Investigating physiological and genetic characteristics of community acquired infections and potential antimicrobial interventions

Abstract

Staphylococcus aureus and Enterococcus sp. infections occur in hospital and, increasingly, in community settings, with the potential of having different susceptibility to antimicrobial agents. The purpose of this study was to investigate the effect of antimicrobial agents against community acquired S.aureus and investigate antibiotic characteristics, biofilm formation and gene expression following exposure to an antimicrobial agent. The susceptibility of S. aureus isolates and a vancomycin resistant Enterococcus faecium isolate to antibiotics, essential oils and disinfectants were investigated under planktonic conditions using standardised antimicrobial susceptibility tests and the Quantitative suspension and surface tests (EN 1276 and EN 13697) for the disinfectants. Biofilm formation, inhibition and eradication was investigated using the crystal violet (CV) assay while the viability of treated biofilms were investigated using the 2, 3-bis [2-methyloxy-4-nitro-5- sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and CFU/ml assay. Multiplex PCR was used to determine the presence of PVL, icaA and IcaD genes as well as SCCmec typing while RT-PCR used to investigate changes in gene expression in five target genes following treatment of PVL CA MSSA and CA MRSA MW2 biofilms with grapefruit EO. The S. aureus isolates all formed biofilms and had similar molecular characteristics however one isolate (CA MRSA SR) was multidrug resistant and PVL negative. The VRE isolate was negative for biofilm formation. In suspension, household bleach and NaDCC caused >5 log reduction in viable counts and on stainless steel surfaces, there was <3.5 log reduction. Against biofilms, Household bleach at 5000ppm caused 100% biofilm eradication within 10 minutes while NaDCC eradicated <50% of the biofilm within one hour at 10,000ppm.The eco-friendly product did not demonstrate any antimicrobial activity against planktonic cells or biofilms. Antimicrobial activity of six essential oils (EO) (lime, lemon, lemongrass, geranium, grapefruit, bergamot), and two components (limonene and citral) was investigated for the S. aureus isolates. Following exposure to lemongrass EO extensive disruption to S. aureus biofilms was shown under scanning electron microscopy. The most notable changes in gene expression following exposure to grapefruit EO were the /caD, luxS and sodA genes when the PVL CA MSSA biofilms was compared to the prototype community acquired strain, CA MRSA MW2. The S. aureus isolates were susceptible to the essential oils with the exception of limonene and lemon EO. Lemongrass EO inhibited biofilm formation, metabolic activity and viability. No anti-biofilm activity was observed for Grapefruit EO against S. aureus except for one isolate (PVL positive CA MSSA), where an increase in metabolic activity was observed following treatment. Lemongrass EO was effective as an antibacterial and antibiofilm agent and could be a potential alternative to chemical based antimicrobial agents in both healthcare and non-healthcare environments.

Date of Award	2013
Original language	English
Awarding Institution	University of Northampton
Supervisor	Stuart C H Allen (Supervisor) & Carol A Phillips (Supervisor)