| 1. |
- Alalam, Hanna, et al.
(author)
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A High-Throughput Method for Screening for Genes Controlling Bacterial Conjugation of Antibiotic Resistance.
- 2020
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In: mSystems. - 2379-5077. ; 5:6
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Journal article (peer-reviewed)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.
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| 2. |
- Alalam, Hanna, et al.
(author)
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Conjugation factors controlling F-plasmid antibiotic resistance transmission
- 2018
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In: BioRxiv. - : Cold Spring Harbor Laboratory.
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Journal article (other academic/artistic)abstract
- The rapid horizontal transmission of many antibiotic resistance genes between bacterial host cells on conjugative plasmids is a major cause of the accelerating antibiotic resistance crisis. Preventing understanding and targeting conjugation, there currently are no experimental platforms for fast and cost-efficient screening of genetic effects on antibiotic resistance transmission by 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 explored chromosomal genes controlling F plasmid donation within E. coli populations, by screening the Keio deletion collection at high replication. We recover all six known chromosomal gene mutants affecting conjugation and identify >50 novel factors, all of which diminish antibiotic resistance transmission. We verify 10 of the novel genes' effects in a liquid mating assay. The new framework holds great potential for exhaustive disclosing of candidate targets for helper drugs that delay resistance development in patients and societies and improves the longevity of current and future antibiotics.
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| 3. |
- Thongsomboon, W., et al.
(author)
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Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose
- 2018
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In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 359:6373, s. 334-338
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Journal article (peer-reviewed)abstract
- Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.
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| 4. |
- Urrutia Iturritza, Miren, et al.
(author)
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An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings
- 2024
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In: Micromachines. - : MDPI AG. - 2072-666X. ; 15:6
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Journal article (peer-reviewed)abstract
- Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.
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