Antibacterial Activity of Cinnamomum camphora Essential Oil on Escherichia coli During Planktonic Growth and Biofilm Formation

Abstract

Bacterial biofilms are believed to be principal virulence factors for many localized chronic infectious diseases. Escherichia coli is one of the most common microbial pathogens and frequently causes biofilm-associated opportunistic infections, such as diarrhea, endometritis and mastitis. Cinnamomum camphora essential oil (CCEO) has shown potential in treating intractable chronic endometritis in dairy cows. There is little scientific evidence regarding the effect of CCEO on bacterial biofilms. The objective of this study was to investigate the effect of CCEO on E. coli biofilm formation and how CCEO affects E. coli in suspension and in a biofilm. CCEO killed all clinical E. coli strains in either planktonic or biofilm state isolated from dairy cows with clinical endometritis. The minimum inhibitory concentration (MIC) for 90% of the organisms was 4.297 μL/mL, the minimum bactericidal concentration for 90% of the organisms was 6.378 μL/mL, the minimum biofilm inhibitory concentration for 90% of the organisms was 6.850 μL/mL, and the minimum biofilm eradication concentration (MBEC) for 90% of the organisms was 8.467 μL/mL. The MBECs were generally two times higher than the MICs. Flow cytometry analysis confirmed that significant bacterial killing occurred during the first 1 h after exposure to subinhibitory concentrations of CCEO. In addition, CCEO exerted a significant inhibitory effect on E. coli biofilm formation, and bacterial killing occurred during the first 30 min of exposure to subinhibitory biofilm concentrations of CCEO. The biofilm yield of E. coli was significantly reduced after CCEO treatment, along with an increased dead/live microbial ratio in biofilms compared with that in the non-treated control, as confirmed by scanning electron microscopy images and confocal laser scanning microscopy images. These data revealed that CCEO efficiently kills E. coli during planktonic growth and biofilm formation.

Keywords: Cinnamomum camphora essential oil; Escherichia coli; bactericidal effect; biofilm; planktonic growth.

FIGURE 1

Cumulative minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of C. camphora essential oil (CCEO) against clinical strains of E. coli isolated from dairy cows affected with clinical endometritis in China, expressed as a percentage of the tested strains. MIC and MBC, n = 44; MBIC and MBEC, n = 39.

FIGURE 2

Time-kill curves of C. camphora essential oil (CCEO) at 0, 1, 2, 4, or 8 μL/mL against planktonic E. coli ATCC 25922 for 24 h (A) and for 2 h (B). A sample of 0 μL/mL CCEO was used as a control. Data indicates confidence interval at 95% of three independent experiments.

FIGURE 3

Flow cytometry (FCM) dot plots for E. coli ATCC 25922: (A) control; (B) treated with C. camphora essential oil (CCEO) at 2 μL/mL for 0.5, 1, 4, and 24 h; (C) treated with CCEO at 4 μL/mL for 0.5, 1, 4, and 24 h; and (D) treated with CCEO at 8 μL/mL for 0.5, 1, 4, and 24 h. A sample of 0 μL/mL CCEO was used as a control.

FIGURE 4

Effect of C. camphora essential oil (CCEO) on E. coli ATCC 25922 biofilms determined by crystal violet assays. A sample of 0 μL/mL CCEO was used as a control. Data represents the mean ± SD of three independent experiments, ** indicates p < 0.01 compared with the control group.

FIGURE 5

Scanning electron microscopy (SEM) images of E. coli ATCC 25922 biofilms: (A) control; (B) treated with C. camphora essential oil (CCEO) at 2 μL/mL for 6, 12, and 24 h; (C) treated with CCEO at 4 μL/mL for 6, 12, and 24 h. A sample of 0 μL/mL CCEO was used as a control.

FIGURE 6

Time-kill curves of C. camphora essential oil (CCEO) at 1, 2, 4, or 8 μL/mL against E. coli ATCC 25922 biofilms. Data represents the mean ± SD of three independent experiments.

FIGURE 7

Analyses of live/dead bacteria in E. coli ATCC 25922 biofilms: (A) Confocal laser scanning microscopy (CLSM) images of C. camphora essential oil (CCEO) at 0 μL/mL for 6, 12 and 24 h; (B) CLSM images of CCEO at 2 μL/mL for 6, 12 and 24 h; Green, live bacteria, red, dead bacteria; Images from three independent replicates with 20 μm bars are representative; (C) Biomass of live cells in bilfilm; (D) Ratio between dead and live cells in biofilm. A sample of 0 μL/mL CCEO was used as a control. Data represents the mean ± SD of three independent experiments, ** indicates p < 0.01 compared with the control at the same time.