Analysis of essential gene changes in MRSA and non-MRSA strains inhibited by methanolic extract correlated to membrane permeabilising peptide

Abstract number: 1734_87

Shamsudin M., Alipiah N., Vasantha N., Yusoff F., Arshad A.

Objective: In this study, a methanolic extract derived from a marine organism was investigated for the effect on the growth of MRSA resistant to b-lactam antibiotics. The nucleotide sequence changes of essential genes inclusive of mecA, mecR1, 16sRNA, mprF and msR of the inhibited MRSA strains were determined. A potential antibacterial peptide with activity on permeabilising the membrane of MRSA strains was investigated through a cellular bioassay.

Methods:S. aureus isolates (MRSA and non-MRSA) obtained from patients of hospitals in Malaysia and environmental isolates were studied. Anti-MRSA activity was determined through MIC and disc diffusion assay. Membrane permeabilising property of the methanolic extract was investigated by the penetration of the fluorescence Sytox green dye into MRSA cells. The RT-PCR analysis and nucleotide sequencing of essential genes, followed by gene sequence analysis were conducted for determining molecular mechanism of inhibition of S. aureus isolates.

Results: The methanolic marine extract showed variation in nucleotide sequence changes of several essential genes namely the mprF membrane gene in S. aureus (gene encoding for peptides involved in transportation of lysine to phospholipids bilayer in membrane). The intensities of SYTOX green fluorescence dye in S. aureus cells treated with the extract at the MIC values increases with time, similar to treatment with polymyxin, the positive control antibiotic. The negative control fucidic acid accordingly did not affect bacterial cell membrane permeabilisation and thus showed constant fluorescence intensity even after 6 hour of incubation in the antibiotic. The fluorescence intensities increase drastically first 120 minutes treatments indicating the membrane permeabilising ability is affected and the dye coloured the nucleic acid.

Conclusions: This study demonstrates the anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of methanolic marine extract by exhibiting substantial effect at both the molecular and cellular levels elucidated through nucleotide sequence changes and Sytox green dye penetration. Molecular correlation to cellular activity of extract is a viable strategy to explore alternative anti-MRSA agents.