A sequenced strain of Cutibacterium acnes (C. acnes), sampled from facial acne (ATCC® 6919TM, LGC standards s.r.l., Milano, Italy), was grown by solid culture and used to infect HaCaT cells. The bacteria were stored at −80 °C, at the optical density (O.D.) of 5, in cryovials containing reinforced clostridial medium 30% (Oxoid, Hampshire, UK), glycerol 20%, and defibrinated sheep blood 50% (TCS Biosciences Ltd., Oxoid, Hampshire, UK). The solid culture was achieved using agar-blood Petri dishes, containing RCM, agar 1% (Merck Life Science, Milan, Italy), and defibrinated sheep blood 5%. Petri dishes were inoculated with 100 μL of the store aliquots of bacteria, sealed in plastic bags containing an anaerobic atmosphere generated by CO2 GasPakTM EZ (Becton Dickinson, Sparks, MD, 21152 USA), and then placed in a cell incubator (37 °C) for 48 h before HaCaT cell infection. At the time of infection, bacteria were collected and suspended in PBS 1X solution, counted by the optical method at 600 nm in 1 mL cuvette (Biochrom Libra S22 UV/VIS spectrophotometer), and directly diluted in the medium of cultured HaCaT cells, with a final O.D. of 0.1.
Reinforced clostridial medium
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Reinforced clostridial medium is a culture medium used for the growth and isolation of anaerobic bacteria, particularly Clostridium species. It provides the necessary nutrients and growth factors required for the cultivation of these microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Brucella broth, by BD (2 mentions)
Defibrinated sheep blood, by Merck Group (1 mentions)
Libra s22 uv vis spectrophotometer, by Harvard Bioscience (1 mentions)
Anaeropack, by Mitsubishi (1 mentions)
Insulin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Daejung Chemicals (1 mentions)
Propionate, by Merck Group (1 mentions)
Butyrate, by Merck Group (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Avantor (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
9 protocols using reinforced clostridial medium
1
Cutibacterium acnes Infection of HaCaT Cells
2
Quantifying Anaerobic Bacterial Growth
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The CFU assay was performed as described previously (McInturff et al., 2005 (link)). P. acnes strains (Table 1 ) were grown under anaerobic conditions in Reinforced Clostridial Medium (Oxoid, Basingstroke, England) and collected in mid-log phase. The bacteria were washed with the assay buffer (10mM Tris pH 7.4, supplemented with 1% volume Trypticase soy broth, Tris-TSB), and enumerated by applying a conversion factor of 7.5 x 107 bacteria per mL=1 OD unit at 600 nm. Various concentrations of Pentobra, pen peptide or tobramycin were incubated with 3.75 x105 bacteria in a final volume of 100 μL at 37°C for 3 h. After incubation, 103–104-fold dilutions were prepared and plated on solid media comprised of Brucella broth (BD Biosciences, San Diego, California) with 5% sheep red blood cells (Remel, Lenexa, Kansas). Plates were incubated for 4 days at 37°C under anaerobic conditions, then individual colonies were counted and the number of CFU per tube was calculated.
3
Quantifying Anaerobic Bacterial Growth
4
ACAT Model of JBC 1847 Antimicrobial Properties
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JBC 1847 was synthesized at University of Copenhagen, Denmark, as previously described and initial investigations on the antimicrobial and physicochemical properties of JBC 1847 reported [10 (link)]. In this study, an advanced compartment and transit (ACAT) model using GastroPlusTM software (Simulations Plus, Inc., Lancaster, CA) was used. This model incorporates physicochemical (i.e., solubility and permeability), physiological (i.e., regional pH and transit time along the gastrointestinal tract), and pharmacokinetic (i.e., clearance and volume of distribution) parameters and other factors (such as dose and P-glycoprotein [Pgp] substrate data) to predict exposure. The input was set to 100 mg JBC 1847 in tablet form administrated to a 70 kg human individual.
C. difficile ATCC 43255, previously isolated from an abdominal wound, was used to assess the anti-clostridioides activity of JBC 1847. The strain was stored in reinforced clostridial medium (Oxoid, Roskilde, Denmark) in 15% glycerol at -80⁰C until use and grown anaerobically on ChromID® C. difficile plates (bioMérieux, Ballerup, Denmark).
C. difficile ATCC 43255, previously isolated from an abdominal wound, was used to assess the anti-clostridioides activity of JBC 1847. The strain was stored in reinforced clostridial medium (Oxoid, Roskilde, Denmark) in 15% glycerol at -80⁰C until use and grown anaerobically on ChromID® C. difficile plates (bioMérieux, Ballerup, Denmark).
5
Antimicrobial Activity of Th17 Cytokines
6
Anaerobic Culture of PA Strain 6919
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PA strain 6919 obtained from American Type Culture Collections (ATCC; Manassas, VA, USA) and grown on brucella agar with 5% sheep blood, hemin, and vitamin K (Thermo Fisher Scientific Remel Productions, Lenexa, KS, USA) at 37℃ for 3~5 days in sealed chambers with Anaero Packs (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). Cultures grown under same conditions in reinforced clostridial medium (Oxoid, Basingstoke, England). Spectrophotometer OD600 used to determine bacterial log phase. Cultures diluted to final concentration of 0.5 multiplicity of infection (MOI) to stimulate cells.
7
Reagents for Cell Culture Experiments
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Reinforced clostridial medium (RCM) and De Man, Rogosa and Sharpe (MRS) medium were purchased from Oxoid Ltd. (Hampshire, United Kingdom) and BD Biosciences (Bergen County, NJ, USA), respectively. Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), penicillin/streptomycin (P/S), and trypsin-EDTA were purchased from HyClone (Logan, UT, USA). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from DAEJUNG (Siheung, Korea).
8
Cultivation of Cells with Standard Media
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RPMI-1640 medium, foetal bovine serum (FBS), and penicillin/streptomycin for the cultivation of cells were obtained from HyClone (Logan, UT, USA). Reinforced clostridial medium (RCM) was purchased from Oxoid (Hampshire, UK), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Amresco (Solon, OH, USA), and dextran sodium sulfate (DSS, MW 36,000–50,000 Da) was purchased from MP Biomedicals (Solon, OH, USA). High-performance liquid chromatography (HPLC)-grade acetate, propionate, and butyrate; dimethyl sulfoxide (DMSO); and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (St. Louis, MO, USA).
9
Antimicrobial Susceptibility Testing of GTE
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Staphylococcus aureus ATCC 700699, ET199 and LMG10147, Staphylococcus epidermidis ET086 and P. aeruginosa ATCC9027 were cultured aerobically in Mueller-Hinton broth (MH; Oxoid, Basingstoke, England), at 37°C. Propionibacterium acnes LMG 16711 was cultured anaerobically in Reinforced clostridial medium (RCM; Oxoid), at 37°C. Candida albicans ATCC MYA-2876 was cultured aerobically in RPMI 1640 medium (Sigma-Aldrich, Diegem, Belgium), at 37°C. Determination of the Minimal Inhibitory Concentration (MIC): MIC of GTE alone or in the presence of WPI (50 or 800 µg/ml) was determined according to the EUCAST broth microdilution protocol, using f lat-bottom 96-well microtiter plates (TPP, Trasadingen, Switzerland). The inoculum was standardized to approximately 5 × 10 5 colony forming units (CFU)/ml. The concentration of GTE tested ranged from 0.5 -1024 µg/ml. The plates were incubated at 37°C for 24 h, and the optical density at 590 nm was determined using a multilabel microtiter plate reader (Envision; Perkin-Elmer LAS, Waltham, MA). MIC was recorded as the lowest concentration of GTE, alone or in combination with WPI, which displayed a similar optical density as that observed in inoculated and blank wells.
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