Senior Honors Projects, 2020-current

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Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Date of Graduation

5-8-2020

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

James B. Herrick

Abstract

Use of antibiotics in the agricultural industry introduces selective pressure and, consequently, could increase the presence of antibiotic resistant organisms in surrounding environments. One such environment is litter (manure and bedding) produced during large-scale poultry production in the Shenandoah Valley. Litter, with its microorganisms, is commonly applied to fields within the Shenandoah River watershed. Antibiotic resistance (AR) and virulence genes are potentially transmissible between organisms through horizontal gene transfer of genetic mobile elements, for which poultry litter could be a reservoir. The typical, culture-based approach to detecting and analyzing AR plasmids and other mobile genetic elements is limited due to the inability to culture, isolate, and analyze all bacteria in nearly all environments. In addition, the expense and time of extracting and sequencing plasmids from culturable isolates is great. The goals of this study were (i) to use a non-culture-dependent plasmid isolation method to isolate AR plasmids directly from poultry litter, (ii) to sequence and assemble the whole genome of the plasmid capture strain E. coli LA61RifR, and (iii) use a combination of short- and long-read sequencing and computational methods to assemble and annotate one of the captured plasmids. It was also wished to determine the antibiotic susceptibility of the captured plasmids. An exogenous plasmid capture method was used to isolate tetracycline-resistance plasmids EH1-12, some of which conferred phenotypic resistance to a range of late-generation, clinically-significant antibiotics. Of the 12 transconjugants, 11 conferred resistance to more than one antibiotic (excluding tetracycline), the most common were resistances to piperacillin and piperacillin/tazobactam. Perhaps most striking was the resistance conferred by plasmid EH11 to aztreonam, a monobactam antibiotic effective against gram negative aerobic organisms, which has rarely been observed. Other surprising resistance phenotypes included ceftazidime and ciproflaxocin which are members of the cephalosporin and quinolone drug classes, respectively. The whole genomes of both the plasmid capture strain LA61RifR and one of the multidrug resistant transconjugants, LA61RifR::pEH11, were sequenced. SPAdes and Canu were used to assemble the genomes of LA61RifR and of LA61RifR::pEH11, respectively. Ninety-seven contigs assembled from short-read sequencing data comprised the LA61RifR genome and 5 contigs assembled from long-read data comprised the LA61RifR::pEH11 genome. One contig of LA61RifR::pEH11was identified as plasmid EH11. Genes encoding antibiotic resistance, bacteriocins, and aerobactin siderophore systems were annotated with ARGannot, RAST, and Prokka . Eight repeat regions, 47 transposase genes, and two regions responsible for plasmid replication and transfer were also identified. Overall this study, through phenotypic and genotypic analyses, demonstrated that poultry litter can act as reservoir for transmissible multidrug-resistant plasmids. Genome analysis also demonstrated the potential to transfer genes that contribute to a host’s virulence. Such resistances and virulence genes, encoded on transmissible plasmids, provide advantages to infectious agents and enable their survival in poultry litter and other environments, thus possibly complicating treatment of resulting infections.

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Microbiology Commons

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