Bovine thrombin, Fluorescein isothiocyanate (FITC), dialysis tubing, wortmannin, TFLLR-NH2 and AYPGKF-NH2 were obtained from Sigma (Poole, UK). Bisindolylmaleimide I (Bis-1) and primary 4G10 antibody were purchased from Merck Millipore (Darmstadt, Germany). RWJ56110 and TCY-NH2 were purchased from R&D Systems (Minneapolis, MN, USA). Primary FITC labeled PAC-1 and Cy5 labelled anti-CD62P were purchased from BD Biosciences (New Jersey, NJ, USA). Staphostatin A was purchased from Biocentrum (Krakow, Poland). Fast flow DEAE and CF sepharose columns were purchased from GE Healthcare (Buckinghamshire, UK). Brain heart infusion media was purchased from Oxoid (Basingstoke, UK). All other reagents were from previously described sources [26 (link)].
Brain heart infusion media
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Brain heart infusion media is a general-purpose microbiological culture medium used for the cultivation and isolation of a wide variety of microorganisms, including fastidious and anaerobic bacteria. It provides essential nutrients for the growth of these organisms.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna broad range kit, by Thermo Fisher Scientific (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Bovine thrombin, by Merck Group (1 mentions)
Rwj 56110, by R&D Systems (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Tfllr nh2, by Merck Group (1 mentions)
Aypgkf nh2, by Merck Group (1 mentions)
Haemophilus test media, by Thermo Fisher Scientific (1 mentions)
Anaerobic chamber, by Coy Laboratory Products (1 mentions)
6 protocols using brain heart infusion media
1
Platelet Activation and Signaling Assay
2
Bacterial Strain Characterization for Respiratory Pathogens
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The bacterial reference strains used in this study included type strain H. influenzae DSM 4690, a range of H. influenzae capsular subtypes (a–f), and closely related Haemophilus strains, with N. meningitidis NCTC 10025 and S. pneumoniae DSM 20566 type strains (Table S1 ). Haemophilus test media (Oxoid, Hampshire, UK) was used to culture all Haemophilus strains, and brain heart infusion media (Oxoid) was used to culture the N. meningitidis and S. pneumoniae type strains. Bacterial incubation was carried out at 37 °C for 18 h under microaerophilic conditions. The DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and Qubit dsDNA broad range kit (Life Technologies, Warrington, UK) were used as per manufacture instructions to extract and quantify DNA from all cultured strains. All bacterial strains and extracted DNA were stored at −80 °C prior to use. Genome copy number values for DNA extracted from each strain were generated using resulting DNA concentrations with approximate genome size standards of 1.83 Mb, 2.2 Mb and 2.1 Mb for Haemophilus, N. meningitidis, and S. pneumoniae strains, respectively.
3
Characterization of MRSA Strains
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SH1000 is a laboratory strain provided by Simon Foster (University of Sheffield, UK), expressing a chloramphenicol resistance plasmid (pSK5487) with a gfp gene. MRSA strain JE2 is derived from USA300, a well characterised clinical isolate of community acquired multi-drug resistant Staphylococcus aureus. It was produced by removing a macrolide resistance plasmid and a cryptic plasmid and was kindly provided by Andrew Liew Tze Fui (University of Technology, Sydney, Australia). S235 is a virulent clinical isolate of Community Associated-MRSA (CA-MRSA) kindly donated by the Sheffield School of Clinical Dentistry. Bacteria were routinely grown in Brain Heart Infusion Media (Oxoid, Basingstoke, UK), and bacteria were grown in log phase (as determined by both optical density at 600nm (OD600) and CFU counts) for infection assays.
4
Extraction and Quantification of E. coli DNA
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All E. coli isolates used for assay development and evaluation (Table S3) were stored at −80°C on glycerol bead stocks or in extracted DNA form. Culturing of animal and environmental E. coli isolates was performed using brain heart infusion media (Oxoid, Hampshire, UK) with incubation at 37°C for 18 h, aerobically. Bacterial DNA extraction and quantification was carried out using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) and Qubit dsDNA broad range kit (Life Technologies, Warrington, UK), as per manufacturer’s instructions. All human E. coli isolates were supplied in extracted DNA form from the Royal College of Surgeons, Ireland, and the National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, National Institute of Health Dr. Ricardo Jorge, Portugal. These isolates were selected from human strain collections recovered from urine, blood, pus, and biopsy specimens of different hospitals. All patient samples were anonymized and not analyzed for human DNA; thus, ethical approval was not required. Gene target copy number values for each isolate were determined using resulting DNA concentrations with the assumption that 0.5 ng E. coli blaCTX-M-1 or E. coli blaCTX-M-15 purified genomic DNA extract corresponds to 90,000 copies with one copy per bacterium (54 (link), 55 (link)).
5
Anaerobic Cultivation of Erysipelotrichaceae and Ruminococcaceae
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The presence of the PnuC gene in species within Erysipelotrichaceae and Ruminococcaceae was verified by running a BLAST for the gene in the NCBI database showing that some members of the enriched families did have the transporter. Clostridium innocuum (strains DSM 26113 and DSM 1286) and Pseudoflavonifractor sp (strains DSM 107456 and DSM 23940) were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). All bacterial strains were cultured in Brain heart infusion media (Oxoid, Basingstoke, UK) and under anaerobic conditions (85% N2, 10% CO2 and 5% H2 at 37 °C in an anaerobic chamber (Coy Laboratory Products, Michigan, USA).
6
Preparation of Caries-Relevant Bacteria
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Inocula of S. mutans [American Type Culture Collection (ATCC) 35668], L. acidophilus (ATCC 9224), and A. naeslundii (ATCC 12104), some of the main organisms known to be involved in the caries process, were prepared from a 24-hour anaerobic incubation on blood-agar. Organisms were harvested to produce suspensions in sterile brainheart infusion media (Oxoid, Unipath, Basingstoke, England) to a MacFarland optical density of 0.5 at 660 nm.
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