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Spectinomycin

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Spectinomycin is a broad-spectrum antibiotic that inhibits bacterial protein synthesis. It is commonly used in laboratory settings for various research and testing applications.

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Lab products found in correlation

Yeast extract, by Merck Group (3 mentions) Erythromycin em, by Merck Group (1 mentions) Tetracycline tc, by Merck Group (1 mentions) Phusion polymerase, by New England Biolabs (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Chloramphenicol, by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Gapdh, by Merck Group (1 mentions) Glucose 6 phosphate (g6p), by Merck Group (1 mentions) Nadph, by Merck Group (1 mentions) Pyruvate, by Merck Group (1 mentions) Phenylboronic acid, by Merck Group (1 mentions) Pyruvate kinase, by Merck Group (1 mentions) Lactate dehydrogenase, by Merck Group (1 mentions) 1 3 propanediol, by Merck Group (1 mentions) Rubisco, by Merck Group (1 mentions) Phosphocreatine, by Merck Group (1 mentions) Acetoin, by Merck Group (1 mentions)

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Spectinomycin sulfate (8 citations)

9 protocols using spectinomycin

1

Enterococcus faecalis Growth Conditions

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All experiments were performed in strain OG1RF, a fully sequenced E. faecalis oral isolate [38 (link)]. Overnight cultures were grown at 37°C in sterile REMEL™ BHI Broth–Brain Heart Infusion (37g in 1L demineralized water) following isolation of a single colony from BHI Agar plates–Fisher BioReagents™ Agar Powder/Flakes (15g/L). Media and plates were supplemented with Spectinomycin (120 μg/ml) (Spec120) (Spectinomycin sulfate; MP Biomedicals).
Hallinen K.M., Guardiola-Flores K.A, & Wood K.B. (2020). Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis. PLoS ONE, 15(5), e0232539.
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2

Selective growth of Streptococcus mutans

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Streptococcus mutans strain UA159 (JM10 : : pJM1-ldh, luc+, SpcR, luc under the control of the ldh promoter) was utilized for this project. The selection of antibiotic-resistant colonies was performed on TH plates (Todd-Hewitt, BD Difco, USA) supplemented with 0.3 % yeast extract (EMB, Germany) and 800 μg/mL of spectinomycin (MP Biomedicals, USA). The plates were incubated under anaerobic conditions at 37°C for 48 h.
Florez F.L., Hiers R.D., Larson P., Johnson M., O’Rear E., Rondinone A.J, & Khajotia S.S. (2018). Antibacterial dental adhesive resins containing nitrogen-doped titanium dioxide nanoparticles. Materials science & engineering. C, Materials for biological applications, 93, 931-943.
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3

Genetic Manipulation of S. aureus Strains

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The S. aureus strains tested in the murine gastrointestinal colonization model are listed in Table 3; each was resistant to Sm. Mutations in the S. aureus ica locus, spa, and atl were moved into Sm-resistant Newman by transduction with phage 80α or phage 85 from the original antibiotic marked mutant strains [42 (link)–44 (link)]. The tagO::tet mutation was moved from S. aureus RN4220 into Newman Δatl as described previously [22 (link)]. Mutants were confirmed by PCR or Southern blot analysis and were phenotypically identical to the parental strains in terms of the growth rate, hemolysis on sheep blood agar plates, and the metabolic profile on API Staph test strips (Biomerieux, Inc., Durham, NC). S. aureus strains were cultivated in TSB to the logarithmic growth phase, unless otherwise noted. Sm (0.5 mg/ml; Sigma Chemical Co., St. Louis, Mo.), erythromycin (Em; 5 μg/ml; Sigma), tetracycline (Tc; 2.5 μg/ml; Sigma), or spectinomycin (Spc; 100 μg/ml; MP Biomedicals, Solon, OH) was added to culture medium for selection where appropriate.
Misawa Y., Kelley K.A., Wang X., Wang L., Park W.B., Birtel J., Saslowsky D, & Lee J.C. (2015). Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid, Capsule, and Surface Proteins. PLoS Pathogens, 11(7), e1005061.
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4

Metabolic Pathway Analysis with Isotopic Labeling

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The following reagents were obtained from Sigma-Aldrich: glucose, 1,3-propanediol, phenylboronic acid, cycloheximide, 23BD, acetoin, pyruvate, PEP, 6PG, G6P, F6P, Ru5P, R15P, ATP, dithiothreitol (DTT), NADH, NADPH, NADP+, phosphocreatine, RuBisCO, pyruvate kinase, lactate dehydrogenase, G6P dehydrogenase, 3PGA kinase, GAPDH and creatine kinase. U-13C glucose and 13C-NaHCO3 were obtained from Cambridge Isotope Laboratories. IPTG and chloramphenicol were obtained from Fischer Scientific. Gentamycin was purchased from Teknova. Spectinomycin was purchased from MP Biomedicals. Kanamycin was purchased from IBI Scientific. Phusion polymerase was purchased from New England Biolabs. All oligonucleotide synthesis and DNA sequencing were performed by Eurofins MWG Operon Inc.
Kanno M., Carroll A.L, & Atsumi S. (2017). Global metabolic rewiring for improved CO2 fixation and chemical production in cyanobacteria. Nature Communications, 8, 14724.
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5

Bioluminescent Streptococcus mutans Biofilm Growth

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A genetically modified and bioluminescent strain of Streptococcus mutans (JM10), a derivative of wild type UA159 constructed by Merritt et al. [12 (link)] was used. Colonies of JM10 were grown on TH agar plates (Todd-Hewitt, BD Difco, USA) supplemented with 0.3% yeast extract (EMD Millipore, USA) and 800 μg/mL of Spectinomycin (MP Biomedicals, USA) under anaerobic conditions at 37 °C for 48 h for a total of two passages. A single colony was inoculated in 4.0 mL of THY broth with 32 μL of Spectinomycin (100 mg/mL), then incubated at 37 °C for 16 h (static cultures, anaerobic conditions). Planktonic cultures having optical densities (OD600) equal to or higher than 0.900 (corresponding to 6.43 e+12 CFU/mL) were used for biofilm growth. Four dilutions of bacterial inoculum [1:50, 1:100, 1:250 and 1:500 (v/v)], three concentrations of THY biofilm growth media (0.35x, 0.65x, and 1x) and three concentrations of sucrose [0.1, 0.5, or 1.0% (w/v)] were the growth conditions tested. Each combination (inoculum + growth medium + sucrose) was then used to grow biofilms (n = 6/combination, total n = 10,368) in the wells of sterile white 24-well plates (catalog #6005816; Perkin-Elmer, USA) for either 24 or 48 h in anaerobic conditions at 37 °C, as described in Table 1.
Esteban Florez F.L., Hiers R.D., Zhao Y., Merritt J., Rondinone A.J, & Khajotia S.S. (2020). Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives. Dental materials : official publication of the Academy of Dental Materials, 36(3), 353-365.
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6

Cultivation and Induction of B. anthracis Strains

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B. anthracis strains were cultivated at 37°C in Brain Heart Infusion (BHI) (Becton, Dickson and Company) or Casamino Acid medium containing 0.8% sodium bicarbonate (CA CO3) (Thorne and Belton, 1957; Hadjifrangiskou, 2007). BHI broth cultures were incubated with agitation (200 r.p.m.) in air. CA broth cultures were shaken in 5% atmospheric CO2. Cells from stationary phase BHI cultures were subcultured into fresh CACO3 to an optical density at 600 nm (OD600) of 0.08. For strains harboring atxA, acpA, and acpB alleles under control of the hyperspank promoter (Phyperspank) (Britton 2002), expression was induced with isopropyl β-D-thiogalactoside (IPTG) during early exponential phase at 2 h and harvested at early stationary phase at 4 h. Optical densities for early exponential phase cultures ranged from OD600 0.25–0.35, and early stationary phase cultures ranged from 1.2 to 1.7.
Cultures were supplemented with antibiotics when appropriate at the following concentrations: spectinomycin (MP Biomedicals, Solon, OH) (50 μg ml−1 for E. coli and 100 μg ml−1 for B. anthracis), erythromycin (Fisher Bioreagents, Fair Lawn, NJ) (150 μg ml−1 for E. coli and 10 μg ml−1 for B. anthracis), and carbenicillin (Research Products International Corp, Mt. Prospect, IL) (100 μg ml−1 for E. coli).
Raynor M.J., Roh J.H., Widen S.G., Wood T.G, & Koehler T.M. (2018). Regulons and Protein-Protein Interactions of PRD-containing Bacillus anthracis Virulence Regulators Reveal Overlapping but Distinct Functions. Molecular microbiology.
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7

Bioluminescent Streptococcus mutans Biofilm Growth

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A genetically modified and bioluminescent strain of Streptococcus mutans (JM10), a derivative of wild type UA159 constructed by Merritt et al. [12 (link)] was used. Colonies of JM10 were grown on TH agar plates (Todd-Hewitt, BD Difco, USA) supplemented with 0.3% yeast extract (EMD Millipore, USA) and 800 μg/mL of Spectinomycin (MP Biomedicals, USA) under anaerobic conditions at 37 °C for 48 h for a total of two passages. A single colony was inoculated in 4.0 mL of THY broth with 32 μL of Spectinomycin (100 mg/mL), then incubated at 37 °C for 16 h (static cultures, anaerobic conditions). Planktonic cultures having optical densities (OD600) equal to or higher than 0.900 (corresponding to 6.43 e+12 CFU/mL) were used for biofilm growth. Four dilutions of bacterial inoculum [1:50, 1:100, 1:250 and 1:500 (v/v)], three concentrations of THY biofilm growth media (0.35x, 0.65x, and 1x) and three concentrations of sucrose [0.1, 0.5, or 1.0% (w/v)] were the growth conditions tested. Each combination (inoculum + growth medium + sucrose) was then used to grow biofilms (n = 6/combination, total n = 10,368) in the wells of sterile white 24-well plates (catalog #6005816; Perkin-Elmer, USA) for either 24 or 48 h in anaerobic conditions at 37 °C, as described in Table 1.
Esteban Florez F.L., Hiers R.D., Zhao Y., Merritt J., Rondinone A.J, & Khajotia S.S. (2020). Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives. Dental materials : official publication of the Academy of Dental Materials, 36(3), 353-365.
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8

Biofilm Formation of Streptococcus mutans

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Streptococcus mutans strain UA159 (JM10:pJM1-ldh, luc+, SpcR, luc under the control of the ldh promoter) was utilized as the model organism in the present study. [34 (link),35 (link),42 (link)] The selection of antibiotic-resistant colonies was performed on two passages of TH plates (Todd-Hewitt, BD Difco, New Jersey, NJ, USA), supplemented with 0.3% yeast extract (EMD Millipore Sigma, Burlington, MA, USA) and 800 µg/mL of spectinomycin (MP Biomedicals, Santa Ana, CA, USA). The plates were incubated under anaerobic conditions at 37 °C for 48 h. Planktonic cultures of S. mutans (JM10) were grown in THY culture medium at 37 °C for 16 h. Cultures having optical density (OD600) levels equal to or higher than 0.900 (corresponding to 6.43 e+12 CFU/mL) were used as inoculum to grow biofilms. Optimal biofilm growth parameters identified during a previous study from our group [1:50 dilution, 0.65× THY + 1% (w/v) sucrose, 1000 µL] [6 (link)] were then used to grow the biofilms. Aliquots (1.0 mL) of inoculated biofilm growth media were dispensed into the wells of sterile 24-well microtiter plates (Falcon, Corning, NY, USA), containing sterile specimens. Biofilms were grown for 24 h (static cultures, microaerophilic conditions, 37 °C). An additional set of specimens fabricated with OPTB was treated with 2% chlorhexidine gluconate (CHX) for 2 min and served as the control group.
Hiers R.D., Huebner P., Khajotia S.S, & Florez F.L. (2022). Characterization of Experimental Nanoparticulated Dental Adhesive Resins with Long-Term Antibacterial Properties. Nanomaterials, 12(21), 3732.
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9

Constructing Bioluminescent S. mutans Strain

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Bioluminescent S. mutans strain JM10 [25 (link)], a derivative of wild type UA159 was used. Details about the strain construction were reported by Merritt et al. [25 (link)]. Briefly, the strain was constructed by transforming plasmid pJM-1 (Φ::(ldh-luc), SpcR) into UA159 selecting for chromosomal integration of the plasmid via its spectinomycin resistance, which is only expressed when the plasmid integrates into the chromosomal region via homologous recombination at the ldh locus. The presence of the reporter fusion was confirmed by selection of antibiotic-resistant colonies on TH (Todd-Hewitt, BD Difco, USA) plates supplemented with 0.3% yeast extract (EMD Millipore, USA) and 800μg/mL of spectinomycin (MP Biomedicals, USA). Colonies were cultivated under anaerobic conditions at 37°C for 48 hours. It is important to note that the manipulation of recombinant strains requires trained researchers and certified laboratory facilities.
Florez F.L., Hiers R.D., Smart K., Kreth J., Qi F., Merritt J, & Khajotia S.S. (2016). Real-time Assessment of Streptococcus mutans Biofilm Metabolism on Resin Composite. Dental materials : official publication of the Academy of Dental Materials, 32(10), 1263-1269.
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