A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance

ABSTRACT The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on...

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Autores principales:	Hanna Alalam, Fabrice E. Graf, Martin Palm, Marie Abadikhah, Martin Zackrisson, Jonas Boström, Alfred Fransson, Chris Hadjineophytou, Linnéa Persson, Simon Stenberg, Matilda Mattsson, Payam Ghiaci, Per Sunnerhagen, Jonas Warringer, Anne Farewell
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	high-throughput screening antibiotic resistance conjugation horizontal transmission plasmid horizontal gene transfer Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ec18ddae15a9475aa05aa8f10371186f
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spelling	oai:doaj.org-article:ec18ddae15a9475aa05aa8f10371186f2021-12-02T19:46:19ZA High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance10.1128/mSystems.01226-202379-5077https://doaj.org/article/ec18ddae15a9475aa05aa8f10371186f2020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.01226-20https://doaj.org/toc/2379-5077ABSTRACT The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes’ effects in liquid mating assays and complement one of the novel genes’ effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains. IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.Hanna AlalamFabrice E. GrafMartin PalmMarie AbadikhahMartin ZackrissonJonas BoströmAlfred FranssonChris HadjineophytouLinnéa PerssonSimon StenbergMatilda MattssonPayam GhiaciPer SunnerhagenJonas WarringerAnne FarewellAmerican Society for Microbiologyarticlehigh-throughput screeningantibiotic resistanceconjugationhorizontal transmissionplasmidhorizontal gene transferMicrobiologyQR1-502ENmSystems, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	high-throughput screening antibiotic resistance conjugation horizontal transmission plasmid horizontal gene transfer Microbiology QR1-502
spellingShingle	high-throughput screening antibiotic resistance conjugation horizontal transmission plasmid horizontal gene transfer Microbiology QR1-502 Hanna Alalam Fabrice E. Graf Martin Palm Marie Abadikhah Martin Zackrisson Jonas Boström Alfred Fransson Chris Hadjineophytou Linnéa Persson Simon Stenberg Matilda Mattsson Payam Ghiaci Per Sunnerhagen Jonas Warringer Anne Farewell A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
description	ABSTRACT The rapid horizontal transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. Plasmid-carrying donor strains are constructed in high-throughput. We then mix the resistance plasmid-carrying donors with recipients in a design where only transconjugants can reproduce, measure growth in dense intervals, and extract transmission times as the growth lag. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within Escherichia coli populations, by screening the Keio deletion collection in high replication. We recover all seven known chromosomal gene mutants affecting conjugation as donors and identify many novel mutants, all of which diminish antibiotic resistance transmission. We validate nine of the novel genes’ effects in liquid mating assays and complement one of the novel genes’ effect on conjugation (rseA). The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improve the longevity of current and future antibiotics. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains. IMPORTANCE The rapid transmission of antibiotic resistance genes on conjugative plasmids between bacterial host cells is a major cause of the accelerating antibiotic resistance crisis. There are currently no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by conjugation, which prevents understanding and targeting conjugation. We introduce a novel experimental framework to screen for conjugation-based horizontal transmission of antibiotic resistance between >60,000 pairs of cell populations in parallel. As proof-of-principle, we exhaustively explore chromosomal genes controlling F-plasmid donation within E. coli populations. We recover all previously known and many novel chromosomal gene mutants that affect conjugation efficiency. The new framework holds great potential for rapid screening of compounds that decrease transmission. Further, the platform can easily be adapted to explore interspecies conjugation, plasmid-borne factors, and experimental evolution and be used for rapid construction of strains.
format	article
author	Hanna Alalam Fabrice E. Graf Martin Palm Marie Abadikhah Martin Zackrisson Jonas Boström Alfred Fransson Chris Hadjineophytou Linnéa Persson Simon Stenberg Matilda Mattsson Payam Ghiaci Per Sunnerhagen Jonas Warringer Anne Farewell
author_facet	Hanna Alalam Fabrice E. Graf Martin Palm Marie Abadikhah Martin Zackrisson Jonas Boström Alfred Fransson Chris Hadjineophytou Linnéa Persson Simon Stenberg Matilda Mattsson Payam Ghiaci Per Sunnerhagen Jonas Warringer Anne Farewell
author_sort	Hanna Alalam
title	A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
title_short	A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
title_full	A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
title_fullStr	A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
title_full_unstemmed	A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance
title_sort	high-throughput method for screening for genes controlling bacterial conjugation of antibiotic resistance
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/ec18ddae15a9475aa05aa8f10371186f
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A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance

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