Rapid, Low-Technology MIC Determination with Clinical
            <i>Mycobacterium tuberculosis</i>
            Isolates by Using the Microplate Alamar Blue Assay

Franzblau, Scott G.; Witzig, Richard; McLaughlin, James C.; Torres, Patricia; Madico, Guillermo; Hernández, A.M.; Degnan, Michelle T.; Cook, Mary B.; Quenzer, Virginia K.; Ferguson, Robert M.; Gilman, Robert H.

doi:10.1128/jcm.36.2.362-366.1998

Cited by 860 publications

(345 citation statements)

References 10 publications

(13 reference statements)

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“…Plates were not counted if microscopic growth was so heavy that individual colonies could not be identified. The microplate Alamar blue assay was used to determine the efficacy of STD and DPPC LE-doxy in a cell-free system as a comparison (Franzblau et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model ofMycobacterium smegmatisInfection

et al. 2015

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Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 mg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.

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Section: Methodsmentioning

confidence: 99%

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model ofMycobacterium smegmatisInfection

et al. 2015

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“…The compounds were tested at concentrations of 1, 5, 10, 20 and 40 lg/mL. The minimal inhibitory concentrations (MIC 99 ) of compounds 1-20 were determined using the proportion method in the BACTEC MGIT 960 system (9) in the TB Exist mode (24). Compounds 2, 3, 6, 7, 10, 15-17, and 19 all had a negative effect on the growth of mycobacterial cells, with 99% inhibition observed at concentrations between 5 and 40 lg/mL.…”

Section: Resultsmentioning

confidence: 99%

5‐Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis

et al. 2015

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Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, and 1,3-di-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

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“…All the newly synthesized peptides 1-11 were evaluated in vitro antitubercular screenings against MTBH37Ra using the microplate Alamar blue assay (MABA) method. The activity is expressed as % inhibition at various concentrations used for the assay (Franzblau et al, 1998;O'Brien, Wilson, Orton, & Pognan, 2000). Compounds were suspended in Milli-Q water (Stock con.…”

Section: In Vitro Antimycobacterial Activitymentioning

confidence: 99%

Synthesis and biological activity of Ub2 derived peptides as potential host‐directed antitubercular therapy

et al. 2019

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The correlation of mycobactericidal property of macrophages with its potential to deliver bacteria to hydrolytic lysosomes, augmented with ubiquitin‐derived peptides (Ub2), activates the process of autophagy. This leads to the formation of phagolysosomes supported by factor involving increased cationic charges which regulate the acidic pH causing elimination of Mycobacterium. To better understand this interaction of cationic‐rich ubiquitin‐derived peptides with mycobacteria and to identify putative mycobacterial intrinsic resistance mechanisms for phagolysosome formation, we have synthesized a new series of Ub2 peptides, wherein the Gly residues are replaced with azaGly with the aim to improve metabolic stability. In addition to that a new methodology is reported for the synthesis of heteroaryl tethered peptides using azaGly as a linker. We have demonstrated that positive puncta (directly proportional to the acidification of lysosome) in cytosol was significantly increased after 6 hours on the treatment of macrophage with Ub2 peptide derivatives (1, 6, 10, and 11) causing the higher intensity of lysosome observed through LysoTracker Red Dye. The circular dichroism spectral studies are carried out in water and water:TFE mixture and demonstrated that the Ub2 peptides have helix‐forming tendency in the presence of TFE. The recognizable intracellular killing of Mycobacterium tuberculosis by Ub2 peptides provides a new approach for host‐directed therapy.

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Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by Using the Microplate Alamar Blue Assay

Cited by 860 publications

References 10 publications

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model ofMycobacterium smegmatisInfection

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model ofMycobacterium smegmatisInfection

5‐Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis

Synthesis and biological activity of Ub2 derived peptides as potential host‐directed antitubercular therapy

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