Mycobacterium smegmatis mc2 155 was used as a drug-susceptible strain for all experiments. The MDR Mycobacterium smegmatis strains (referred to as erythromycin-resistant Mycobacterium smegmatis A and B) were generated in this study, under in vitro conditions, and were used as drug-resistant strains. Middlebrook 7H10 agar base (Sigma Aldrich) and Middlebrook 7H9 broth base (Sigma Aldrich) were used for preparing agar plates and liquid broth, respectively, according to the manufacturer’s instructions. The M7H10 agar base was supplemented with 0.085% NaCl and 0.5% dextrose and the M7H9 broth base was supplemented with 0.085% NaCl, 0.44% glycerol, and 0.25% Tween 80. All commercially available antibiotics that were used for this study are listed in Tables S2 and S3 . The amount or concentration of each antibiotic used for specific assays are indicated in the text of the results.
Middlebrook 7h9 broth base
Manufactured by Merck Group
Sourced in United States
Middlebrook 7H9 broth base is a culture medium used for the growth and cultivation of mycobacteria, including the causative agent of tuberculosis, Mycobacterium tuberculosis. It provides the necessary nutrients and growth factors required for the optimal growth of these slow-growing bacteria.
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Lab products found in correlation
Middlebrook 7h10 agar base, by Merck Group (2 mentions)
Glycerol, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
Bacitracin, by Merck Group (1 mentions)
Sputasol, by Thermo Fisher Scientific (1 mentions)
Columbia blood agar base, by Thermo Fisher Scientific (1 mentions)
N methylpyrrolidine, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Phosphate buffered saline tablet, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Nh4pf6, by Merck Group (1 mentions)
Dextrose, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Isoniazid inh, by Merck Group (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Dmso d6, by Cambridge Isotopes (1 mentions)
7 protocols using middlebrook 7h9 broth base
1
Generation of Antibiotic-Resistant Mycobacterium Strains
2
Antimicrobial Susceptibility Testing of CF Isolates
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Brain heart infusion broth (CM1136), brain heart infusion agar (CM1135), Columbia blood agar base (CM0331), CLED medium with Andrades indicator (CM0423), Sabouraud dextrose agar (CM0041), bacteriological agar no. 1 (LP001B), and Sputasol (SR0233A) were all purchased from Oxoid, Basingstoke, UK. Bacitracin (11072), chloramphenicol (C0378), Middlebrook 7H9 broth base (M0178), glycerol (G5516), 85 μm polyacrylate solidphase micro-extraction fibres, and N-methyl pyrrolidine (NMP) (328634) were purchased from Sigma, Gillingham, UK. Defribrinated horse blood (5% v/v, HB035) was purchased from TCS biosciences, Buckingham, UK. Burkholderia cepacia selective agar (PP0160) was purchased from EO laboratories, Bonnybridge, UK. Yeast extract (03904110) was purchased from bioMérieux, Marcy-l'Étoile, France. All bacteria used in this study were acquired from the Freeman Hospital Microbiology Department, Newcastle upon Tyne, UK. All P. aeruginosa strains (except NCTC 12903) were all strains isolated from patients with cystic fibrosis globally. All Burkholderia strains were part of the Burkholderia cepacia complex.
3
Mycobacterium tuberculosis Strain Preparation
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A multidrug-resistant (MDR) strain of M. tuberculosis from the collection kept at the Reference Laboratory for Mycobacteria at the INEI-ANLIS “Carlos G. Malbrán” in Buenos Aires and identified on the basis of their spoligotype IS6110 RFLP fingerprint pattern was used: outbreak M strain which belongs to the Haarlem family (isolate 6548). Besides, the reference H37Rv strain (T family) kindly provided by I.N. de Kantor (TB laboratory, INPPAZ PAHO/WHO) was used. Both strains were grown in Middlebrook 7H9 broth base (Sigma Aldrich, St. Louis, MO, USA) with ADC enrichment in agitation for 15–21 days at 37°C in 5% CO2 humidified atmosphere until log phase, clumps were disaggregated using glass beads and after 2 h of settling, and the supernatant was harvested, aliquoted, and stored at −80°C until use. In some experiments, H37Rv bacteria were killed by gamma irradiation (2.4 Mrads from a 137Cs source) and sonicated and suspended in pyrogen-free phosphate-buffered saline (PBS) 1X at an optical density of 1 OD 600 nm (108 bacilli/ml approximately).
4
Synthesis and Characterization of Ruthenium Complex
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Cis-[Ru(bpy)2Cl2] (bpy = 2,2′-bipyridine) was synthesised as described.26 Isoniazid (INH) and NH4PF6 were purchased from Sigma-Aldrich and used without further purification. The solvents used for UV-visible absorption spectroscopy were reagent grade. The NMR spectroscopy solvent DMSO-d6 was purchased from Cambridge Isotope Laboratories Inc and D2O was purchased from Sigma-Aldrich. Middlebrook 7H9 Broth Base, glycerol, dextrose, sodium chloride, bovine serum albumin, Tween® 80, phosphate-buffered saline tablets and tryptic soy broth were purchased from Sigma Aldrich. The bacteria, B. subtilis 168 and E. coli C43 (DE3), were kindly provided by Mrs Anne Smith from the Chemical Biology Laboratory, Department of Chemistry, University of Warwick. The M. smegmatis was obtained from the National Collection of Type Cultures (NCTC). The normal lung fibroblast cell line (MRC-5) was purchased from ATCC and cultured in DMEM medium (Gibco) supplemented with FCS (10%; Gibco), penicillin (100 U mL–1), and streptomycin (100 mg mL–1) in a humidified atmosphere at 37 °C and 5% CO2.
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Cis-[Ru(bpy)2Cl2] (bpy = 2,2′-bipyridine) was synthesised as described.26 Isoniazid (INH) and NH4PF6 were purchased from Sigma-Aldrich and used without further purification. The solvents used for UV-visible absorption spectroscopy were reagent grade. The NMR spectroscopy solvent DMSO-d6 was purchased from Cambridge Isotope Laboratories Inc and D2O was purchased from Sigma-Aldrich. Middlebrook 7H9 Broth Base, glycerol, dextrose, sodium chloride, bovine serum albumin, Tween® 80, phosphate-buffered saline tablets and tryptic soy broth were purchased from Sigma Aldrich. The bacteria, B. subtilis 168 and E. coli C43 (DE3), were kindly provided by Mrs Anne Smith from the Chemical Biology Laboratory, Department of Chemistry, University of Warwick. The M. smegmatis was obtained from the National Collection of Type Cultures (NCTC). The normal lung fibroblast cell line (MRC-5) was purchased from ATCC and cultured in DMEM medium (Gibco) supplemented with FCS (10%; Gibco), penicillin (100 U mL–1), and streptomycin (100 mg mL–1) in a humidified atmosphere at 37 °C and 5% CO2.
5
Macrophage Polarization Induction Protocol
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glycerol tripalmitate was purchased from Thermo Fisher Scientific (MA, USA). Rifampicin (RIF) was purchased from Tokyo Chemical Industry (TCI, Japan). Octadecylamine (SA), d -(+)-mannose, Tween 80, SpectraPor® 12–14 kDa dialysis membranes, Triton X-100, phorbol myristate acetate (PMA), Middlebrook 7H9 broth base, Middlebrook 7H10 agar base, oleic acid, bovine serum albumin, bovine liver catalase, and glycerol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dextrose and sodium chloride were purchased from Duksan general science (Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from VWR Life Sciences (Radnor, PA, USA). Interleukin-4 (IL-4) and interleukin-13 (IL-13) used for macrophage differentiation and polarization were obtained from PeproTech (Cranbury, NJ, USA).
6
Ayurvedic Herbal Formulation for Antimicrobial Efficacy
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The raw materials like Zingiber officinale, Piper longum, Piper nigrum, Dryopteris filix-mas, Aconitum ferox, and red orpiment were procured from Rajan & Co., Chennai, Tamil Nadu, India. The herbals used were identified and authenticated by Dr. Ravichandran, Botanist, SASTRA Deemed University, Thanjavur by standard pharmacognosy screening procedures and the herbarium of the same has been archived at the department (Herbarium no: CARISM 00157-00161). The experimentation using plants was performed in accordance with the International Union for Conservation of Nature (IUCN). Moreover, the plants used were not categorized in the IUCN endangered or extinct species list. Sulphur, Mercury, Borax, Mueller-Hinton Agar, Middlebrook 7H9 broth base, and Alamar reagent were obtained from Sigma-Aldrich, Mumbai, and verified for their identity and purity. All the chemicals and reagents used were of analytical grade.
7
Mycobacterium tuberculosis Suspension Preparation
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3–4 drops of 0.50% Tween 80 and resistant MTB colonies grown on L-J medium for 3–4 weeks were placed into a thick large glass tube. After grinding, 6–8 drops of physiological saline were dropped into the tube and then stood to precipitate large particles of bacteria. Finally, supernatants were aspirated, and Middlebrook 7H9 Broth Base (Sigma, San Francisco, California, United States) was used to adjust the supernatants to 3.0 × 108 CFU/ml.
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