Real-time monitoring of antimicrobial activity with the multiparameter microplate assay

Lehtinen, Janne; Järvinen, Suvi; Virta, Marko; Lilius, Esa‐Matti

doi:10.1016/j.mimet.2006.01.002

Cited by 46 publications

(50 citation statements)

References 19 publications

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“…It should also be noted that other microplate-compatible methods have previously been developed for screening for antimicrobial compounds. [19][20][21] However, this method may be advantageous for some applications. First, it does not require transformation or genetic engineering of bacterial cells with foreign reporter genes (e.g., green fluorescent protein, luciferase, or β-galactosidase).…”

Section: Figmentioning

confidence: 99%

High-Throughput Screening for Antimicrobial Compounds Using a 96-Well Format Bacterial Motility Absorbance Assay

Malapaka¹,

Barrese²,

Tripp³

2007

SLAS Discovery

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There is a pressing need to develop new antimicrobial drugs because of the increasing resistance of pathogenic bacteria to existing antibiotics. The preliminary development and validation of a novel methodology for the high-throughput screening of antimicrobial compounds and inhibitors of bacterial motility is described. This method uses a bacterial motility swarming agar assay, combined with the use of offset inoculation of the wells in a standard, clear, 96-well plate, to enable rapid screening of compounds for potential antibiotic and antimotility properties with a standard absorbance microplate reader. Thus, the methodology should be compatible with 96-well laboratory automation technology used in drug discovery and chemical biology studies. To validate the screening method, the Genesis Plus structurally diverse library of 960 biologically active compounds was screened against a motile strain of the gram-negative bacterial pathogen Salmonella typhimurium. The average Z′ value for the positive and negative motility controls on all 12 compound plates was 0.67 ± 0.14, and the signal-to-baseline ratio calculated from the positive and negative controls was 5.9 ± 1.1. A collection of 70 compounds with well-known antimicrobial properties was successfully identified using this assay. (Journal of Biomolecular Screening 2007:849-854)

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Section: Figmentioning

confidence: 99%

High-Throughput Screening for Antimicrobial Compounds Using a 96-Well Format Bacterial Motility Absorbance Assay

Malapaka¹,

Barrese²,

Tripp³

2007

SLAS Discovery

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“…Measurements of optical density are often used to assess antibiotic efficacy and pharmacokinetics prior to testing in mouse models. Bacteria expressing bioluminescent [9,10], or fluorescent [11,12] genetic reporters have been incorporated in these basic in vitro systems as a way of enhancing the sensitivity of the toxicity assays. More sophisticated in vitro models of infection employ a mixed culture of mammalian and bacteria cells.…”

Section: Imaging In Vitro Models Of Bacterial Infectionmentioning

confidence: 99%

Optical imaging of bacterial infection models

Leevy¹,

Serazin²,

Smith³

2007

Drug Discovery Today: Disease Models

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Over the last thirteen years, the field of optical imaging has expanded from in vitro fluorescence microscopy of cells to in vivo imaging of living animals. Recent advances in optical imaging of bacterial infection have been propelled by the invention of genetic methods that produce fluorescent and bioluminescent bacteria, and also the discovery of synthetic fluorescent probes that selectively target bacterial cell surfaces. Optical imaging is an effective method of conducting longitudinal studies of bacterial infection in small animals such as nude mice. It can be used to address questions in medical microbiology concerning migration and colonization and it is an attractive method for determining the efficacy of antibiotic therapies.

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“…Endotoxin quantification assays (Tamura, 1990(Tamura, , 1991(Tamura, , 1992(Tamura, , 1993, biological activity studies (Barret, 1997;Holowachuk, 2003;Lethinen, 2006), microbiological assays (Bamba, 1997;Burton, 2007;Pettit, 2005) and molecular biology studies (Borisevich, 2008;Mayo, Curnutte 1990;Zhong, Smith 1994) employ kinetic reading microplate systems, allowing analysis of a large number of samples both quickly and reliably. Bacteriostatic, bactericidal and bacteriolytic tests have been performed using microplate systems with kinetic reading of optic density fluorescence bioluminescence (Lethinen, 2006;Smith, 2008). Microplate systems have also been used in the assessment of microbiological activity of biological fluids (Holowachuk, 2003) and in the detection of antibiotic residues in food (Lamar, Petz, 2007).…”

Section: Introductionmentioning

confidence: 99%

Antibiotic microbial assay using kinetic-reading microplate system

Lourenço

Pinto

2011

Braz. J. Pharm. Sci.

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The aim of this study was to determine the optimal experimental conditions to develop a methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of selectivity, linearity, linear range, limits of detection and quantification, accuracy and precision. The turbidimetric assay principle is simple: the test solution is added to a suspension of test microorganism in culture media, the mixture is incubated under appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is of considerable interest since it allows reduction of material and analysis time and enables a large number of samples to be analyzed simultaneously, with automated reading and calculating. Established conditions considered the standard-curve of apramycin at concentrations from 5.0 to 35.0 mg mL -1 , and tryptic soy broth inoculated with 5% Escherichia coli (ATCC 8739) suspension. Satisfactory results were obtained with 2 hours of incubation. The developed method showed appropriate selectivity, linearity in the range from 5.0 to 35.0 mg mL , respectively, as well as satisfactory accuracy (recuperation = 98.5%) and precision (RSD = 6.0%). Microplate assay combined the characteristics of microbiological (evaluation of antibiotic activity against sensitive test microorganism) and physico-chemical (operationally straightforward and faster results) assays.Uniterms: Antibiotics/microbial assay. Apramycin/microbial assay. Apramycin sulfate. Turbidimetric assay/medicines analysis.O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e quantificação, exatidão e precisão. O ensaio turbidimétrico é simples: a solução-teste é adicionada à suspensão de microrganismo-teste em meio de cultura, a mistura é incubada em condições apropriadas e o crescimento microbiano é medido por meio de leitura fotométrica. O emprego de método de microplacas com leitura cínética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizados. As condições estabelecidas abrangem curva-padrão de apramicina com concentrações entre 5 e 35 mg/mL, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporção de 5%. Foram obtidos resultados satisfatórios após 2 horas de incubação. O método desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 mg/mL, limite de detecção e quantificação de 0,1 e 0,4 mg/mL, respectivament...

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Real-time monitoring of antimicrobial activity with the multiparameter microplate assay

Cited by 46 publications

References 19 publications

High-Throughput Screening for Antimicrobial Compounds Using a 96-Well Format Bacterial Motility Absorbance Assay

High-Throughput Screening for Antimicrobial Compounds Using a 96-Well Format Bacterial Motility Absorbance Assay

Optical imaging of bacterial infection models

Antibiotic microbial assay using kinetic-reading microplate system

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