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Antimicrobial activity and mode of action of 1,8-cineol against carbapenemase-producing Klebsiella pneumoniae - PubMed

  • ️Fri Jan 01 2021

Antimicrobial activity and mode of action of 1,8-cineol against carbapenemase-producing Klebsiella pneumoniae

Chew-Li Moo et al. Sci Rep. 2021.

Abstract

Antimicrobial resistance remains one of the most challenging issues that threatens the health of people around the world. Plant-derived natural compounds have received considerable attention for their potential role to mitigate antibiotic resistance. This study was carried out to assess the antimicrobial activity and mode of action of a monoterpene, 1,8-cineol (CN) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Results showed that resazurin microplate assay and time-kill analysis revealed bactericidal effects of CN at 28.83 mg/mL. Zeta potential showed that CN increased the surface charge of bacteria and an increase of outer membrane permeability was also detected. CN was able to cause leakage of proteins and nucleic acids in KPC-KP cells upon exposure to CN and ethidium bromide influx/efflux experiment showed the uptake of ethidium bromide into the cell; this was attributed to membrane damage. CN was also found to induce oxidative stress in CN-treated KPC-KP cells through generation of reactive oxygen species which initiated lipid peroxidation and thus damaging the bacterial cell membrane. Scanning and transmission electron microscopies further confirmed the disruption of bacterial cell membrane and loss of intracellular materials. In this study, we demonstrated that CN induced oxidative stress and membrane damage resulting in KPC-KP cell death.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Killing kinetics of KPC-KP treated with CN at 28.83 mg/mL and 14.42 mg/mL for 24 h.

Figure 2
Figure 2

Membrane-related assays performed showing CN disrupts the bacterial membrane of KPC-KP. (a) Outer membrane permeability of KPC-KP cells. Comparative absorbance of KPC-KP cells exposed to 0.1% SDS or saline after treatment with CN at 14.42 mg/mL (b) Membrane zeta potential of untreated and CN-treated KPC-KP. (c) Leakage of UV-absorbing materials; nucleic acids and (d) proteins of KPC-KP treated with CN. (e) EtBr influx assay with untreated KPC-KP and CN-treated KPC-KP cells exposed to 1 mg/L of EtBr for 60 min at 5 min intervals followed by (f) EtBr efflux assay with EtBr removed, treated with glucose or CN only, and combination of glucose with CN. The mean ± SD for three replicates is illustrated. Data were analysed by Student's t-test with *P < 0.05 being significantly different from the untreated KPC-KP cells.

Figure 3
Figure 3

Oxidative stress assessment via (a) ROS measurement using DCF-DA and (b) lipid peroxidation assay in treated and untreated KPC-KP cells. The mean ± SD for three replicates is illustrated. Data were analysed by Student's t-test with *P < 0.05 being significantly different from the untreated KPC-KP cells.

Figure 4
Figure 4

Scanning electron micrographs of KPC-KP. (a) Untreated; (b) CN-treated KPC-KP cells (14.42 mg/mL). The treated KPC-KP cells with corrugated cell membrane was indicated by the white arrow. Scale bar in scanning electron micrographs represents 1 μm. Tranmission electron micrographs of KPC-KP. (c) Untreated; (d) CN-treated KPC-KP cells (14.42 mg/mL). The treated cells with cytoplasmic clear zones and lacked of staining zones were indicated by the white arrows. Scale bar in scanning electron micrographs represents 0.5 μm.

Figure 5
Figure 5

The proposed mode of action of CN against KPC-KP cells. (i) CN reacted with the bacterial membrane which produced ROS. (ii) ROS attacks membrane lipid to initiate lipid peroxidation, causing chain reaction which disrupts the bacterial membrane. (iii) The disrupted membrane leads to the influx of ROS into the intracellular region of KPC-KP. (iv) ROS degrades intracellular materials such as nucleic acids, proteins and lipids. (v) Intracellular materials leak out due to membrane disruption.

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