All mouse experiments were approved by the IACUC committee of Indiana University School of Medicine (IUSM) under the protocol number #11339. Four-week-old C3H/HeN mice (Harlan, Indianapolis, IN) were subcutaneously inoculated with doses of spirochetes as indicated. Mice were euthanized at the end of the experiments, and multiple tissues (joint, heart, skin) were harvested. All tissues were cultivated in 2 ml of the BSK-II medium (Sigma-Aldrich, St. Louis, MO) containing an antibiotic mixture of phosphomycin (2 mg/ml), rifampin (5 mg/ml), and amphotericin B (250 mg/ml) (Sigma-Aldrich) to inhibit bacterial and fungal contamination. All cultures were maintained at 37°C and examined for the presence of spirochetes by dark-field microscopy beginning from 5 days after inoculation. A single growth-positive culture was used as the criterion to determine positive mouse infection.
Phosphomycin
Manufactured by Merck Group
Sourced in United States
Phosphomycin is a broad-spectrum antibiotic compound. It functions by inhibiting the early stage of bacterial cell wall synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Merck Group (6 mentions)
Rifampin, by Merck Group (5 mentions)
Bsk 2 medium, by Merck Group (5 mentions)
Rifampicin, by Merck Group (5 mentions)
Penicillin, by Merck Group (3 mentions)
Piperacillin, by Merck Group (2 mentions)
2 2 bipyridyl, by Merck Group (2 mentions)
Mecillinam, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Amphotericin, by Merck Group (2 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by AppliChem (1 mentions)
Deferoxamine, by Merck Group (1 mentions)
Novobiocin, by Merck Group (1 mentions)
Clavulanic acid, by Merck Group (1 mentions)
Vancomycin, by AppliChem (1 mentions)
Teicoplanin, by Merck Group (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
15 protocols using phosphomycin
1
Murine Model of Borrelia Infection
2
Production and Characterization of Anti-Waddlia Antibodies
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Polyclonal rabbit antibodies against W. chondrophila were produced in our laboratory as described previously [44 (link)]. Secondary antibody, Alexa Fluor® 488 donkey anti-rabbit IgG, was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Clavulanic acid, deferoxamine, mecillinam, novobiocin, penicillin, phosphomycin, piperacillin, teicoplanin, and 2,2′-bipyridyl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin was obtained from AppliChem (Darmstadt, Germany). MP265 was purchased from American Custom Chemicals Corporation (San Diego, CA, USA). All drugs were diluted in deionized water with the exception of MP265 and 2,2′-bipyridyl, which were diluted in dimethyl sulfoxide (DMSO, AppliChem) and in ethanol, respectively, in order to obtain the specific concentrations described in Table 1 . Finally, the solutions were filtered through a 0.22 μm pore filter and stored at −20 °C.
3
Evaluating Lyme Disease Infection in Mice
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All tick-mouse experiments were approved by the IACUC committee of Indiana University School of Medicine under the protocol number #19792. Four-week-old C3H/HeN mice (Harlan, Indianapolis, IN) were subcutaneously inoculated with 1 × 105 spirochetes. For the mice infected with the complemented strain of the ltpA mutant, IPTG was given via IP injection (100 µl of 10 mM IPTG) every two days. The mice were killed, and ear, skin (inoculation site), bladder, heart, and joint tissues were collected 7 weeks after infection and cultivated in 2 ml of BSK-II medium (Sigma-Aldrich, St. Louis, MO) containing an antibiotic mixture of phosphomycin (2 mg/ml), rifampin (5 mg/ml), and amphotericin B (250 mg/ml) (Sigma-Aldrich, St. Louis, MO). All cultures were maintained at 37 °C and examined for the presence of spirochetes by dark-field microscopy beginning 5 days after inoculation. A single growth-positive culture was used as the criterion to determine the presence of mouse infection.
4
Immunofluorescence Assay for W. chondrophila
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Polyclonal mouse and rabbit antibodies against W. chondrophila were produced in-house as previously described48 (link). Antibodies against actin were purchased from Sigma-Aldrich (St Louis, MO). MitoTracker Red CMXRos (M7512) as well as secondary antibodies Alexa Fluor 488 goat anti-rabbit, 488 anti-mouse, 594 anti-rabbit and 594 anti-mouse were all obtained from Molecular Probes (Grand Island, NY). A22, phosphomycin, penicillin, mecillinam and piperacillin were purchased from Sigma-Aldrich. MP265 was synthesized by American Custom Chemicals Corporation (San Diego, CA). Vero cells were treated with 10 μM nocodazole (Sigma-Aldrich) or 10 μM cytochalasin D (Sigma-Aldrich). Primers and probes used in this study are described in supplementary Table 3 .
5
Isolation and Characterization of Streptomyces
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The isolation and phylogenetic characterization of Streptomyces sp. IB2014/011-12 were reported in (Axenov-Gribanov et al., 2016 (link)). Streptomyces strains were grown on solid nutrient medium MS (mannitol soy flour agar) and in liquid TSB medium (Kieser, 2000 ). For secondary metabolite production, NL19 (MS medium without agar) and SG (glucose, yeast, Bacto Soytone, and calcium carbonate) medium have been used. Escherichia coli XL1Blue (Agilent, United States) was used for routine cloning, and E. coli MW 6026 was used as a donor in the intergenic conjugation (Blodgett et al., 2007 (link)). E. coli strains were grown in Luria-Bertani (LB) broth. For MW 6026, diaminopimelic acid was added. When required, antibiotics were added to the cultures at the following concentrations: 50 μg ml−1 apramycin, 100 μg ml−1 spectinomycin, 100 μg ml−1 phosphomycin, and 100 μg ml−1 carbenicillin (Sigma, United States; Roth, Germany).
6
Immunofluorescence Assay for W. chondrophila
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Polyclonal mouse antibodies against W. chondrophila were produced by our group, as previously described [16 (link)]. Secondary antibodies, Goat anti-mouse green Alexa 488, and Goat anti-rabbit red Alexa 594 were purchased from Thermo Fischer Scientific (Waltham, MA, USA). DAPI was obtained from Molecular Probes (Grand Island, NY). The antibiotics penicillin and phosphomycin, and 2, 2-Bipyridyl were purchased from Sigma-Aldrich (St Louis, MO, USA).
7
Borrelia Detection in Infected Mice
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Tissues (~3 mm2) or 100 μl blood from infected mice were incubated in complete BSK‐II medium supplemented with 100 μg/ml gentamycin (GCB726 only), 20 μg/ml phosphomycin (Sigma), 50 μg/ml rifampicin, and 2.5 μg/ml amphotericin (Bioshop) for 1–4 weeks. Following incubation, tissues were identified as positive or negative based on the presence or absence of Borrelia detected in media by dark‐field microscopy. One piece of each tissue was cultivated in a single (non‐replicate) culture for each sample.
8
Cultivation and Preparation of Borrelia burgdorferi
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All Borrelia samples (B. burgdorferi strain:
ATCC 35210) were cultured in the modified Barbour-Stoenner-Kelly medium,
BSK-H, with 6% rabbit serum22 (link) (Dalynn Biologicals)
with antibiotics (amphotericin B at 2.5 μg/mL, rifampicin at
0.05 mg/mL, and phosphomycin at 0.02 mg/mL) from Sigma-Aldrich, for
at least 4 weeks at 34–35 °C with 5% CO2. Borrelia cells were killed by exposure to a dilute salt
environment (0.1× PBS) and freezing for >48 h at −80
°C. Borrelia cell extracts were stored at −20
°C
until use.
The incubation of fibronectin-modified electrodes
in analytes involved drop-casting of the Borrelia samples onto the electrode surface and subsequent incubation in
a humidity chamber. After analyte incubation, electrodes were rinsed
with distilled water prior to electrochemical measurement.
E. coli was chosen as a negative control in all
electrochemical tests due to similarity in the size of species (B. burgdorferi that is approximately 0.3 μm wide and
5–20 μm long and the E. coli with approximately
1.1–1.5 μm wide and 2–6 μm long).
ATCC 35210) were cultured in the modified Barbour-Stoenner-Kelly medium,
BSK-H, with 6% rabbit serum22 (link) (Dalynn Biologicals)
with antibiotics (amphotericin B at 2.5 μg/mL, rifampicin at
0.05 mg/mL, and phosphomycin at 0.02 mg/mL) from Sigma-Aldrich, for
at least 4 weeks at 34–35 °C with 5% CO2. Borrelia cells were killed by exposure to a dilute salt
environment (0.1× PBS) and freezing for >48 h at −80
°C. Borrelia cell extracts were stored at −20
°C
until use.
The incubation of fibronectin-modified electrodes
in analytes involved drop-casting of the Borrelia samples onto the electrode surface and subsequent incubation in
a humidity chamber. After analyte incubation, electrodes were rinsed
with distilled water prior to electrochemical measurement.
E. coli was chosen as a negative control in all
electrochemical tests due to similarity in the size of species (B. burgdorferi that is approximately 0.3 μm wide and
5–20 μm long and the E. coli with approximately
1.1–1.5 μm wide and 2–6 μm long).
9
Cultivation of Lyme Disease Spirochete
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First passage B. burgdorferi strain B31 clone 5A19 spirochetes, isolated from an ear biopsy of a previously infected mouse, were grown in Barbour-Stoenner-Kelly-H medium supplemented with 6% rabbit serum and antibiotics (rifampicin at 45.4 μg/mL, phosphomycin at 193 μg/ml, and amphotericin at 0.25 μg/ml; Sigma-Aldrich, St. Louis, MO) to late logarithmic phase under microaerophilic conditions. An inoculum containing 1 × 108 spirochetes/ml in RPMI 1640 medium (Invitrogen, USA) was prepared as previously described [11 (link)].
10
Detecting Borrelia burgdorferi Infection
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Ear, heart, tibiotarsal joint, and urinary bladder samples were collected to assess the B. burgdorferi infection status. All instruments were disinfected in ethanol between dissections of different samples. The tissue samples were cultured in BSK II medium supplemented with phosphomycin (50 μg/ml; Sigma-Aldrich, Steinheim, Germany) and rifampicin (100 μg/ml; Sigma-Aldrich) at 33 °C for 6 weeks. Growth was monitored every 2 weeks with a dark-field microscope.
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