Isolation, sequencing, and characterization of four transmissible antibiotic resistance plasmids captured from bacteria in stream sediments

Author

Curtis J. KapsakFollow

Preferred Name

Curtis J Kapsak

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

ORCID

https://orcid.org/0000-0002-8735-1190

Date of Graduation

Summer 2018

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Department of Biology

Advisor(s)

James B. Herrick

Morgan Steffen

Timothy Alan Bloss

Stephen Turner

Abstract

Self-transmissible plasmids are key vectors in the transfer of resistance, catabolic, and other genes among bacteria native to environments such as streams and wetlands. The evolution of antibiotic resistance in particular is known to be powerfully affected by conjugative plasmid transfer due to the ease in which some plasmids can be horizontally transferred into a broad range of host bacteria and their ability to exchange mobile genetic elements that often contain antibiotic resistance genes.

In this study, we captured tetracycline resistance plasmids from stream sediments impacted by agricultural runoff. We selected for resistance plasmids using tetracycline, an antibiotic commonly used in agricultural operations, due to the numerous neighboring cattle pastures and poultry farms. We hypothesized that stream sediment is a “hot spot” for horizontal gene transfer due to the use of antibiotics in agricultural operations combined with runoff into streams. Selective pressures exerted on gut and fecal bacteria of farm animals may select for antibiotic resistance genes that can be horizontally transferred to native stream sediment bacteria when runoff events occur.

We characterized four transmissible, tetracycline resistance plasmids: the 71 kb IncP-1β plasmid pEG1-06, the 121 kb IncA/C₂ plasmid pCCRT11-6, and the 59 kb IncP-9 plasmids pCCP1 and pCCP2. We built upon and improved the methods developed for the preparation of plasmid DNA for sequencing using 2^nd and 3^rd generation DNA sequencers, hybrid genome assembly, annotation, and analysis. We demonstrated this process by assembling the four plasmid genomes into single, circular contiguous sequences and compared them to the closest related plasmids allowing us to classify their respective incompatibility groups, reveal the essential backbone and accessory genes present on the plasmid genomes including antibiotic resistance genes, and determine their similarity to the closest related known, existing plasmids.

Recommended Citation

Kapsak, Curtis J., "Isolation, sequencing, and characterization of four transmissible antibiotic resistance plasmids captured from bacteria in stream sediments" (2018). Masters Theses, 2010-2019. 577.
https://commons.lib.jmu.edu/master201019/577