Metronidazole (M1547; Sigma-Aldrich; final concentration 5 mM) was dissolved in water, the relatively high concentration of Metronidazole seems to be a function of bioavailability in zebrafish as higher concentrations are routinely required for nitroreductase mediated cellular ablation studies38 (link). Pazopanib (sc-364564; Santa Cruz Biotechnology; final concentration 250 nM for larvae, 1 μM for adults) and SU5416 (S8442; Sigma-Aldrich; final concentration 250 nM) were dissolved in DMSO. Metronidazole, Pazopanib, SU5416 were added immediately after infection and refreshed every two days for the duration of the experiment. Randomisation into drug treatment groups was achieved by random selection of infected zebrafish from a single pool prior to addition of drugs.
Metronidazole
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, India, Japan, Switzerland, Spain, Australia, Czechia, Sao Tome and Principe, Brazil
Metronidazole is a synthetic antimicrobial agent. It is a white to pale yellow crystalline powder that is slightly soluble in water and freely soluble in alcohol, acetone, and dimethyl sulfoxide. Metronidazole can be used as a reference standard or in the manufacture of pharmaceutical preparations.
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Lab products found in correlation
Vancomycin, by Merck Group (173 mentions)
Ampicillin, by Merck Group (166 mentions)
Neomycin, by Merck Group (109 mentions)
Neomycin sulfate, by Merck Group (47 mentions)
Gentamicin, by Merck Group (39 mentions)
Ciprofloxacin, by Merck Group (35 mentions)
Kanamycin, by Merck Group (30 mentions)
Colistin, by Merck Group (26 mentions)
Amoxicillin, by Merck Group (16 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (16 mentions)
Clindamycin, by Merck Group (15 mentions)
Streptomycin, by Merck Group (14 mentions)
Clarithromycin, by Merck Group (13 mentions)
Levofloxacin, by Merck Group (10 mentions)
Vancomycin, by MP Biomedicals (9 mentions)
Ceftriaxone, by Merck Group (8 mentions)
Tetracycline, by Merck Group (8 mentions)
Amphotericin b, by Merck Group (7 mentions)
Fluconazole, by Merck Group (7 mentions)
Doxycycline, by Merck Group (7 mentions)
451 protocols using metronidazole
1
Zebrafish Drug Treatment Protocol
2
Antimicrobial Phytochemical Evaluation
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Phytochemicals (berberine chloride, 8-hydroxyquinoline, salicylic acid, tannic acid, and sanguinarine chloride) and their synthetic analogs [chloroxine (5,7-dichloroquinolin-8-ol), nitroxoline (5-nitroquinolin-8-ol), ferron (7-iodo-8-hydroxyquinoline-5-sulfonic acid), bismuth subsalicylate, and zinc pyrithione], as well as antibiotics (ceftriaxone sodium, ciprofloxacin, chloramphenicol, metronidazole, tetracycline, and vancomycin hydrochloride), used in this study were purchased from Sigma-Aldrich (Prague, Czech Republic). Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Prague, Czech Republic) was used to prepare the stock solutions of all test compounds, except those of metronidazole, salicylic acid, vancomycin, and zinc pyrithione, which were prepared using distilled water. Stock solutions of chloramphenicol, tannic acid, and tetracycline were prepared using 96% ethanol (Sigma-Aldrich, Prague, Czech Republic). The chemical structures of individual compounds tested are shown in Figure 3 .
3
Zebrafish Prey Capture Behavior
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Individual 7-8 dpf zebrafish larva were head-mounted in 2% low-melting-point agarose (Fisher Scientific, BP1360-100) in a 35 mm Petri dish with the eyes and tail free to move and filmed under infrared illumination (940 nm) using a Raspberry Pi camera at 30 Hz based on a previous design (Maia Chagas et al., 2017 (link)). An Arduino-microcontroller was used to iteratively switch top-illumination of the dish between UV (374 ± 15 nm) or yellow (507 ± 10 nm) LED light in periods of 1 minute. The peak power of both LEDs was set to be equal at 0.12 W m-2. The same fish was filmed continuously for three such cycles (total of 12 minutes per n = 12 fish wild-type and another n = 12 fish with UV cones ablated), and behavioral performance was manually annotated offline as either a “full prey capture bout” (eye convergence plus tail movement) or “tracking” (single or bilateral eye movements in the absence of tail movements). To ablate UV-cones, Tg(opn1sw1:nfsBmCherry) larval zebrafish were treated with 10 mM Metronidazole (Sigma, M3761) for 2 hours and thereafter transferred to fresh fish water without Metronidazole. Behavioral assays were performed one day after the Metronidazole treatment to ensure that UV-cone ablation was complete (Yoshimatsu et al., 2016 (link)).
4
Metronidazole-Iodine Cream Formulation
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Metronidazole (MTZ, analytical standard, Mw = 171.15 g/mol), polyvinylpyrrolidone-iodine complex (PVP-I, European Pharmacopoeia Reference Standard with average Mw = 365 g/mol), high molecular weight polyvinylpyrrolidone (PVP, Mw = 360,000 g/mol), polycaprolactone (PCL, Mw = 80,000 g/mol), polyethylene glycol (PEG, BioUltra, Mw = 35,000 g/mol), dichloromethane (DCM), dimethylformamide (DMF) and ethanol (EtOH) were purchased from Sigma Aldrich (Poole, Dorset, UK) and used as received.
MTZ and Metronidazole-iodine (MTZ-I) in aqueous cream BP (British Pharmacopoeia) base were prepared as follows: 10 g of the MTZ cream comprised of 0.1 g of MTZ (batch number MKBZ3056V, Sigma Aldrich), 3 g of emulsifying ointment (batch number NR 10043, Pinewood Healthcare, Dublin, Ireland), 0.1 g of phenoxyethanol (batch number STBD 3014V, Sigma Aldrich) and made up to the target weight with purified water. The MTZ-I cream contained the same ingredients as the MTZ cream with the addition of 0.5 g PVP-I, amounting to 0.5% elemental Iodine (equivalent to a 2:1 MTZ: PVP-I weight ratio). Lysogeny broth (LB) and LIVE/DEADTM BacLightTM Bacterial Viability and Counting Kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA), whilst LB agar was purchased from Sigma Aldrich.
MTZ and Metronidazole-iodine (MTZ-I) in aqueous cream BP (British Pharmacopoeia) base were prepared as follows: 10 g of the MTZ cream comprised of 0.1 g of MTZ (batch number MKBZ3056V, Sigma Aldrich), 3 g of emulsifying ointment (batch number NR 10043, Pinewood Healthcare, Dublin, Ireland), 0.1 g of phenoxyethanol (batch number STBD 3014V, Sigma Aldrich) and made up to the target weight with purified water. The MTZ-I cream contained the same ingredients as the MTZ cream with the addition of 0.5 g PVP-I, amounting to 0.5% elemental Iodine (equivalent to a 2:1 MTZ: PVP-I weight ratio). Lysogeny broth (LB) and LIVE/DEADTM BacLightTM Bacterial Viability and Counting Kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA), whilst LB agar was purchased from Sigma Aldrich.
5
Antibiotics Induced Microbiome Depletion
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To deplete the commensal microbiota, four- to six-week old mice received drinking water ad libitum containing 1 mg/ml Cefoxitin (Santa Cruz Biotechnology, Inc), 1 mg/ml gentamycin sulfate (Sigma-Aldrich), 1 mg/ml metronidazole (Sigma-Aldrich) and 1 mg/ml vancomycin (HEXAL®) for 4 weeks. For experiments at Washington University, six-week old mice received drinking water with 1 mg/ml ampicillin, 1 mg/ml metronidazole, 1 mg/ml neomycin, 0.5 mg/ml vancomycin (Sigma, St. Louis, MO) in 20 mg/ml grape Kool-Aid (Kraft Foods, Northfield, IL), or with Kool-Aid alone, ad libitum for 9–14 days. In most experiments we confirmed the absence of live bacteria in fecal samples by plating the material on suitable agar plates.
6
Microbiome Depletion for Viral Infection Study
7
Zebrafish Drug Treatment Protocol
8
Antimicrobial Susceptibility of Gardnerella vaginalis
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Clinical G. vaginalis isolates were evaluated for antimicrobial susceptibilities in vitro to metronidazole and clindamycin (Sigma-Aldrich, St. Louis, MO, US) targeting planktonic cells; to achieve this, we used the anaerobic agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI).
One-hundred microliters of the prepared bacterial suspension (106 CFU/mL) was added to different concentrations of metronidazole and clindamycin at 37°C. After 48 h, the bacterial growth was evaluated by taking an endpoint reading at OD595 with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The minimum inhibitory concentration (MIC) for metronidazole and clindamycin was defined as the lowest antibiotic concentration yielding marked reduction in the growth or no growth at all. Under the same conditions, G. vaginalis ATCC®14018 was tested using the broth microdilution assay. The microbiological susceptibility and resistant breakpoints for metronidazole (<8 μg/mL and ≥32 μg/mL) and clindamycin (<2 μg/mL and ≥8 μg/mL), as defined by CLSI, were used for interpreting MIC results [21 ].
One-hundred microliters of the prepared bacterial suspension (106 CFU/mL) was added to different concentrations of metronidazole and clindamycin at 37°C. After 48 h, the bacterial growth was evaluated by taking an endpoint reading at OD595 with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The minimum inhibitory concentration (MIC) for metronidazole and clindamycin was defined as the lowest antibiotic concentration yielding marked reduction in the growth or no growth at all. Under the same conditions, G. vaginalis ATCC®14018 was tested using the broth microdilution assay. The microbiological susceptibility and resistant breakpoints for metronidazole (<8 μg/mL and ≥32 μg/mL) and clindamycin (<2 μg/mL and ≥8 μg/mL), as defined by CLSI, were used for interpreting MIC results [21 ].
9
Antibiotic Combination Treatments in Mice
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We initially treated four different groups of mice (4–5 per group) separately with either metronidazole (5 mg/mL) or a combination of metronidazole (5 mg/mL) + clindamycin (10 mg/mL), metronidazole (5 mg/mL) + imipenem (5 mg/mL), or metronidazole (5 mg/mL) + imipenem (5 mg/mL) + clindamycin (10 mg/mL) (Sigma Aldrich, USA) (Figure 1 A). All mice received a daily dose of 200 µL of antibiotic preparations via the oral gavage for four weeks. The metronidazole (5 mg/mL) + clindamycin (10 mg/mL) treatment was repeated in two additional independent experiments using different sets of mice (Figure 1 B,C). Fresh antibiotics were prepared at every 3-day interval.
10
Conditional Cell Ablation via Nitroreductase
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