Unless otherwise specified, E. faecalis strains OG1RF and its derivatives were grown overnight on Brain Heart Infusion (BHI) broth (BD Company) supplemented with rifampin (25 μg/ml) (Sigma-Aldrich) and fusidic acid (25 μg/ml) (Sigma-Aldrich) and were inoculated from a single bacterial colony grown on BHI agar plates supplemented with rifampin and fusidic acid. Liquid cultures were grown statically at 37°C for 18 hours. E. coli strains were grown in LB broth or agar (Becton, Dickinson and Company) supplemented with ampicillin (100 μg/ml) and kanamycin (100 μg/ml). Bacterial strains are listed in table S1 .
Rifampin
Manufactured by Merck Group
Sourced in United States, Germany, Spain, Macao
Rifampin is a laboratory product manufactured by Merck Group. It is a chemical compound used in various scientific and research applications. Rifampin is a widely used antibiotic that has been extensively studied and utilized in various fields of research.
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Lab products found in correlation
Isoniazid, by Merck Group (22 mentions)
Vancomycin, by Merck Group (9 mentions)
Gentamicin, by Merck Group (8 mentions)
Ciprofloxacin, by Merck Group (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Erythromycin, by Merck Group (6 mentions)
Chloramphenicol, by Merck Group (6 mentions)
Ampicillin, by Merck Group (6 mentions)
Phosphomycin, by Merck Group (5 mentions)
Bsk 2 medium, by Merck Group (5 mentions)
Amphotericin b, by Merck Group (5 mentions)
Tetracycline, by Merck Group (5 mentions)
Kanamycin, by Merck Group (5 mentions)
Streptomycin, by Merck Group (5 mentions)
Levofloxacin, by Merck Group (4 mentions)
Clofazimine, by Merck Group (4 mentions)
Pyrazinamide, by Merck Group (4 mentions)
Omeprazole, by Merck Group (4 mentions)
Ethambutol, by Merck Group (4 mentions)
Miconazole, by Merck Group (3 mentions)
115 protocols using rifampin
1
Culturing Enterococcus faecalis and Escherichia coli
2
Isolation and Cultivation of Thauera aminoaromatica
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The T. aminoaromatica MZ1T wild-type strain was originally isolated from the wastewater treatment plant of the Eastman Chemical Company in Kingsport, TN (7 (link)). A rifampin-resistant (Rifr) mutant of MZ1T was isolated by repeated plating on Stoke's agar plates (1.5% Bacto agar in Stoke's broth [5 g polypeptone, 0.2 g MgSO4·7H2O, 0.15 g Fe NH4SO4), 0.1 g sodium citrate, 0.05 g CaCl2, 0.05 g MnSO4, 0.01 g FeCl3·6H2O]) amended with 100 μg/ml rifampin (Sigma) and was used for all studies. T. aminoaromatica MZ1T wild type and mutants defective in floc formation were routinely grown in Stoke's broth. The medium was sterilized by autoclaving, and the following filter-sterilized vitamin solutions were added to yield the indicated final concentrations: cyanocobalamin, 0.5 mg/liter; thiamine hydrochloride, 0.4 mg/liter; and biotin, 0.4 mg/liter. Otherwise, Thauera defined medium (TDM), modified from that used by Rabus and Widdel (8 (link)), was used for growing the MZ1T and mutant strains for EPS extraction and purification. All MZ1T strains were grown aerobically at 30°C in a shaking incubator at 225 rpm.
3
Antibiotic Susceptibility Screening
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Ofloxacin, kanamycin, rifampin, tetracycline, and gentamicin were obtained from Sigma-Aldrich Chemical Co., and were used at 5, 30, 100, 50, 20 μg/ml, respectively. The Escherichia coli single gene deletion mutant library Keio collection and the parent strain BW25113 were used for the screen (see below). All experiments were conducted at 37°C in Luria-Bertani (LB) medium.
4
Antibiotic Sensitivity of M. tuberculosis
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Middlebrook 7H11 plates were prepared to contain rifampin (Sigma; 0.1 μg/ml) or 0.2 μg/ml isoniazid (Sigma; 0.2 μg/ml). Phage lysate diluted to 105 PFU in 0.1 ml was spread onto 7H11 plates with or without antibiotics and allowed to dry in a laminar flow biosafety cabinet; 0.1 ml of an M. tuberculosis H37Rv culture was then spread into plates and incubated for 6 weeks at 37°C.
5
Antibiotic Susceptibility of MDR Isolates
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Twenty-three MDR clinical isolates, of which eight were E. coli, seven were P. aeruginosa, and seven were A. baumannii, which differed in their clinical origins and were epidemiologically unrelated, were used in this study. The control strains used were E. coli ATCC 25922, P. aeruginosa ATCC 27853 and PAO1, and A. baumannii ATCC 17978. The strains were cultured 24 h at 37 °C in tryptone soy agar, tryptone soy broth, and Mueller–Hinton broth, all of which were purchased from Sharlau (Sentmenat, Barcelona, Spain).
In this study, three different antibiotic substances were used. Colistin was kindly supplied by Zhejiang Shenghua Biok Biology Co., Ltd., (Shangai, China). Linezolid and rifampin were purchased from Sigma–Aldrich Chemicals (Madrid, Spain).
In this study, three different antibiotic substances were used. Colistin was kindly supplied by Zhejiang Shenghua Biok Biology Co., Ltd., (Shangai, China). Linezolid and rifampin were purchased from Sigma–Aldrich Chemicals (Madrid, Spain).
6
Cultivating E. coli in Minimal Medium
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We grew E. coli cultures in liquid M9 minimal medium with 1% glucose and a mixture of amino acids (10 µg/ml each) (21 (link)). We plated cells from these cultures on rich LB agar plates as we have described previously (21 (link)). We isolated plasmids from cells grown in LB medium.
We obtained IPTG (isopropyl-β-d -thiogalactopyranoside), phosphate-buffered saline (PBS), rifampin, and trypsin from Sigma (St. Louis, MO). We obtained ampicillin from Biochemie GmbH (Kundl, Austria). We purchased primers for cloning and mutant generation from Hy-labs (Rehovot, Israel) and from Integrated DNA Technologies (IDT, Hudson, NH, USA). We used a chemically synthesized EDF peptide (98% purity) synthesized by GenScript Corporation (Piscataway, NJ). We purchased our plasmid DNA isolation kit from Qiagen (Hilden, Germany), avian myeloblastosis virus (AMV) reverse transcriptase from Promega (Madison, WI, USA), an AmpliScribe T7-Flash transcription kit from Epicenter (Madison, WI, USA), a C-18 Sep-Pak cartridge from Waters (Milford, MA, USA), and methanol and acetonitrile (CH3CN) from BioLab (New York, NY, USA).
We obtained IPTG (isopropyl-β-
7
Measuring mRNA Decay Kinetics
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Cultures from strains NA1000 (wt), rho::Tn5, and rhlE::Tn5 were grown in M2 medium at 30°C with agitation until the OD600 value reached 0.5. Samples (1 ml) were centrifuged for 1 min; the pellets were resuspended in 0.5 ml of TRIzol Reagent (Invitrogen Life Technologies) and frozen in a dry ice/ethanol bath (t0). Then, 200 μg/ml rifampin (Sigma-Aldrich) was added to each culture kept at 30°C with agitation; samples were taken at several time points and immediately treated and frozen in the same manner. Determination of the mRNA decay at 10°C was carried out in the same way, except that the cultures were incubated for 2 h at 10°C before taking the t0 aliquot and adding rifampin.
Total RNA was isolated and converted to cDNA as described above. The mRNA decay was assessed by RT-qPCR using equal amounts of cDNA for each point, with primer pairs for the CCNA_02070 and kdpA genes (Table S3).
Total RNA was isolated and converted to cDNA as described above. The mRNA decay was assessed by RT-qPCR using equal amounts of cDNA for each point, with primer pairs for the CCNA_02070 and kdpA genes (Table S3).
8
Multi-drug Resistant Acinetobacter Evaluation
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All work was carried out under biosafety level II or II+ conditions. All the A. baumannii strains used in this study can be found in Table 1 . Routine growth and strain maintenance was carried out in Luria-Bertani Lennox (LB) broth and agar. Bacterial identification, antibiograms, and MIC were determined using the Vitek 2 (bioMérieux, France) and Phoenix (Becton, Dickinson and Co., Franklin Lakes, NJ) automated systems according to the manufacturer’s instructions. Rifampin was obtained from Sigma-Aldrich (St. Louis, MO), prepared in dimethyl sulfoxide (DMSO), and then further diluted in sterile saline.
9
Coxiella burnetii Growth and Antibiotic Preparation
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The Coxiella burnetii strain used in this study was Nine Mile Phase 2, clone 4 (NMP2), a low virulence strain that has a large genomic deletion that removes genes responsible for the complexity of LPS side chains29 (link). Stocks of NMP2 were prepared by growth in ACCM-2 media at pH 4.75, and freezing in sodium phosphate glutamate (SPG) buffer. Antibiotics were purchased as follows: doxycycline hyclate, ciprofloxacin, rifampin, hydroxychloroquine, erythromycin, and levofloxacin (Sigma, St. Louis, MO), azithromycin dihydrate, and moxifloxacin hydrochloride (United States Pharmocopeia, Rockville, MD). doxycycline hyclate, moxifloxacin hydrochloride, ciprofloxacin, and hydroxychloroquine stocks were prepared by dissolving in deionized water at a concentration of 1280 μg/ml. levofloxacin was dissolved in an aqueous solution of 0.05 M NaOH at 1280 μg/ml. erythromycin and azithromycin were dissolved in 95% ethanol at 1280 μg/ml.
10
First-Line Anti-TB Drug Susceptibility
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According to the WHO instructions (2018 ), the susceptibility of isolates to first-line anti-TB drugs was tested using the proportion method on the LJ medium. In brief, critical concentrations of 0.2, 40, and 2 µg/ml were prepared for isoniazid, rifampin, and ethambutol (Sigma-Aldrich, Germany), respectively. Twenty-nine cultures that showed 1% or more growth on a drug-containing medium compared with the growth on a control without the same drug were considered resistant. The M. tuberculosis H37Rv was used for quality control.
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