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.
Project description:Comparison of probe-target dissociations of probe Eub338 and Gam42a with native RNA of P. putida, in vitro transcribed 16s rRNA of P. putida, in vitro transcribed 16S rRNA of a 2,4,6-trinitrotoluene contaminated soil and an uncontaminated soil sample. Functional ANOVA revealed no significant differences in the dissociation curves of probe Eub338 when hybridised to the different samples. On the opposite, the dissociation curve of probe Gam42a with native RNA of P. putida was significantly different than the dissociation curves obtained with in vitro transcribed 16S rRNA samples. Keywords: Microbial diversity, thermal dissociation analysis, CodeLink microarray
Project description:Abstract: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets.
Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.
Project description:Abstract: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets. Two drip flow biofilm conditions with three replicates each: (1) baseline control at 72hrs, (2) no treatment for 12 hours past baseline. Data from these two conditions were pooled
Project description:Low concentrations of pharmaceutical compounds were shown to induce transcriptional responses in isolated microorganisms, which could have consequences on ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two distinct rivers and supplemented with environmentally relevant doses of four pharmaceutical products (erythromycin-ER, gemfibrozil-GM, sulfamethazine-SN and sulfamethoxazole-SL). To follow the expression of functional genes, we constructed a 9,600 features anonymous DNA microarray platform onto which cDNA from the various biofilms was hybridized. The reactor design for biofilm development has been previously described (Lawrence et al., 2004; Lawrence et al., 2000). Two duplicate experiments were carried out, with reactors being inoculated with either water from the WC (nutrient rich) or the SSR (nutrient poor). Treatments consisted in the addition of various pharmaceutical compounds: 1 µg l-1 erythromycin (ER), 1 µg l-1 gemfibrozil (GM), 0.5 µg l-1 sulfamethazine (SN), 0.5 µg l-1 sulfamethoxazole (SL). Nothing was added to control reactors (CO). All treatments were replicated independently three times. A reference sample (composite sample from Wascana Creek reactors used to construct the microarray) was hybridized (Cy5) on each slide.
Project description:Primary outcome(s): Analysis of the diversity and composition of the gut microbiome by 16S rRNA sequencing
Study Design: Observational Study Model : Others, Time Perspective : Prospective, Enrollment : 60, Biospecimen Retention : Collect & Archive- Sample with DNA, Biospecimen Description : Blood, Stool
Project description:Transcriptome analysis was applied to characterize the physiological activities of Psuedomonas aeruginosa cells grown for three days in drip flow biofilm reactors when compared to the activities of P. aeruginosa grown planktonically to exponential phase in the same media. Here, rather than examining the effect of an individual gene on biofilm antibiotic tolerance, we used a transcriptomics approach to identify regulons and groups of related genes that are induced during biofilm growth of Pseudomonas aeruginosa. We then tested for statistically significant overlap between the biofilm-induced genes and independently compiled gene lists corresponding to stress responses and other putative antibiotic protective mechanisms. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance.