Project description:Liquid cultures of Enterococcus faecalis OG1RF and OG1RF Δbph were grown in tryptic soy broth without added dextrose (TSB-D) for 2 and 4 hr. At each time point, the transcriptomes were compared to identify differentially expressed genes in the Δbph mutant.
Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 7 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of one of the following CW inhibitors: ampicillin (20 μg ml-1), bacitracin (80 μg ml-1), or cephalothin (80 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition. RNA was extracted from four replicate samples of each condition of interest (control cells grown to OD600 = 0.3 in FMC medium supplemented with 10mM glucose, then treated with ampicillin, bacitracin, or cephalothin for 30 or 60 minutes) and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from E. faecalis OG1RF cells grown in BHI medium to mid-log.
Project description:Changes in Enterococcus faecalis OG1RF gene expression during infection in a rabbit model of subdermal abscess formation were studied using microarray analysis.
Project description:Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 7 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of one of the following CW inhibitors: ampicillin (20 μg ml-1), bacitracin (80 μg ml-1), or cephalothin (80 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition.
Project description:Changes in Enterococcus faecalis OG1RF(pCF10) gene expression at 4 hours post-infection in a rabbit model of subdermal abscess formation were studied using RNA-seq analysis.
Project description:Changes in Enterococcus faecalis OG1RF gene expression during infection in a rabbit model of subdermal abscess formation were studied using microarray analysis. Samples assayed in arrays consisted of the input inoculum and total RNA collected from the subdermal chambers at 2 and 8 hours post-inoculation. Alexa647-labeled samples were co-hybridized with 0.5 micrograms of sheared OG1RF genomic DNA labeled with Cy3 as a standard reference between chips. Four biological replicates were performed for each of the 3 time points.
Project description:Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants.
