Development of a high-throughput platform to measure plasmid transfer frequency

Frontiers in Cellular and Infection Microbiology

Cellular and Infection Microbiology

Lide Arana¹  Itziar Alkorta²  Ana Rey-Sogo²  Oihane Altube²  Nagore Santos-Fernández²  Kepa Arbé-Carton²  Carlos Garbisu³ 

[1] Department of Applied Chemistry, University of the Basque Country (UPV/EHU), Donostia, Spain;Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain;Department of Conservation of Natural Resources, NEIKER-Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Derio, Spain;

关键词: antibiotic resistance;    bacterial conjugation;    conjugation frequency;    conjugation inhibitors;    Escherichia coli;    high-throughput screening platform;    horizontal gene transfer;   

DOI  :  10.3389/fcimb.2023.1269732

 received in 2023-07-30, accepted in 2023-09-25,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Arbé-Carton, Rey-Sogo, Santos-Fernández, Altube, Garbisu, Arana and Alkorta