Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology

Major Professor

Daniel Lim, Ph.D.

Co-Major Professor

Andrew Cannons, Ph.D.

Committee Member

Valarie Harwood, Ph.D.

Committee Member

Mylein Dao, Ph.D.

Keywords

Salmonella, Ribotyping, PFGE, Antibiotic resistance, Integrons

Abstract

The genus Salmonella, comprised of 2400 serotypes, is one of the leading causes of foodborne illnesses in the US and has been used for the deliberate contamination of food. A rapid system for detection, isolation, typing and antibiotic susceptibility profiling is essential for diagnosis and source tracking in natural outbreaks or a bioterrorism event. Pure culture is essential for molecular typing and antibiotic resistance testing. The virulence and the resistance mechanisms of Salmonella are rapidly evolving and many are still unexplained. The first aim of the study was to rapidly detect and isolate Salmonella from intentionally contaminated food. The second aim was to build a DNA fingerprinting database for accurate identification of the subtype. The third objective was to study the antibiotic susceptibility patterns and the underlying mechanisms of resistance. A correlation between the DNA subtypes and antibiograms was hypothesized. An association between the resistance determinants and pathogenicity genes was expected. A total of 114 isolates including environmental and clinical sources were tested. General and selective enrichments and immunomagnetic separation (IMS) were tested for rapid detection and isolation of Salmonella from eight food groups. Isolates were subtyped by pulsed field gel electrophoresis (PFGE) and automated RiboPrinter®. Resistance to 31 drugs was tested by the Sensititre® system and integrons were identified by PCR. The association between virulence and resistance was verified by Southern hybridization. Of the three genes tested, ompF was found to be the most reliable target for identifying Salmonella subspecies I, III and IV. Detection by real time PCR after enrichment in buffered peptone water and isolation by IMS provided the fastest results. Sixty two ribotypes and 74 pulsotypes were observed for the 100 isolates subtyped. Sixty isolates were resistant to one or more antimicrobials and 12 had class-1 integrons. In conclusion, pure culture was achieved in 25 hours by IMS. Ribotyping, a comparatively rapid technique was found to be ideal for initial identification. PFGE, which was more discriminatory, was appropriate for source tracking. Contrary to the original hypothesis, no correlation between subtyping and antibiograms was observed and no association of integrons with the virulence genes tested was demonstrated

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