Date of Completion

11-30-2018

Embargo Period

11-30-2019

Keywords

antibiotic resistance, hirudotherapy, Aeromonas, Flavobacterium, minION, assembly, microbiome

Major Advisor

Joerg Graf

Associate Advisor

Jonathan Klassen

Associate Advisor

J Peter Gogarten

Associate Advisor

Victoria Robinson

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

For this thesis work, the possibilities of next-generation sequencing were explored in the context of two different host-bacterial symbioses. The first concerns the symbiotic Aeromonas population living in the gut of the medicinal leech, an FDA-approved medical device used on patients post-surgery. Despite reports of infections caused by fluoroquinolone (FQ) resistant Aeromonas, a thorough study linking the gut symbionts to infectious strains was lacking. Genome and gyrA sequencing along with wet lab experiments allowed us to track changes in Aeromonas in response to low FQ contamination. This work provides evidence that Aeromonas pathogens followed a nosocomial route of infection and addresses important ecological questions about the effects of low-level antibiotics.

The second symbiosis studied was that of two fast-spreading pathogenic species in the genus Flavobacterium that cause devastating losses in rainbow trout farms. One farm in the U.S. was used as a case study for a USDA consumer safety grant. The goal was to develop a high-throughput amplicon sequencing method to detect these two species at the farm and determine their in situ origins. We categorized sequenced 16S rRNA gene reads into well-separated species groups, which simultaneously provided a way to characterize the microbial community composition in these samples. We established a large collection of farm samples including swabs from farm surfaces. The species’ reads were detected at the farm and interestingly, at the upstream spring which supplies this farm with water.

Both of these thesis studies have benefited from high-quality Aeromonas and Flavobacterium genome assemblies, which are especially important for annotating difficult genetic regions, including GC-rich and long repetitive areas. A third study incorporated long-read sequencing technology, the Oxford Nanopore MinION, to acquire long-reads and improve assemblies created by short-read only assemblers.

This thesis work contributes to a growing body of knowledge on the in vivo effects of antibiotic contamination in our daily environments and how amplicon sequencing can be used to detect pathogenic species in aquaculture. It is an example of how the ongoing advancements in high-throughput sequencing and bioinformatics can help us explain pathogenicity, determine a likely origin of infectious agents, and improve the genomes of difficult-to-assemble microbes.

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