Antibiotic powders were purchased from Sigma-Aldrich, Inc. (St. Louis, MO): ampicillin sodium (product number: A0166), penicillin G potassium salt (product number: 46609), and ceftriaxone sodium (product number: PHR1382). Experiments were performed using cation adjusted (calcium, 25 μg/mL; magnesium, 12.5 μg/mL) Mueller-Hinton broth (MHB; BD Difco, Sparks, MD). All viable cell count samples and subcultures were plated on brain heart infusion agar (BHIA; BD Difco, Sparks, MD) (15 (link), 28 (link), 29 (link)).
Ampicillin sodium
Manufactured by Merck Group
Sourced in Switzerland, Canada, United Kingdom, Spain, United States
Ampicillin sodium is a broad-spectrum antibiotic medication used as a laboratory reagent. It is a crystalline white to off-white powder that is soluble in water. Ampicillin sodium is commonly used in microbiology and molecular biology research settings.
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Lab products found in correlation
Tetracycline hydrochloride, by Merck Group (3 mentions)
Rifampicin, by Merck Group (3 mentions)
Streptomycin sulfate, by Merck Group (2 mentions)
Kanamycin sulfate, by Merck Group (2 mentions)
Ceftriaxone sodium, by Merck Group (1 mentions)
Penicillin g potassium salt, by Merck Group (1 mentions)
Vancomycin hydrochloride, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Filmtracer sypro ruby biofilm matrix stain, by Thermo Fisher Scientific (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Ofloxacin, by Merck Group (1 mentions)
Carbenicillin disodium, by Merck Group (1 mentions)
Cefalexin, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Ciprofloxacine, by Merck Group (1 mentions)
Doxycycline hyclate, by Merck Group (1 mentions)
Phenformin, by Merck Group (1 mentions)
Fluconazole, by Merck Group (1 mentions)
13 protocols using ampicillin sodium
1
Antibiotic Susceptibility Testing Protocol
2
Antibiotic Treatment in Mcl1 Knockout Mice
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Ciprofloxacin (200 mg/L) (Fluka; Buchs, Switzerland), ampicillin sodium (1 g/L) (Sigma; St. Louis, MO), metronidazole (1 g/L) (Fluka), and vancomycin HCl (500 mg/L) (Fluka) were added to the drinking water of cohoused, 1-month-old wild-type control and Mcl1ΔIEC mice for 4 weeks.18 (link)
3
Fluorescent and Bioluminescent Bacterial Assays
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White flat bottom 96-well microplates were purchased from Thermo Scientific. Sodium alginate and calcium chloride (CaCl2), phosphate buffered saline (PBS), cation-adjusted Mueller Hinton broth 2 (CMHB), powered LB broth, and tryptone soy powder were purchased from Sigma Aldrich (St. Louis, MO). Red fluorescent protein tagged E. coli RP437/pRSH103 were obtained from Dr. Dacheng Ren at Syracuse University (Syracuse, NY) [66 (link)]. Green fluorescent protein tagged P. aeruginosa PAO1/pTdK-GFP was obtained from Dr. Joy at The University of Akron (Akron, OH) [67 (link)]. Green fluorescent protein tagged S. aureus AH2547/pCM29 were provided by Dr. Alexander Horswill at The University of Iowa (Iowa City, IA) [68 (link)]. The bacterial plasmids were encoded with fluorescence proteins. Bioluminescent E. coli Xen14 was purchased from Perkin Elmer. The powder form of antibiotics used in the study, including ampicillin sodium, tetracycline hydrochloride, chloramphenicol, vancomycin hydrochloride, and ciprofloxacin were supplied by Sigma Aldrich. LIVE/DEAD™ BacLight™ Bacterial Viability kit, FilmTracer™ SYPRO® Ruby biofilm matrix stain, and FilmTracer™ FM® 1–43 Green biofilm cell stain was purchased from ThermoFisher Scientific (Waltham, MA). All other chemicals can be purchased from major suppliers.
4
Antibiotic Stock Solution Preparation
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Antibiotics: ampicillin sodium, carbenicillin disodium, tetracycline, doxycycline hyclate, kanamycin sulfate, streptomycin sulfate, ofloxacin, cefalexin, ciprofloxacine were purchased from Sigma Aldrich, Oakville, Ontario, Canada.
Antibiotic stock solutions: All antibiotics were dissolved to yield a stock solution of 10 mM. Ampicillin, carbenicillin, tetracycline, doxycycline, kanamycin, streptomycin were dissolved in distilled H2O (pH = 6.5); ofloxacin, ciprofloxacin and cefalexin in an aqueous solution at a pH of 1.5 using HCl.
Antibiotic stock solutions: All antibiotics were dissolved to yield a stock solution of 10 mM. Ampicillin, carbenicillin, tetracycline, doxycycline, kanamycin, streptomycin were dissolved in distilled H2O (pH = 6.5); ofloxacin, ciprofloxacin and cefalexin in an aqueous solution at a pH of 1.5 using HCl.
5
Antibiotic Effects on Drosophila Gut Microbiome
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Wolb+ and Wolb− flies were raised at 25°C, collected as 0- to 1-day-old adults, and placed on fly food with 100 mg/ml of tetracycline hydrochloride, rifampicin, ampicillin sodium, or streptomycin sulfate (all from Sigma) or control food with antibiotic solvent (water or ethanol). At day 10 of treatment, flies were challenged with DCV and placed at 18°C, and survival was followed for an additional 20 days. Food vials were changed every 3 days. At day 0, day 10, and day 30, flies were collected to assay Wolbachia levels by qPCR. At day 31, guts from three Wolb+ flies per condition were dissected to assess the effect of antibiotics on the gut-associated bacteria. Guts were homogenized in 250 μl of sterile LB, and 30 μl was plated on mannitol agar plates. This medium sustains growth of the main gut-associated bacteria in lab D. melanogaster , Acetobacter , and Lactobacillus species (72 (link)). CFU were counted after 5 days of incubation at 26°C.
6
Antifungal and Biguanide Compound Testing
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Metformin hydrochloride was purchased from Cayman Chemicals. Fluconazole, ampicillin sodium, rapamycin, and other biguanide hydrochlorides including phenformin and buformin were purchased from Sigma. Clinical grade formulations of the antifungal agents voriconazole, amphotericin B deoxycholate, and micafungin were purchased from the Massachusetts General Hospital (MGH) clinical pharmacy. All reagents were dissolved in distilled water or DMSO according to the manufacturer’s recommendation, and stored at −20°C. Cell culture media include liquid YPD (1% yeast extract, 2% peptone, 2% dextrose); YPD agar (1% yeast extract, 1% peptone, 2% dextrose, 2% agar); RPMI-MOPS (RPMI 1640 containing 2% glucose and 0.165M MOPS, buffered at pH7); and complete RPMI (RPMI 1640 w/2mM L-glutamine, 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin).
7
Broad-Spectrum Antibiotic Treatment for Toxoplasmosis
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The broad-spectrum antibiotic treatment regimen was followed as described by Hand et al. (2012 (link)) with modifications. Briefly, each animal received a daily combination of 5 mg neomycin trisulfate (Sigma), 2.5 mg vancomycin (Sigma), 5 mg metronidazole (sigma) and 5 mg ampicillin sodium (sigma) diluted in 200 μL water via oral gavage for 2 weeks before and 2 weeks post-infection with T. gondii.
8
Antibiotic Effects on Gut Microbiome
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Wolb+ and Wolb-flies were raised at 25°C, collected as 0-1 days old adults and placed on fly food with 100 mg/ml of tetracycline hydrochloride, rifampicin, ampicillin sodium or streptomycin sulphate (all Sigma) or control food with antibiotics solvent (water or ethanol). At day 10 of treatment, flies were challenged with DCV, placed at 18°C and survival was followed for additional 20 days. Food vials were changed every three days. At day 0, day 10 and day 30 flies were collected to assay Wolbachia levels by qPCR. At day 31, guts from three Wolb+ flies per condition were dissected to assess the effect of antibiotics on the gut-associated bacteria. Guts were homogenized in 250 µl of sterile LB and 30 µl was plated on mannitol agar plates. This medium sustains growth of the main gut-associated bacteria in lab D. melanogaster, Acetobacter and Lactobacillus species (47).
CFUs were counted after five days of incubation at 26°C.
CFUs were counted after five days of incubation at 26°C.
9
Antimicrobial Agent Preparation and Characterization
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Commercially procured powders of quinine sulfate (Sigma-Aldrich, United Kingdom), chloroquine phosphate (Sigma-Aldrich, United Kingdom), ampicillin sodium (Merck, United Kingdom), cloxacillin sodium monohydrate (Sigma-Aldrich, Germany), and nalidixic acid (Merck, United Kingdom) were used in the study. Test antibiotic solutions were prepared as outlined in the Clinical and Laboratory Standards Institute (CLSI) guideline. All test compounds were dissolved in water except nalidixic acid which was dissolved in 0.1 N sodium hydroxide (O’Neil, 2001 ). Stock solutions were prepared at 10 mg/mL for quinine and chloroquine and 1 mg/mL for ampicillin, cloxacillin, and nalidixic acid (Wiegand et al., 2008 (link); Clinical and Laboratory Standards Institute [CLSI], 2018 ). Fresh stock solutions of ampicillin, cloxacillin and nalidixic acid was made for each experiment.
10
Antibiotic Susceptibility Profiling of M. abscessus
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Select edited M. abscessus strains were further phenotypically verified for loss of function by determining MICs against ampicillin sodium (EMD Millipore), rifampicin (Sigma-Aldrich), and capreomycin sulfate (Sigma-Aldrich). Strains were grown to optical density at wavelength 600 nm (OD600) ~0.8–1.0 in media supplemented with KAN and HYG. Cultures were diluted to 100 µL of serially diluted antibiotics in 96-well clear bottom plates (Corning) (final OD600 = 0.0025) in four technical replicates. One percent dimethylsulfoxide (DMSO) (Sigma-Aldrich) was used as the negative untreated control, and 25 µg/mL ciprofloxacin hydrochloride (MP Biomedicals) was used as the positive control. Plates were incubated at 37°C without shaking for approximately 4 days before OD600 measurements. Growth (%) was calculated by normalizing OD600 from treatment wells with OD600 from DMSO control wells and plotted using GraphPad Prism 9.4.0. MIC values were taken as the minimum concentration needed to inhibit growth by 90%.
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