Gene expression analysis of transport channels in Enterococcus faecium (VRE)

Introduction: An important aspect of the bacterial response towards stress and environmental stimuli is alteration of gene expression levels . A combination of carvacrol, cuminaldehyde, and vancomycin has previously been shown to re-sensitise vancomycin-resistant E. faecium (VRE) to vancomycin . The effect of treatment with the novel antimicrobial combination for 60minutes on gene expression in VRE was analysed by microarray analysis. Microarray data showed that 15 genes were differentially regulated and five genes associated with transport channels were chosen for further analysis; bcr, ecfa_1, ecsa1, ylob, and nhac_2. A time course study using qPCR was conducted to further understand the antimicrobial mechanism of action of the novel formula. Methods: qPCR was carried out to validate the microarray data at 60mins. In addition, alterations in the expression levels of the five genes were assessed at 10mins, 30mins, 2hrs and 6hrs, in response to cuminaldehyde and carvacrol alone, in combination, and in combination with the vancomycin. Results: VRE responds to the novel formula in the initial stages of exposure; at 10mins significant changes (p≤0.05) were demonstrated in the expression of the five genes, bcr, ecfa_1, ecsa_1, ylob, nhac_2 with fold changes of -13.5, -1.41, -3.95, -5.67, and -6.31 respectively. At 60mins only nhac_2 showed a significant fold change of -3.09. At 2hrs there were significant fold changes for bcr at 15.03, ecfa-1 at 2.85 and nhac_2 at 4.7, whereas at 6hrs there were no significant changes for any of the five genes tested. Conclusion: This study has demonstrated that treating VRE with EOs alone and in combination with vancomycin has resulted in fold changes in the expression levels of the transport genes of interest. A new EO-vancomycin formulation to combat VRE could be developed through exploiting transport channels in Enterococcus sp.