Development of a high-throughput platform to measure plasmid transfer frequency
Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-10-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1269732/full |
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| author | Kepa Arbé-Carton Ana Rey-Sogo Nagore Santos-Fernández Oihane Altube Carlos Garbisu Lide Arana Itziar Alkorta |
| author_facet | Kepa Arbé-Carton Ana Rey-Sogo Nagore Santos-Fernández Oihane Altube Carlos Garbisu Lide Arana Itziar Alkorta |
| author_sort | Kepa Arbé-Carton |
| collection | DOAJ |
| description | Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD600 value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay. |
| first_indexed | 2024-03-11T18:52:22Z |
| format | Article |
| id | doaj.art-1d1d6840d922408aaa909e8efb02be51 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-03-11T18:52:22Z |
| publishDate | 2023-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-1d1d6840d922408aaa909e8efb02be512023-10-11T07:31:49ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882023-10-011310.3389/fcimb.2023.12697321269732Development of a high-throughput platform to measure plasmid transfer frequencyKepa Arbé-Carton0Ana Rey-Sogo1Nagore Santos-Fernández2Oihane Altube3Carlos Garbisu4Lide Arana5Itziar Alkorta6Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, SpainDepartment of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, SpainDepartment of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, SpainDepartment of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, SpainDepartment of Conservation of Natural Resources, NEIKER-Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Derio, SpainDepartment of Applied Chemistry, University of the Basque Country (UPV/EHU), Donostia, SpainDepartment of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, SpainAntibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD600 value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.https://www.frontiersin.org/articles/10.3389/fcimb.2023.1269732/fullantibiotic resistancebacterial conjugationconjugation frequencyconjugation inhibitorsEscherichia colihigh-throughput screening platform |
| spellingShingle | Kepa Arbé-Carton Ana Rey-Sogo Nagore Santos-Fernández Oihane Altube Carlos Garbisu Lide Arana Itziar Alkorta Development of a high-throughput platform to measure plasmid transfer frequency Frontiers in Cellular and Infection Microbiology antibiotic resistance bacterial conjugation conjugation frequency conjugation inhibitors Escherichia coli high-throughput screening platform |
| title | Development of a high-throughput platform to measure plasmid transfer frequency |
| title_full | Development of a high-throughput platform to measure plasmid transfer frequency |
| title_fullStr | Development of a high-throughput platform to measure plasmid transfer frequency |
| title_full_unstemmed | Development of a high-throughput platform to measure plasmid transfer frequency |
| title_short | Development of a high-throughput platform to measure plasmid transfer frequency |
| title_sort | development of a high throughput platform to measure plasmid transfer frequency |
| topic | antibiotic resistance bacterial conjugation conjugation frequency conjugation inhibitors Escherichia coli high-throughput screening platform |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1269732/full |
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