Presentation Title

An Effective Lysis and Mobile Element Capture Method to Analyze the Antibiotic Resistome of Coastal Microbial Communities

Faculty Mentor

Elinne Becket

Start Date

23-11-2019 12:45 PM

End Date

23-11-2019 1:00 PM

Location

Markstein 205

Session

oral 3

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Microbes have the ability to transfer mobile genetic elements to one another in a process called horizontal gene transfer. These mobile elements often contain antibiotic resistance genes (ARGs), allowing for the rapid spread of antibiotic resistance. In order to study the transfer of ARGs in the coastal microbiome, it is necessary to develop an inexpensive, accessible method to enrich for ARGs and analyze patterns of their spread. To accomplish this, we created an assay which enriches for the most common mobile genetic elements, conjugative transposons and plasmids. We coupled a lysis method (that does not fragment DNA) with a plasmid isolation procedure to enrich for these elements and in turn ARGs. Bacterial communities sampled from the Carlsbad coastal waters were enzymatically lysed using four enzymes that work on a range of bacterial species. Circular DNA elements were then isolated using a plasmid preparation kit. The total extracted DNA underwent next-generation sequencing, revealing high quality sequence reads. We observed >20-fold ARG enrichment using our protocol, compared to sequencing non-enriched total community DNA. We observed antibiotic resistance genes for major classes of antibiotics that are often in urban and agricultural waste. After the development of this assay is complete, it can be used to study ARGs in transit, either naturally or in response to exposure to anthropogenic wastes.

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Nov 23rd, 12:45 PM Nov 23rd, 1:00 PM

An Effective Lysis and Mobile Element Capture Method to Analyze the Antibiotic Resistome of Coastal Microbial Communities

Markstein 205

Microbes have the ability to transfer mobile genetic elements to one another in a process called horizontal gene transfer. These mobile elements often contain antibiotic resistance genes (ARGs), allowing for the rapid spread of antibiotic resistance. In order to study the transfer of ARGs in the coastal microbiome, it is necessary to develop an inexpensive, accessible method to enrich for ARGs and analyze patterns of their spread. To accomplish this, we created an assay which enriches for the most common mobile genetic elements, conjugative transposons and plasmids. We coupled a lysis method (that does not fragment DNA) with a plasmid isolation procedure to enrich for these elements and in turn ARGs. Bacterial communities sampled from the Carlsbad coastal waters were enzymatically lysed using four enzymes that work on a range of bacterial species. Circular DNA elements were then isolated using a plasmid preparation kit. The total extracted DNA underwent next-generation sequencing, revealing high quality sequence reads. We observed >20-fold ARG enrichment using our protocol, compared to sequencing non-enriched total community DNA. We observed antibiotic resistance genes for major classes of antibiotics that are often in urban and agricultural waste. After the development of this assay is complete, it can be used to study ARGs in transit, either naturally or in response to exposure to anthropogenic wastes.