Senior Honors Projects, 2010-current

Date of Award

Summer 2013

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

James B. Herrick

Crystal Scott-Croshaw

Steven Cresawn

Abstract

Streams harbor large numbers of native and introduced bacteria, which can be both recipients and donors of antibiotic resistance genes on mobile elements. Transmissible, plasmid- borne resistance to expanded-spectrum cephalosporins and fluoroquinolones is of increasing concern in clinical settings, but is rare in natural environments, such as streams and soils. Using a method developed in our laboratory, a rifampicin-resistant strain of Escherichia coli was used to capture, without culturing, two plasmids allowing growth on trypticase soy agar supplemented with the beta lactam antibiotics ampicillin and ceftazadime. Plasmids were captured directly from stream sediment taken from Muddy Creek, a stream heavily impacted by agricultural runoff. Transconjugants were also tested – using a modified “Stokes” disk diffusion method – for resistance to tetracycline, gentamycin, ciprofloxacin and ceftriaxone. One transconjugant, originally isolated from a rifampicin/ceftazidime plate, was resistant to ciprofloxacin, ampicillin, and tetracycline, in addition to ceftazadime. A second transconjugant, originally isolated from a rifampicin/ampicillin plate, was also resistant to ciprofloxacin and tetracycline, in addition to ampicillin. To confirm that the transconjugants were indeed the rifampicin-resistant capture strain, they were fingerprinted using PCR amplification of BOX repetitive sequences. The presence of plasmids was confirmed via a plasmid preparation method specifically developed in our lab for large plasmids; the plasmids appear to be in the 50-100 kb range. The recovery of transmissible plasmids encoding resistance to late-generation, human therapeutic antibiotics, such as ceftazadime and ciprofloxacin, from streams is surprising since such antibiotics are not approved for use in agriculture. It also suggests the presence of a reservoir – of unknown size and composition – of transmissible antibiotic resistance genes in stream sediment bacterial communities impacted by agricultural runoff.

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