Attenuation of Listeria monocytogenes virulence by Cannabis sativa L. essential oil

Abstract

Introduction. In recent years, the use of plant products as alternative/adjunct antimicrobial agents to control pathogenic microorganisms has been attracting mounting interest. A major group of plant antimicrobial compounds is represented by essential oils (EOs), complex mixtures of volatile secondary metabolites belonging to different chemical families. Cannabis sativa L. has been grown for thousands of years for a multiplicity of purposes; in recent years, some genotypes containing low cannabinoid concentrations have been selected and used for research purposes. Listeria monocytogenes is a facultative intracellular food pathogen able to persist within biofilms and to develop resistance to sanitizers. In humans, the treatment of listeriosis is hampered by the intracellular location of listeriae and the poor intracellular penetration of some antibiotics. The purpose of this study was to investigate the in vitro and in vivo antibacterial and anti-virulence properties of an EO extracted from a legal C. sativa L. variety against L. monocytogenes isolates collected from patients with invasive listeriosis. Materials and Methods. Eleven L. monocytogenes strains isolated in Italy in 2014-2016 were studied. The antibacterial and anti-virulence activity of the EO, extracted from C. sativa Futura 75 by steam-distillation, was determined by susceptibility tests, biofilm formation assays, Caco-2 invasion assays, motility assays, optical and scanning electron microscopy, Real-time RT-PCR experiments, and Galleria mellonella survival assays. Results. A moderate bactericidal activity of the C. sativa EO was detected (Minimum Bactericidal Concentration >2048 mg/L). In presence of EO at sub-lethal concentrations, the ability to form biofilm and to invade Caco-2 cells was significantly reduced (up to 15% and 73%, respectively). Motility tests demonstrated that, listeriae became non-motile i.e. they didn’t show the typical umbrella-like growth. Real-time RT-PCR assay demonstrated a significant downregulation of motility genes (flaA, motA, and motB) in L. monocytogenes exposed to the EO. In survival experiments with Galleria mellonella, larvae infected with L. monocytogenes grown in presence of EO showed much higher (93%) survival rates compared with controls (50%). Discussion and Conclusions. Anti-virulence strategies are being explored among plant products as a novel approach to combat bacterial pathogens. The significant anti-virulence properties of C. sativa EO against L. monocytogenes suggests that it could be employed as an alternative agent to control L. monocytogenes infectio