We purchased Escherichia coli (KCTC1682), Salmonella typhimurium (KCTC 1926), Staphylococcus aureus (KCTC 1621), Bacillus subtilis (KCTC 1021), and Propionibacterium acnes (KCTC 3314, KCTC 3220, KCTC 5527, and KCTC 5933) from the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience & Biotechnology (Daejeon, Korea). S. aureus (CCARM 0027 and CCARM 3708) was obtained from the Culture Collection of Antimicrobial Resistant Microbes (Seoul, Korea). The MIC of phloretin against each of these bacteria was determined by broth microdilution assay [43 (link)]. We defined the MIC as the lowest concentration of phloretin that completely inhibited bacterial growth; this was calculated as the average of three independent measurements in the range of 0.5 to 512 µM. P. acnes was grown for 48 to 72 h at 37 °C on sheep blood agar under anaerobic conditions in a mixture of H2 and CO2 (95:5, v/v). Microbroth dilution using Muller–Hinton (MH) broth was used to determine the efficacy of phloretin against P. acnes. We diluted a bacterial suspension in MH broth to achieve turbidity equivalent to a McFarland standard of 0.5 (~1 × 108 cells/mL), yielding 1 × 104 cells/mL in each well after inoculation. Phloretin MICs were determined after incubation for 48 h at 37 °C.
Bacillus subtilis
Manufactured by Korean Collection for Type Cultures
Bacillus subtilis is a Gram-positive, spore-forming bacterium commonly found in soil. It is a model organism used in laboratory studies and has diverse applications in research and industry.
Automatically generated - may contain errors
Lab products found in correlation
Escherichia coli, by Korean Collection for Type Cultures (3 mentions)
Staphylococcus aureus, by Korean Collection for Type Cultures (3 mentions)
Salmonella typhimurium, by Korean Collection for Type Cultures (2 mentions)
Staphylococcus epidermidis, by Korean Collection for Type Cultures (2 mentions)
Pseudomonas aeruginosa, by Korean Collection for Type Cultures (2 mentions)
Acinetobacter baumannii, by Korean Collection for Type Cultures (1 mentions)
Staphylococcus aureus atcc 25923, by American Type Culture Collection (1 mentions)
S aureus, by Korean Collection for Type Cultures (1 mentions)
6 protocols using bacillus subtilis
1
Antimicrobial Susceptibility of Common Pathogens
2
Antimicrobial Susceptibility Screening
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Four Gram-negative bacteria species (Escherichia coli KCTC 1682, Acinetobacter baumannii KCTC 2508, Pseudomonas aeruginosa KCTC 1637 and Salmonella typhimurium KCTC 1926) and three Gram-positive bacteria species (Staphylococcus aureus KCTC 1621, Bacillus subtilis KCTC 3028 and Staphylococcus epidermidis KCTC 1917) were procured from the Korean Collection for Type Cultures (KCTC). Multi-drug-resistant Gram-negative bacteria (Salmonella typhimurium CCARM 8003 and 8007, Escherichia coli CCARM 1229 and 1238, Pseudomonas aeruginosa CCARM 2002 and 2095 and Acinetobacter baumannii CCARM 12035 and 12036) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM). To determine MICs, bacteria were cultured in Luria-Bertani (LB) medium for overnight at 37 °C. An aliquot of the culture was incubated in 1% peptone media at 37 °C until mid-log phase. 100 μl of serial 2-fold diluted peptides were treated in 96 well plates and added to 100 μl of 2 × 106 CFU/ml bacterial suspensions in 1% peptone media for 16 h at 37 °C. The lowest concentration of peptide that completely inhibited bacterial growth was determined to be the MIC.
3
Antibacterial Activity Evaluation of Bacterial Strains
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Pseudomonas aeruginosa (ATCC 15692), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923) were purchased from the American Type Culture Collection. Listeria monocytogens (KCTC 3710) and Bacillus subtilis (KCTC 1998) were from the Korean Collection for Type Cultures. Drug-resistant S. aureus (DRSa-001~005) and P. aeruginosa (DRPa-001~005) were collected from Paik Hospital of Inje University in Busan, South Korea. Pre-cultured bacteria grown to mid-log phase were suspended in 10 mM sodium phosphate (SP, pH 7.2) or phosphate-buffered saline (PBS, pH 7.2) with 10% culture media. Bacterial suspensions were added to each peptide dilute (final 5 × 105 colony-forming units (cfu)/mL), after which the plates were incubated at 37 °C for 24 h. The turbidity of each well was measured at the absorbance at 600 nm and the morphology of bacterial growth was observed under light microscopy. MICs (Minimum inhibitory concentrations) were defined as the lowest concentration of peptide that expresses the complete inhibition of bacterial growth [42 ,43 (link)].
4
Microbial Preculture Protocols for Ginseng Extraction
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Lactobacillus plantarum KCCM 11322 was purchased from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea). Leuconostoc mesenteroides subsp. mesenteroides KCTC 3718 and Bacillus subtilis KCTC 2023 were purchased from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea). Saccharomyces bayanus EC-1118 (Lalvin, Montreal, Canada) was also used for comparison.
Before inoculation with ginseng extracts, strains were precultured at 37°C for 48 h in de Man Rogosa Sharpe broth (Difco, Sparks, MD, USA) for L. plantarum and L. mesenteroides, nutrient broth (Difco, Sparks, MD, USA) for B. subtilis, and yeast malt broth (Difco, Sparks, MD, USA) for S. bayanus to obtain a final cell count of above 107 CFU/mL.
Before inoculation with ginseng extracts, strains were precultured at 37°C for 48 h in de Man Rogosa Sharpe broth (Difco, Sparks, MD, USA) for L. plantarum and L. mesenteroides, nutrient broth (Difco, Sparks, MD, USA) for B. subtilis, and yeast malt broth (Difco, Sparks, MD, USA) for S. bayanus to obtain a final cell count of above 107 CFU/mL.
5
Antibacterial Activity Screening of Peptides
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Bacteria, including E. coli (KCTC 1682), Staphylococcus typhimurium (KCTC 1926), Pseudomonas aeruginosa (KCTC 1637), Bacillus subtilis (KCTC 3068), Staphylococcus epidermidis (KCTC 1917), and S. aureus (KCTC 1621) were procured from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology (Deajeon, South Korea). MRSA strains (CCARM 3089, CCARM 3090, and CCARM 3095) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at the Seoul Women's University (Seoul, South Korea). All bacteria were routinely grown at 37°C in Luria-Bertani broth. MICs were measured in 96-well microplates using the micro-broth dilution method. Bacteria were grown in Luria-Bertani broth at 37°C for 18 h and then diluted with 1% peptone to a final concentration of 2 × 10 6 colonyforming units per ml. Subsequently, 100 µl of a bacterial suspension and 100 µl of peptide solution were mixed together in a 96-well plate. The peptide solution was prepared by performing two-fold dilutions in 1% peptone, while the PGP-E solutions were prepared to final peptide concentrations of 0.06-128 µg ml -1 . The bacteria and peptide solution mixtures were incubated at 37°C for 18 h. The peptide MIC was defined as the lowest concentration of peptide that inhibited bacterial growth.
6
Isolation and Cultivation of Diverse Microbes
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Anabaena variabilis NIES 23 and Scenedesmus acutus NIES 94 were obtained from the National Institute for Environmental Studies (NIES), Japan. M. aeruginosa KW was isolated from freshwater in Korea. The strains were grown in BG-11 liquid medium (pH 7.5) under 120 µmol Photons/m 2 /s 1 provided by cool white fluorescent tubes at 27 ± 1°C. Micrococcus luteus (KCTC 1056), Bacillus subtilis (KCTC 1022), Staphylococcus aureus (KCTC 1621), Staphylococcus aureus (KCTC 1916), and Escherichia coli (KCTC 2443) were obtained from Korean Collection for Type Cultures (KCTC), and Erwinia carotovora was provided by Dr. Seung-Hwan Park (KRIBB, Korea). P. polymyxa E681, which was isolated from the roots of winter barley in the Republic of Korea, was cultured in Katznelson and Lochhead medium (KL medium) [9] .
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