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In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation
Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transco...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9410318/ https://www.ncbi.nlm.nih.gov/pubmed/36013391 http://dx.doi.org/10.3390/life12081212 |
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author | Gibert, Marta Jiménez, Carlos J. Comas, Jaume Zechner, Ellen L. Madrid, Cristina Balsalobre, Carlos |
author_facet | Gibert, Marta Jiménez, Carlos J. Comas, Jaume Zechner, Ellen L. Madrid, Cristina Balsalobre, Carlos |
author_sort | Gibert, Marta |
collection | PubMed |
description | Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry. |
format | Online Article Text |
id | pubmed-9410318 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-94103182022-08-26 In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation Gibert, Marta Jiménez, Carlos J. Comas, Jaume Zechner, Ellen L. Madrid, Cristina Balsalobre, Carlos Life (Basel) Article Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry. MDPI 2022-08-10 /pmc/articles/PMC9410318/ /pubmed/36013391 http://dx.doi.org/10.3390/life12081212 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Gibert, Marta Jiménez, Carlos J. Comas, Jaume Zechner, Ellen L. Madrid, Cristina Balsalobre, Carlos In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title | In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title_full | In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title_fullStr | In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title_full_unstemmed | In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title_short | In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation |
title_sort | in situ monitoring and quantitative determination of r27 plasmid conjugation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9410318/ https://www.ncbi.nlm.nih.gov/pubmed/36013391 http://dx.doi.org/10.3390/life12081212 |
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