Effect of Cinnamomum zeylanicum and Origanum onites on the growth and biofilm forming ability of ESBL and non-ESBL producing Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is a leading cause of a wide range of community and hospital-acquired infections. Most K. pneumoniae strains have acquired plasmids containing varying set of antimicrobial resistance genes, conferring resistance to nearly all available classes of antibacterials and placing this species among the most defiant pathogens to treat. An important virulence factor of K. pneumoniae is its ability to form biofilms, which are communities of microbes attached to an abiotic or biotic surface, including native tissues, medical implants and catheters. In fact, bacterial cells in biofilm are found to be up to 1000-fold more resistant to antibacterials as compared to planktonic cells. Hence the need for discovering alternative treatments. Using medicinal plants and/or their essential oil constituents, in order to prevent and inhibit biofilm formation, was suggested. In this study, Cinnamomum zeylanicum and Origanum onites methanolic extracts and their major essential oils, cinnamaldehyde, eugenol, thymol and carvacrol, were tested for their ability to inhibit the K. pneumoniae biofilm formation. Twenty-six biofilm forming clinical isolates of K. pneumoniae were included in this study. Of these isolates, 13 were chosen to produce extended-spectrum β-lactamases (ESBLs), while the remaining 13 were chosen to be non-ESBLs producing. These K. pneumoniae isolates were genetically characterized by pulsed-field gel electrophoresis. The antibacterial effects of different concentrations of cinnamon (0.2, 0.15, 0.05 g/ml) and oregano (0.2, 0.15, 0.1, 0.05 and 0.02 g/ml) methanolic extracts were then detected using the standard well diffusion assay. Sublethal concentrations of 0.02 g/ml of Cinnamomum zeylanicum and Origanum onites methanolic extracts were later used to test for their ability to inhibit the biofilms formed by the test isolates. Both extracts displayed successful inhibition of K. pneumoniae biofilm formation. Gas chromatography-mass spectrometry was then employed to quantify the exact concentrations of the main essential oil constituents of the C. zeylanicum (cinnamaldehyde and eugenol) and O. onites (thymol and carvacrol) methanolic extracts. The anti-biofilm effect of these essential oils was later tested, in order to check if any of them might have been the chemical responsible for the anti-biofilm effect of the plants’ extracts. Moreover, the possibility of demonstrating a synergistic anti-biofilm effect of a combination of cinnamaldehyde and eugenol and another of thymol and carvacrol was tested. The results showed a biofilm inhibitory effect in 46.15% to 62% of the K. pneumoniae isolates, upon exposure to the different essential oil solutions, with no synergism noted upon their combination. These natural plant-derived compounds can potentially be used in coating indwelling medical devices to prevent bacterial biofilm formation. Consequently, using them not only will prevent infections, but will also help in minimizing the emergence of antibiotic resistance among pathogens and thus aid in solving one of the most challenging health problems.