Suvi Manner, Darla M Goeres, Malena Skogman, Pia Vuorela, Adyary Fallarero. Supplementary information. Supplementary table S1. MBIC values determined ...
Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors: critical factors influencing outcomes Suvi Manner, Darla M Goeres, Malena Skogman, Pia Vuorela, Adyary Fallarero Supplementary information Supplementary table S1. MBIC values determined on S. aureus ATCC 25923 by resazurin and crystal violet staining, respectively. The selected antibiotics are marked in bold. Antibiotics were tested in two replicates, three times (three biological replicates). MBIC Antibiotic Penicillin G Clindamycin Rifampicin Doxycycline Tetracycline Oxacillin Ampicillin Dicloxacillin Levofloxacin Vancomycin Fusidic acid Linezolid Ciprofloxacin Erythromycin Tobramycin Trimethoprim Teicoplanin Azithromycin Kanamycin Neomycin Streptomycin Polymyxin B Chloramphenicol Daptomycin Amphotericin B Gramicidin A Metronidazole N/A = not available NO = no inhibition
Viability mg/L 0.0156 0.0313 0.0313 0.125 0.25 0.25 0.25 0.5 0.5 2 2 2 4 4 8 8 8 8 16 32 32 64 128 1024 NO NO NO
Biomass µM 0.04 0.07 0.04 0.12 0.52 0.52 0.62 0.98 1.38 1.37 3.71 5.93 12.07 5.46 17.11 27.56 4.26 10.68 27.46 35.21 43.91 46.19 396.16 631.83 NO NO NO
mg/L N/A 0.0313 0.0625 0.25 0.125 0.125 0.125 0.5 0.5 N/A 2 2 2 2 4 4 8 8 16 16 N/A 32 128 512 NO NO NO
µM N/A 0.07 0.08 0.24 0.26 0.30 0.31 0.98 1.38 N/A 3.71 5.93 6.04 2.73 8.56 13.78 4.26 10.68 27.46 17.60 N/A 23.09 396.16 315.91 NO NO NO
Supplementary table S2. Effects on pre-formed S. aureus ATCC 25923 biofilms using resazurin staining assay. Antibiotics were tested in two replicates, three times (three biological replicates).
Antibiotic Rifampicin Penicillin G Dicloxacillin Oxacillin Clindamycin Ampicillin Fusidic acid Tetracycline Doxycycline Vancomycin Linezolid Ciprofloxacin Tobramycin Teicoplanin Neomycin Erythromycin Kanamycin Levofloxacin Chloramphenicol Polymyxin B Amphotericin B Azithromycin Daptomycin Gramicidin A Metronidazole Streptomycin Trimethoprim NO = no inhibition
Effects on pre-formed biofilms Viability Inhibition-% mg/L µM 0.04 ⩾0.0313 ∼50 ⩾0.0625 0.18 ∼60 ⩾0.125 0.24 ∼50 0.125 0.30 ∼50 ⩾0.25 0.59 ∼50 1.24 ⩾0.5 ∼60 ⩾1 1.86 ∼60 ⩾2 4.16 ∼50 3.90 ⩾4 ∼60 ⩾8 5.38 ∼50 ⩾16 47.42 ∼60 48.29 ⩾16 ∼60 ⩾16 34.22 ∼50 17.04 ⩾32 ∼60 ⩾32 35.21 ∼50 ⩾32 43.60 ∼60 54.93 ⩾32 ∼60 ⩾32 88.55 ∼50 792.32 ⩾256 ∼70 369.51 ⩾512 ∼60 NO NO NO NO NO NO NO NO NO NO NO NO NO NO -
Supplementary table S3. MBIC values of the top antibiotics determined on S. aureus Newman and effects on pre-formed biofilms using resazurin and crystal violet staining assays, respectively. Antibiotics were tested in two replicates, three times (three biological replicates). MBIC Antibiotic Clindamycin Dicloxacillin Rifampicin Penicillin Doxycycline Levofloxacin Oxacillin Ampicillin Tetracycline Vancomycin
Viability mg/L 0.0313 0.0625 0.125 0.125 0.25 0.25 0.5 0.5 1 32
µM 0.07 0.12 0.15 0.35 0.24 0.69 1.18 1.24 2.08 21.54
Biomass mg/L 0.0625 0.0625 0.125 0.125 0.25 0.25 0.5 0.5 1 32
µM 0.15 0.12 0.15 0.35 0.24 0.69 1.18 1.24 2.08 21.54
Effects on pre-formed biofilms InhibitionViability % mg/L µM 2.35 ⩾1 ∼70 1.96 ⩾1 ∼70 0.04 ⩾0.0313 ∼60 44.90 ⩾16 ∼60 0.97 ⩾1 ∼70 88.55 ⩾32 ∼50 37.79 ⩾16 ∼70 39.66 ⩾16 ∼60 16.64 ⩾8 ∼50 86.15 ⩾128 ∼70
Supplementary table S4. MBIC values of the top antibiotics determined on S. epidermidis ATCC 35984 and effects on pre-formed biofilms using resazurin and crystal violet staining assays, respectively. Antibiotics were three times (three biological replicates). MBIC Antibiotic Rifampicin Penicillin Dicloxacillin Clindamycin Oxacillin Doxycycline Ampicillin Levofloxacin Tetracycline Vancomycin
Viability mg/L 0.0156 0.125 0.25 0.25 0.5 0.5 0.5 0.5 2 16
µM 0.02 0.35 0.49 0.59 1.18 0.49 1.24 1.38 4.16 10.77
Biomass mg/L 0.0156 0.125 0.25 0.25 0.5 0.5 0.5 0.5 2 16
µM 0.02 0.35 0.49 0.59 1.18 0.49 1.24 1.38 4.16 10.77
Effects on pre-formed biofilms InhibitionViability % mg/L µM 0.04 ⩾0.0313 ∼50 0.35 ⩾0.125 ∼55 1.96 ⩾1 ∼50 4.71 ⩾2 ∼55 2.36 ⩾1 ∼55 0.97 ⩾1 ∼55 4.96 ⩾2 ∼55 88.55 ⩾32 ∼40 4.16 ⩾2 ∼65 43.08 ⩾64 ∼40
Supplementary table S5. Summary of results reported as log reductions (LR) (mean ± SD) obtained with each test concentration of tested antibiotics in MWP. All the antibiotics were tested in duplicates at least three times (three biological replicates). Effect (LR ± SD) of antibiotics Antibiotic 0.1 Rifampicin 0.36 ± 0.09 Oxacillin 0.35 ± 0.09 Ampicillin 0.15 ± 0.14 Dicloxacillin 0.09 ± 0.17 Levofloxacin 0.00 ± 0.17 Doxycycline 0.05 ± 0.19 Clindamycin -0.11 ± 0.14 Tetracycline 0.17 ± 0.15 Penicillin G 0.09 ± 0.05 Vancomycin 0.09 ± 0.04
Test concentration (µM) 1 10 3.90 ± 0.77 5.45 ± 0.27 1.39 ± 0.33 5.42 ± 0.28 0.78 ± 0.19 5.02 ± 0.29 0.84 ± 0.04 3.90 ± 0.17 0.19 ± 0.10 0.82 ± 0.12 0.40 ± 0.38 2.46 ± 0.24 0.02 ± 0.09 3.95 ± 0.18 0.31 ± 0.15 0.83 ± 0.21 1.54 ± 0.53 1.90 ± 0.21 0.27 ± 0.08 0.35 ± 0.11
100 7.31 ± 0.20 7.20 ± 0.21 5.30 ± 0.14 5.54 ± 0.40 6.89 ± 0.05 4.68 ± 0.40 4.34 ± 0.17 4.19 ± 0.26 2.91 ± 0.22 0.59 ± 0.11
Supplementary table S6. Summary of results reported as log reductions (LR) (mean ± SD) obtained with selected antibiotics at 100 µM in MWP, when washing step was included. Antibiotics were tested in duplicates, three times. Effect (LR) of antibiotics Antibiotic
Test concentration (µM) 100
Rifampicin
6.85 ± 0.16
Oxacillin
6.87 ± 0.15
Doxycycline
4.04 ± 0.38
The mean log density of untreated control biofilms was 7.84 ± 0.12.
Supplementary table S7. List of antibiotics included in this study. Antibiotic Ampicillin Dicloxacillin sodium salt monohydrate Oxacillin sodium salt Penicillin G sodium salt Polymyxin B Teicoplanin Vancomycin Doxycycline hyclate Tetracycline Kanamycin Neomycin Streptomycin Tobramycin Azithromycin Erythromycin Chloramphenicol Clindamycin Linezolid Fusidic acid Trimethoprim Ciprofloxacin Levofloxacin Metronidazole
Supplier Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Cayman chemicals, Ann Arbor, MI, US Sigma-Aldrich Co, St. Louis, MO, US Cayman chemicals, Ann Arbor, MI, US Cayman chemicals, Ann Arbor, MI, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Cayman chemicals, Ann Arbor, MI, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Cayman chemicals, Ann Arbor, MI, US Cayman chemicals, Ann Arbor, MI, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US
Class β-lactam
Mechanism of action Inhibition of cell wall biosynthesis
β-lactam β-lactam β-lactam Polymyxin Glycopeptide Glycopeptide Tetracycline
Protein synthesis inhibition
Tetracycline Aminoglycoside Aminoglycoside Aminoglycoside Aminoglycoside Macrolide Macrolide Lincosamide Lincosamide Oxazolidinone Fucidane Dihydrofolate reductase inhibitor Fluoroquinolone Fluoroquinolone Nitroimidazole
Folic acid synthesis inhibition DNA synthesis inhibition
Rifampicin Amphotericin B Daptomycin Gramicidin A
Sigma-Aldrich Co, St. Louis, MO, US Sigma-Aldrich Co, St. Louis, MO, US
Rifamycin
Cayman chemicals, Ann Arbor, MI, US Sigma-Aldrich Co, St. Louis, MO, US
Lipopeptide
Polyene
Peptide
RNA synthesis inhibition Targets the cell membrane
