membrane. In conclusion, LNA generates ROS, initiates lipid peroxidation, and damages the bacterial membrane, resulting in intracellular leakage and eventually killing the KPC-KP cells. [Display omitted] • Novel antimicrobial, LNA kills KPC-KP cells effectively at 2.5%. • LNA disrupts bacterial membrane by generating reactive oxygen species. • LNA causes intracellular leakage of nucleic acids and proteins, killing the cells. [ABSTRACT FROM AUTHOR]

Antibiotic–adjuvant combinatory therapy serves as a viable treatment option in addressing antibiotic resistance in the clinical setting. This study was carried out to assess and characterize the adjuvant potential and mode of action of linalool against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Linalool exhibited bactericidal activity alone (11,250 μg/ml) and in combination with meropenem (5,625 μg/ml). Comparative proteomic analysis showed significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in linalool-treated KPC-KP cells. Upregulation of oxidative stress regulator proteins and downregulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that linalool increases the bacterial surface charge as well as the membrane permeability. Intracellular leakage of nucleic acid and proteins was detected upon linalool treatment. Scanning and transmission electron microscopies further revealed the breakage of bacterial membrane and loss of intracellular materials. Linalool induced oxidative stress by generating reactive oxygen species (ROS) which initiates lipid peroxidation, leading to damage of the bacterial membrane. This leads to intracellular leakage, eventually killing the KPC-KP cells. Our study demonstrated that linalool possesses great potential in future clinical applications as an adjuvant along with existing antibiotics attributed to their ability in disrupting the bacterial membrane by inducing oxidative stress. This facilitates the uptake of antibiotics into the bacterial cells, enhancing bacterial killing. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (KP) remains the most prevalent nosocomial pathogen and carries the carbapenemase (KPC) gene which confers resistance towards carbapenem. Thus, it is necessary to discover novel antimicrobials to address the issue of antimicrobial resistance in such pathogens. Natural products such as essential oils are a promising source due to their complex composition. Essential oils have been shown to be effective against pathogens, but the overall mechanisms have yet to be fully explained. Understanding the molecular mechanisms of essential oil towards KPC-KP cells would provide a deeper understanding of their potential use in clinical settings. Therefore, we aimed to investigate the mode of action of essential oil against KPC-KP cells from a proteomic perspective by comparing the overall proteome profile of KPC-KP cells treated with cinnamon bark (Cinnamomum verum J. Presl) essential oil (CBO) at their sub-inhibitory concentration of 0.08% (v/v). A total of 384 proteins were successfully identified from the non-treated cells, whereas only 242 proteins were identified from the CBO-treated cells. Proteins were then categorized based on their biological processes, cellular components and molecular function prior to pathway analysis. Pathway analysis showed that CBO induced oxidative stress in the KPC-KP cells as indicated by the abundance of oxidative stress regulator proteins such as glycyl radical cofactor, catalase peroxidase and DNA mismatch repair protein. Oxidative stress is likely to oxidize and disrupt the bacterial membrane as shown by the loss of major membrane proteins. Several genes selected for qRT-PCR analysis validated the proteomic profile and were congruent with the proteomic abundance profiles. In conclusion, KPC-KP cells exposed to CBO undergo oxidative stress that eventually disrupts the bacterial membrane possibly via interaction with the phospholipid bilayer. Interestingly, several pathways involved in the bacterial membrane repair system were also affected by oxidative stress, contributing to the loss of cells viability. [ABSTRACT FROM AUTHOR]