Title

Genomic Resequencing of Iron (II) Resistant Strains of Escherichia Coli

Student Classification

Senior

Faculty Mentor

Dr. Misty Thomas

Department

Biology

Document Type

Poster

Publication Date

Spring 2019

Abstract

Bacterial resistance to antibiotics have caused an increase in usage of heavy metals as antimicrobials in an attempt to control the rate of antibiotic resistance. However, very few studies have been done to address the use of heavy metals and the genetic adaptations that occur as a result. Understanding the genetic changes that result from adaptation will help to elucidate the mechanism of resistance used by gram-negative organisms. Research objective: To assess the genetic changes that result in the genome of E. coli as a result of adaptation of iron (II) resistance. Methods: Evolved strains were pellet by Dr. Ewunkem and sent to us for extraction and sequencing. Extractions were done using the methods outlined in the standard Omega Bio-teck Bacterial DNA Extraction Kit protocol and eluted in 50mL elution buffer. DNA samples were then quantified using the fluorescent based dsDNA kits from Promega and measured using a quantifluor. Library preps were conducted using the DNA flex kit from illumine and sequenced on Illumina MiSeq at JSNN. Fastq files were then processed using the Breseq pipeline to look for variation. Results: Genomic sequencing shows single nucleotide changes in ptsP which is involved in nitrogen metabolism, fecA which is a ferric citrate outer membrane transporter, ilvG involved in valine biosynthesis and finally in rpoB, the beta-subunit of RNA polymerase. Conclusions: The majority of genes identified to carry mutations are primarily involved in metabolism, indicating that these strains may be decreasing metabolic rates in order to potentially prevent entry of excess iron into the cell. In addition, the mutation in fecA may indicate that transport of iron into the cell may be decreased, also in order to prevent survival. Future directions: Due to the requirements of high levels of efflux, metal resistance is often associated with antibiotic resistance. Therefore, the next goal is to take these iron resistant strains in order to see if they are also resistant to common antibiotics. Broader Goals: By understanding mechanisms of metal resistance before they appear in nature makes it easier to develop methods to counteract resistance before it is prominent.

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