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In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptom...

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Autores principales: Filardo, Simone, Di Pietro, Marisa, Pasqualetti, Patrizio, Manera, Martina, Diaco, Fabiana, Sessa, Rosa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112659/
https://www.ncbi.nlm.nih.gov/pubmed/33974662
http://dx.doi.org/10.1371/journal.pone.0251075
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author Filardo, Simone
Di Pietro, Marisa
Pasqualetti, Patrizio
Manera, Martina
Diaco, Fabiana
Sessa, Rosa
author_facet Filardo, Simone
Di Pietro, Marisa
Pasqualetti, Patrizio
Manera, Martina
Diaco, Fabiana
Sessa, Rosa
author_sort Filardo, Simone
collection PubMed
description Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing.
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spelling pubmed-81126592021-05-24 In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials Filardo, Simone Di Pietro, Marisa Pasqualetti, Patrizio Manera, Martina Diaco, Fabiana Sessa, Rosa PLoS One Research Article Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing. Public Library of Science 2021-05-11 /pmc/articles/PMC8112659/ /pubmed/33974662 http://dx.doi.org/10.1371/journal.pone.0251075 Text en © 2021 Filardo et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Filardo, Simone
Di Pietro, Marisa
Pasqualetti, Patrizio
Manera, Martina
Diaco, Fabiana
Sessa, Rosa
In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title_full In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title_fullStr In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title_full_unstemmed In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title_short In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials
title_sort in-cell western assay as a high-throughput approach for chlamydia trachomatis quantification and susceptibility testing to antimicrobials
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112659/
https://www.ncbi.nlm.nih.gov/pubmed/33974662
http://dx.doi.org/10.1371/journal.pone.0251075
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