Gene expression of vancomycin-sensitive Staphylococcus aureus strain EMRSA-15 following exposure to sub-inhibitory levels of vancomycin
Abstract number: P2041
Awad S., Alshami I., Burnie J., Lindsay J., Gould K., Hinds J., Upton M.
Objectives: Vancomycin is the drug of choice for treatment of infection with MRSA. It is possible that MRSA may be exposed to sub-lethal concentrations of vancomycin during therapy. This study aimed to investigate the effect of sub-lethal concentrations of vancomycin on global gene expression in a vancomycin sensitive strain of EMRSA-15.
Methods: A vancomycin sensitive strain of EMRSA-15 was grown in Brain Heart Infusion broth with and without 0.25 mg/l (sub-inhibitory level) of vancomycin. mRNA was extracted from the two samples using Qiagen RNeasy Kits with RNA stabilisation reagent and 'on-column' DNase treatment. Integrity of RNA was checked by electrophoresis in agarose gels and the quantity of RNA recovered was determined using a Bioanalyser. Investigations were performed in three biological replicates. Gene expression was compared for the isolates using microarray slides spotted with PCR products representing every gene from seven S. aureus sequenced genomes. RNA probes were labelled using Cy3 and each prep was hybridised against a common reference of Cy5 labelled genomic DNA extracted from strain EMRSA-15. The slides were scanned by using the Microarray Core Facility at the University of Manchester using a Genepix 4000B scanner (Axon instruments, UK). BlueFuse for microarray 3.3 (BlueGnome, UK) was used to convert all scanned images to raw data. Data analysis was performed in GeneSpring 7.3 (Silicon Genetics).
Results: Gene expression data were analysed using a median normalisation strategy and significant differences in gene expression were identified using ANOVA with Benjamini and Hochberg false discovery rate correction. Comparison of gene expression in the EMRSA-15 strain grown with and without sub-inhibitory levels of vancomycin showed significant differences. Many of the genes with altered expression were involved in cell wall biosynthesis and regulation, but of particular interest was the up regulation of genes in the locus responsible for capsule synthesis.
Conclusion: The data presented support the suggestion that exposure to vancomycin can influence gene expression in MRSA and that growth at sub-lethal concentrations may drive the development of resistance through physiological modifications.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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