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.
Misuse of antibiotics in the clinical and agricultural sectors has caused the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae which contributes a threat to human health. In this study, we assessed the feasibility of lavender essential oil (LVO) as an antimicrobial agent in combinatory therapy with meropenem in suppressing the growth of carbapenemase-producing K. pneumoniae (KPC-KP). Synergistic interactions between LVO and meropenem were detected, which significantly reduce the inhibitory concentration of both LVO and meropenem by 15 and 4-fold respectively. Comparative proteomic profiling identified a disruption in the bacterial membrane via oxidative stress that was indicated by loss of membrane and cytoplasmic proteins and the upregulation of oxidative regulators. As a proof of concept, zeta potential measurements showed a change in cell surface charge while outer membrane permeability measurement indicated an increase in membrane permeability following exposure to LVO. This was indicative of a disrupted outer membrane. Ethidium bromide influx/efflux assays demonstrated no significant efflux pump inhibition by LVO, and scanning electron microscopy revealed irregularities on the cell surface after exposure to LVO. Oxidative stress was also detected with increased level of ROS and lipid peroxidation in LVO-treated cells. In conclusion, our data suggest that LVO induced oxidative stress in K. pneumoniae which oxidizes the outer membrane, enabling the influx of generated ROS, LVO and meropenem into the bacterial cells, causing damage to the cells and eventually death.