Project description:EPEC wild type E2348/69 (strain O127:H6) bacteria were grown overnight (ON) in LB medium (Sigma-Aldrich) at 37°C without shaking, diluted 1:40 into DMEM-HEPES (Gibco, Israel) medium and incubated for 3h at 37°C without shaking to exponential phase (optical density (OD) ~ 0.6). Bacteria were plated on LB plate with streptomycin (50 µg/ml), incubated at 32°C for 15h (for aerobic LB) or for 12h (for aerobic butyrate and infant stool metabolites conditions) or at 37°C (for anaerobic condition) for 12 h and colonies of two sizes (Virulent & Avirulent) were sampled, RNA was extracted and sequencing libraries were constructed based on the RNAtag-Seq protocol (Shishkin et al., 2015) with several modifications.
Project description:Increasing importance in the onset and progression from colonic adenomatous polyps (AP) to colorectal cancer (CRC) has been attributed to the gut microbiota and the oncometabolites they may produce. To comprehensively study the microbial spatial variations and role of microbiota in CRC progression, multiple niches from the gastrointestinal system have to be investigated. We collected saliva, tissue and stool samples from 61 patients, including 46 CRC patients and 15 AP patients, well matched in age and sex, who were undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). For all samples and locations we surveyed microbial composition through 16S ribosomal RNA and metabolites using NMR, and compared them across tissues and disease state, also considering CRC TNM staging. Our result suggest the importance of microbiota communities and derived oncometabolites in CRC development. Such association can be a forerunner for future studies on CRC/AP management.