A-060. The Localisation of a Bacteriolytic Cell Wall Enzyme in MRSA Following Treatment with Manuka Honey

R. A. Cooper, R. E. Jenkins, N. F. Burton;
Univ. of Wales Inst. Cardiff, Cardiff, UNITED KINGDOM.

Background: Manuka honey has been shown to impede the cell cycle of MRSA by affecting the activity of bacteriolytic enzymes involved in the cleavage of peptidoglycan. The aim of this study was to localise the cellular distribution of one of those enzymes (beta-N-endoacetylglucosaminidase ) in MRSA cells with and without exposure to a bactericidal concentration of manuka honey. Methods: EMRSA-15 (NCTC 13142) was cultivated at 37°C for 24 hours in TSB with and without 10% (w/v) manuka honey, as well as in TSB with 10% (w/v) artificial honey (to determine effect of osmolarity). For the extracellular localisation of beta-N-endoacetylglucosaminidase cells were fixed, washed and incubated overnight with primary antibody (anti-beta-N-endoacetylglucosaminidase, rabbit antibody; Nordic Immunology). After washing and incubation for 30 minutes with secondary antibody (anti rabbit conjugated to 10 nm 1:30 gold particles; Amersham, UK), cells were washed and visualised by transmission electron microscopy. For intracellular beta-N-endoacetylglucosaminidase localisation cells were fixed, dehydrated and processed in a Leica EM automated freeze substitution system. Ultra thin sections were cut before overnight incubation with primary antibody, then washed and incubated with secondary antibody. Sections were counter stained using uranyl acetate and lead citrate before visualisation by transmission electron microscopy. Results: No significant differences in the localisation of beta-N-endoacetylglucosaminidase were observed between cells treated with 10% (w/v) artificial honey and untreated cells. Decreased levels of enzyme were observed on the surface of whole cells exposed to manuka honey compared to control cultures (p = 0.003). In thin sections the distribution patterns of the enzyme in honey treated cells were distinct from control cultures Conclusion: Manuka honey seems to induce altered levels and distribution patterns of beta-N-endoacetylglucosaminidase in MRSA cells.