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A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within e...

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Autores principales: Sarshar, Meysam, Scribano, Daniela, Tranquilli, Giulia, Di Pietro, Marisa, Filardo, Simone, Zagaglia, Carlo, Sessa, Rosa, Palamara, Anna Teresa, Ambrosi, Cecilia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849193/
https://www.ncbi.nlm.nih.gov/pubmed/31718545
http://dx.doi.org/10.1186/s12866-019-1625-1
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author Sarshar, Meysam
Scribano, Daniela
Tranquilli, Giulia
Di Pietro, Marisa
Filardo, Simone
Zagaglia, Carlo
Sessa, Rosa
Palamara, Anna Teresa
Ambrosi, Cecilia
author_facet Sarshar, Meysam
Scribano, Daniela
Tranquilli, Giulia
Di Pietro, Marisa
Filardo, Simone
Zagaglia, Carlo
Sessa, Rosa
Palamara, Anna Teresa
Ambrosi, Cecilia
author_sort Sarshar, Meysam
collection PubMed
description BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R(2)), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.
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spelling pubmed-68491932019-11-15 A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria Sarshar, Meysam Scribano, Daniela Tranquilli, Giulia Di Pietro, Marisa Filardo, Simone Zagaglia, Carlo Sessa, Rosa Palamara, Anna Teresa Ambrosi, Cecilia BMC Microbiol Methodology Article BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R(2)), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches. BioMed Central 2019-11-12 /pmc/articles/PMC6849193/ /pubmed/31718545 http://dx.doi.org/10.1186/s12866-019-1625-1 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Sarshar, Meysam
Scribano, Daniela
Tranquilli, Giulia
Di Pietro, Marisa
Filardo, Simone
Zagaglia, Carlo
Sessa, Rosa
Palamara, Anna Teresa
Ambrosi, Cecilia
A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title_full A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title_fullStr A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title_full_unstemmed A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title_short A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
title_sort simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849193/
https://www.ncbi.nlm.nih.gov/pubmed/31718545
http://dx.doi.org/10.1186/s12866-019-1625-1
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