Date of Graduation

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Mary C. Savin

Committee Member

Young M. Kwon

Second Committee Member

Kristen Gibson

Third Committee Member

Mack Ivey

Keywords

Biological sciences; Health and environmental sciences; Antibiotic resistant genes; Dissemination; Integron; Persistence; Plasmid; WWTP

Abstract

Transmissible plasmid-mediated integrons play important role in the persistence and dissemination of antibiotic resistance throughout the environment. Plasmids from 139 multi-antibiotic resistant Escherichia coli recovered from wastewater treatment plant effluent and upstream and downstream receiving stream water in Northwest Arkansas were extracted and profiled. Genes of class 1 and class 2 integrase (intI), mobilization (mob), sulfamethoxazole resistance (sul), and trimethoprim resistance (dfr) were detected using PCR and confirmed through DNA sequencing. Plasmids from almost half of the isolates (47%) were transmissible with mobF12 gene as the most frequently detected mobilization gene. Plasmid-borne class 1 with and without class 2 integrons were prevalent (46%). More isolates possessed plasmid DNA carrying single or coexistence of two or three sul genes (99.3%), and single or a combination up to five dfr genes (89.3%). The presence of integron, more than one mob gene, three sul genes, and three to five dfr genes all increased the proportion of E. coli possessing higher multiple antibiotic resistance numbers. Over an 11-day experiment, isolates persisted at concentrations greater than log 8.99 mL-1 in the presence of low (sub-inhibitory) concentrations of sulfamethoxazole regardless of integron and mobilization gene designation. In the presence of trimethoprim, isolates harboring plasmids mob+int+ were less persistent compared to isolates without either or with a gene from either group individually. Approximately 77% of E. coli possessing mobF12 disseminated plasmids into a recipient under controlled conditions at mean transfer rates of 2.14 x 10-3 (transconjugants/donors) or 7.90 x 10-3 (transconjugants/recipients). Isolates recovered from further downstream and possessing plasmid-mediated-resistance to three antibiotics with three sul genes had significantly higher transfer frequency than their counterparts. Effluent may be an important source of introduction MAR bacteria possessing transmissible plasmids and integron serving as a mechanism for accumulating antibiotic resistance genes. Escherichia coli possessing three sul genes, regardless of integron or mob gene designation appear to be highly persistent and prone to dissemination of resistance genes, and thus may contribute to the spread and persistence of multiple antibiotic resistances among bacteria in the stream environment.

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