Development of Novel Synergistic Therapeutic Strategies to Combat Antimicrobial Resistance: Critical Roles for Natural Products

The increasing prevalence of Antimicrobial Resistant (AMR) bacteria in healthcare and community settings has brought about a need for new antimicrobial treatments. Essential Oils (EOs) and EO compounds inhibit a range of pathogens and can synergistically enhance the antibiotic susceptibility of AMR bacteria, indicating that EO-antibiotic combinations could extend the utility of antibiotics. The antimicrobial activity of cumin, oregano and rosewood EOs was determined against Enterococcus faecium, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, before the antimicrobial EO compounds therein were identified by Thin Layer Chromatography-Direct Bioautography (TLC-DB). Minimum Inhibitory Concentrations (MICs) of the EOs ranged from 0.29-35.20 mg/mL and MICs of the EO components ranged from 0.99-500.92 mM. A 3D quantitative structure-activity relationship was also constructed for the EO components, which suggested that ligand efficiency and electrostatic distribution were important determinants of antimicrobial activity. The EOs and EO components were screened for synergistic interactions with antibiotics using the checkerboard method followed by a time-kill assay. EO/EO component-antibiotic combinations were generally indifferent, however synergistic interactions were identified between ciprofloxacin (0.25 mg/L) and cuminaldehyde (1.05 mM) against Ciprofloxacin Resistant (CR) E. coli and vancomycin (0.031 mg/L), carvacrol (1.98 mM) and cuminaldehyde (4.20 mM) against Vancomycin Resistant E. faecium (VRE) which resulted in 5.57 and 4.73 log10 reductions, respectively. These combinations reduced the antibiotic MICs below the clinical resistance breakpoint, suggesting antibiotic resistance attenuation. The synergistic mechanism of action of the EO-vancomycin combination against VRE was investigated using transcriptomic analysis coupled with β-galactosidase leakage and salt tolerance assays. The results suggested that cell envelope damage contributes to the synergistic bactericidal effect against VRE, for example, a 2.0-fold up-regulation of a lysM-domain containing protein (42912_B02_00848) indicates peptidoglycan structure alteration. Finally, the in vivo antimicrobial activity of the EO-vancomycin combination against VRE was determined by a Galleria mellonella larvae assay, however no antimicrobial action was observed, indicating that further drug development is required for the EO-vancomycin combination to be clinically useful for treatment of VRE infections. The results of this investigation demonstrate that antibiotics and EO components are able to interact synergistically and subsequently re-establish the antibiotic susceptibility of AMR bacteria. The work also considers such combinations from a drug development perspective, which is required to drive the development of such formulations for future clinical use to combat AMR. A formulation of 1.98 mM carvacrol, 4.20 mM cuminaldehyde and 0.031 mg/L vancomycin could be useful as a novel combination therapy against VRE infections, however further drug development is required to increase the in vivo efficacy and amaeliorate the observed toxicity.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2019

Author dc:creator

• Owen, Lucy

Licence

• https://dora.dmu.ac.uk/bitstreams/d3b8428e-5fdf-43c9-82f7-0d85232343a3/download