High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts
The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the r...
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2018-02-01
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doaj-5262826854a1484582a1630dbd39fb6d2020-11-24T22:29:40ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882018-02-01810.3389/fcimb.2018.00043323874High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony CountsStefanie Hoffmann0Steffi Walter1Anne-Kathrin Blume2Stephan Fuchs3Christiane Schmidt4Annemarie Scholz5Roman G. Gerlach6Project Group 5, Robert Koch Institute, Wernigerode, GermanyProject Group 5, Robert Koch Institute, Wernigerode, GermanyDepartment of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle, GermanyDivision 13: Nosocomial Pathogens and Antibiotic Resistances, Robert Koch Institute, Wernigerode, GermanyProject Group 5, Robert Koch Institute, Wernigerode, GermanyProject Group 5, Robert Koch Institute, Wernigerode, GermanyProject Group 5, Robert Koch Institute, Wernigerode, GermanyThe quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays.http://journal.frontiersin.org/article/10.3389/fcimb.2018.00043/fullSalmonellainvasionadhesionintracellular replicationgentamicin protection assayvirtual colony count |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Stefanie Hoffmann Steffi Walter Anne-Kathrin Blume Stephan Fuchs Christiane Schmidt Annemarie Scholz Roman G. Gerlach |
spellingShingle |
Stefanie Hoffmann Steffi Walter Anne-Kathrin Blume Stephan Fuchs Christiane Schmidt Annemarie Scholz Roman G. Gerlach High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts Frontiers in Cellular and Infection Microbiology Salmonella invasion adhesion intracellular replication gentamicin protection assay virtual colony count |
author_facet |
Stefanie Hoffmann Steffi Walter Anne-Kathrin Blume Stephan Fuchs Christiane Schmidt Annemarie Scholz Roman G. Gerlach |
author_sort |
Stefanie Hoffmann |
title |
High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts |
title_short |
High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts |
title_full |
High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts |
title_fullStr |
High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts |
title_full_unstemmed |
High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts |
title_sort |
high-throughput quantification of bacterial-cell interactions using virtual colony counts |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular and Infection Microbiology |
issn |
2235-2988 |
publishDate |
2018-02-01 |
description |
The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays. |
topic |
Salmonella invasion adhesion intracellular replication gentamicin protection assay virtual colony count |
url |
http://journal.frontiersin.org/article/10.3389/fcimb.2018.00043/full |
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