Cells of P. aeruginosa PAO1 or mutants (107 to 109 CFU) were grown in L medium (1.0% polypeptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.0) and spread onto L agar plates (1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar, pH 7.0) containing 1 × , 2 × , 4 × MIC for one of the following seven antimicrobial agents: carbenicillin, imipenem, Amikacin, gentamicin, ciprofloxacin, levofloxacin, and erythromycin. We obtained many colonies that appeared on the plates after an incubation at 37 ℃ for 24–36 h. After single-colony isolation, the drug resistance patterns of the mutants were investigated. Amikacin (Wako, cat. 014-24941), carbenicillin (Wako, cat. 037-23693), ciprofloxacin (Wako, cat. 032-18731), gentamicin (Wako, cat. 079-02973), imipenem (Wako, cat. 098-07283), levofloxacin (Fluka, cat. 28266) were purchased from indicated manufactures.
Imipenem
Manufactured by Fujifilm
Sourced in Japan
Imipenem is a broad-spectrum antibiotic used in the treatment of various bacterial infections. It is a carbapenem-class drug that functions by inhibiting bacterial cell wall synthesis, thereby disrupting the integrity of the cell and leading to cell death. Imipenem is commonly used in hospital settings to manage severe or resistant bacterial infections.
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Lab products found in correlation
Carbenicillin, by Fujifilm (2 mentions)
Gentamicin, by Fujifilm (2 mentions)
Ciprofloxacin, by Fujifilm (2 mentions)
Chloramphenicol, by Fujifilm (2 mentions)
Ampicillin, by Fujifilm (2 mentions)
Amikacin, by Fujifilm (1 mentions)
Falcon 3047, by BD (1 mentions)
Lincomycin, by Merck Group (1 mentions)
Fosfomycin, by Merck Group (1 mentions)
Vancomycin, by Fujifilm (1 mentions)
Enrofloxacin, by Tokyo Chemical Industry (1 mentions)
Levofloxacin, by Fujifilm (1 mentions)
Minocycline, by Fujifilm (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Ofloxacin, by Merck Group (1 mentions)
Metronidazole, by Fujifilm (1 mentions)
Ampicillin, by Nacalai Tesque (1 mentions)
Bacitracin, by Fujifilm (1 mentions)
Vancomycin, by Merck Group (1 mentions)
6 protocols using imipenem
1
Screening of Antibiotic Resistant Mutants in P. aeruginosa
2
Adherent Bacteria Antibiotic Susceptibility Assay
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Susceptibility testing for the adherent bacteria assay following the method of Murakami et al. [24 (link)]. The bacterial suspension which concentration of 2 × 106 CFU/mL was divided into each well of 96-well plates (Falcon 3047, Becton Dickinson, Lincoln Park, NJ, USA; each well contained 50 µL of bacterial suspension) and then centrifuged at 450× g for 15 min at 25 °C. The plate was incubated at 37 °C for 1 h, then saline was removed and 100 µL of serially diluted imipenem (Wako Pure Chemical Industries, Ltd., Osaka, Japan) solutions were transferred to each well in the plate. The final imipenem concentration was used from 0.125 µg/mL to 256 µg/mL. MICAD was determined as the lowest concentration of antibiotic at which there was no bacterial growth after 24 h incubation at 37 °C. The antibiotic solutions were removed, and 200 µL of fresh LB medium was added to each well. MBCAD was determined as the lowest concentration of antibiotic at which there was no bacterial growth after 24 h incubation at 37 °C. Comparison between the populations was performed by Pearson’s chi-square test. Statistical analysis was performed by JMP software 13 (SAS Institute, Inc., Tokyo, Japan) and a p value of <0.01 was considered as statistically significant.
3
Antimicrobial Susceptibility Testing Protocols
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Minimum inhibitory concentrations (MICs) were determined using the agar doubling-dilution
method, in accordance with the criteria proposed by the Clinical and Laboratory Standards
Institute (CLSI) [5 ]. The following antimicrobial
agents were used: ampicillin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), oxacillin
(Sigma-Aldrich, St. Louis, MO, USA), cephalexin (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan), imipenem (FUJIFILM Wako), fosfomycin (Sigma-Aldrich), enrofloxacin (Tokyo Chemical
Industry), levofloxacin (FUJIFILM Wako), erythromycin (Sigma-Aldrich), lincomycin
(Sigma-Aldrich), gentamicin (FUJIFILM Wako), minocycline (FUJIFILM Wako), chloramphenicol
(FUJIFILM Wako), and vancomycin (FUJIFILM Wako). The breakpoints of these antimicrobial
agents were determined using the interpretation criteria proposed by the CLSI [6 ].
method, in accordance with the criteria proposed by the Clinical and Laboratory Standards
Institute (CLSI) [5 ]. The following antimicrobial
agents were used: ampicillin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), oxacillin
(Sigma-Aldrich, St. Louis, MO, USA), cephalexin (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan), imipenem (FUJIFILM Wako), fosfomycin (Sigma-Aldrich), enrofloxacin (Tokyo Chemical
Industry), levofloxacin (FUJIFILM Wako), erythromycin (Sigma-Aldrich), lincomycin
(Sigma-Aldrich), gentamicin (FUJIFILM Wako), minocycline (FUJIFILM Wako), chloramphenicol
(FUJIFILM Wako), and vancomycin (FUJIFILM Wako). The breakpoints of these antimicrobial
agents were determined using the interpretation criteria proposed by the CLSI [6 ].
4
Antibacterial Activity Evaluation Protocols
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Two methods were used for the evaluation of antibacterial activity. To measure the MIC value of nisin A (Sigma–Aldrich, St. Louis, USA), the microdilution method was used as described elsewhere [23 (link)]. Bacitracin (Fujifilm Wako chemicals, Osaka, Japan), vancomycin (Sigma–Aldrich), Metronidazole (Fujifilm Wako chemicals), ampicillin (Nacalai Tesque, Kyoto, Japan), chloramphenicol (Wako chemicals), gentamicin (Nacalai Tesque), ofloxacin (Sigma–Aldrich) and imipenem (Fujifilm Wako chemicals) were also used for MIC evaluations.
To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23 (link)]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37°C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (108 cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37°C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.
To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23 (link)]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37°C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (108 cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37°C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.
5
Carbapenems Absorbance Spectroscopy
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The absorbance of carbapenems, including imipenem (Wako Pure Chemical Industries, Osaka, Japan), meropenem (Tokyo Chemical Industry, Tokyo, Japan), doripenem, biapenem, and ertapenem (Merck, Kenilworth, NJ, USA), was measured using a Corona plate reader SH-9000 (Hitachi, Tokyo, Japan) at room temperature (approximately 25 °C) in a UV-transparent 96-well plate (Zell-kontakt, Nörten-Hardenberg, Germany). Measurements with bandwidths of 1 nm and 5 nm were implemented for analysing absorption spectra.
6
Antimicrobial Compounds Procurement and Synthesis
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TPPC was prepared as reported previously, with slight modifications. 6 BM-TPPC was synthesized as reported previously. 11 We purchased Penicillin G, ampicillin, amoxicillin, carbenicillin, oxacillin (OXA), meropenem, imipenem, cefotaxime, cefaclor, kanamycin, ciprofloxacin and tetracycline from Wako Pure Chemical Industries (Osaka, Japan). Chloramphenicol and cefoxitin were purchased from Sigma-Aldrich (St Louis, MO, USA). Erythromycin was purchased from Nacalai Tesque (Kyoto, Japan). Piperacillin was purchased from Toyama Chemical (Tokyo, Japan). Cefotiam (CTM) was purchased from Takeda Pharmaceutical Company Limited (Osaka, Japan). Ceftizoxime and teicoplanin were purchased from Asteras Pharma (Tokyo, Japan). Flomoxef and vancomycin (VAN) were purchased from Shionogi (Osaka, Japan). DAP and linezolid were purchased from Funakoshi Corporation (Tokyo, Japan). Arbekacin was kindly provided by Meiji Seika Pharma (Tokyo, Japan).
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