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.
Highlights
MATERIALS AND METHODSBacterial biofilms, an emergent form of bacterial life, are “aggregates of microorganisms in which cells are frequently embedded in a self-produced matrix of extracellular polymeric substances that are adherent to each other and/or a surface” (Vert et al, 2012; Flemming et al, 2016)
The National Institutes of Health revealed that 65% of all microbial infections and 80% of all chronic infections are associated with biofilm formation
The minimum biofilm eradication concentration (MBEC) of Cinnamomum camphora essential oil (CCEO) against E. coli was determined by the microdilution method with minor modifications (Ramage et al, 2001)
Summary
An emergent form of bacterial life, are “aggregates of microorganisms in which cells are frequently embedded in a self-produced matrix of extracellular polymeric substances that are adherent to each other and/or a surface” (Vert et al, 2012; Flemming et al, 2016). The minimum biofilm inhibitory concentration (MBIC) of CCEO against 39 E. coli isolates and E. coli ATCC25922 was determined using the microdilution method with minor modifications (AI-Shabib et al, 2017). The minimum biofilm eradication concentration (MBEC) of CCEO against E. coli was determined by the microdilution method with minor modifications (Ramage et al, 2001). E. coli ATCC 25922 suspensions (1 × 105 CFU/mL) were mixed with CCEO at a final concentration of 0 (control), 1, 2, 4, or 8 μL/mL and with 1% DMSO (v/v) in all tubes. After the E. coli ATCC 25922 suspensions (1 × 107 CFU/mL) were incubated with CCEO concentrations of 0 and 2 μL/mL for 24 h, the biofilms were stained with SYTO 9 and PI from the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Invitrogen, France).
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