Growing and Characterizing Biofilms Formed by Streptococcus pneumoniae

The serine protease HtrA plays a key role in heat-induced dispersal of pneumococcal biofilms

“…Cell culture media and supplements as well as trypsin were purchased from GE Healthcare Life Sciences. Reagents for chemically-defined medium for pneumococcal growth (CDM 16 ), proteinase K (from Tritirachium alba ), papain (from papaya latex), phenylmethanesulfonyl fluoride (PMSF; broad serine protease inhibitor), leupeptin (serine and cysteine protease inhibitor), E-64 (cysteine protease inhibitor), lysozyme, mutanolysin, β-casein, sodium dodecyl sulfate (SDS), TRI reagent, Isopropyl-β- d -thiogalactoside (IPTG), NaCl, and imidazole were purchased from Sigma-Aldrich. Gentamicin, HEPES, and DNase I were purchased from Thermo Fisher Scientific.…”

“…Biofilm formation was performed as previously described 16 . Briefly, frozen stocks of pneumococci were diluted in CDM to approximately 10 5 CFU/ml, seeded onto PFA-fixed epithelial cells in 24-well plates, and grown at 34 °C, 5% CO 2 over 72 h, replacing with fresh media every 12 h. A temperature of 34 °C simulates the measured nasopharyngeal temperature 48 .…”

“…The SEM protocol used here has been previously described in detail 16 . In brief, biofilms were formed as described above except pneumococci were seeded onto round glass coverslips covered with a pre-fixed substratum of confluent NCI-H292 cells.…”

“…RNA was isolated from biofilm, supernatant (released bacteria), and planktonic samples. A detailed protocol for RNA isolation from pneumococcal cells is available 16 . Briefly, samples were subjected to enzymatic, chemical, and mechanical lysis.…”

“…Biofilm bacteria are encased within an extracellular matrix composed of DNA, proteins, carbohydrates, and other structural molecules 15 . Bacterial lysis is important for pneumococcal biofilm formation as pneumococci lacking the major autolysin LytA form poor biofilms with no matrix 8 , indicating that the material from lysed pneumococcal cells makes an important contribution to the biofilm matrix and its associated protection from antimicrobial agents 16 . It seems likely that molecules involved in the degradation of biofilm matrix macromolecules are responsible for and potentiate the dispersal process.…”