Date of Award

2005

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Department of Biological Sciences

First Advisor

Dr. Séamus Fanning

Abstract

Rapid and sensitive methods for detecting Escherichia coli 0157 in contaminated food, water and other biological samples are important in limiting and preventing the spread of this pathogen. Traditional culture based methods for bacterial identification and detection can be time consuming and labour intensive. Modem alternative methods should be faster whilst maintaining sensitivity. Immunomagnetic separation (IMS) methodology has been investigated and appears promising for rapid bacterial detection in unrelated food and environmental samples. In this study, a commercial sensor which combined IMS with electrochemiluminescence (ECL) was evaluated for the detection of E. coli 0157 in unrelated food matrices. Emitted signals were detected and measured by the ORIGENTM Analyser (IGEN, Inc.). A ruthenium labelled antibody which binds to a surface antigen when electrically stimulated, emits light of a particular wavelength and this signals the presence of the 0157 antigen. Confirmation of the culture identity after spiking included growth on selective agars (Cefixime Tellurite Sorbitol MacConkey agar [CT-SMAC] and Rainbow agar along with serological and biochemical evaluation. As a further confirmatory step, all isolates were DNA fingerprinted using PCR. Broth studies were carried out to assess the sensitivity of the ORIGENTM Analyser by spiking a non-VTEC E. coli 0157 strain (NCTC 12900) at varying bacterial dilutions. Results indicated a detection level of 1.7 x l04 and approximately 10 cfu/ml after 6 and 22 h incubations respectively, at 37°C. Sixteen unrelated food matrices were tested and six were selected for discussion on the basis of their matrix type and also interesting findings. The ORIGEN™ Analyser was able to detect E. coli 0157 initially inoculated at 1 x l04 cfu/ml in raw minced beef after 6 h incubation at 37°C, however the organism was undetectable in commercial apple juice and dry baby at these conditions. E. coli 0157 detection levels were 1 x 103 , 1 x 102 and 3 x 101 cfu/ml in mince beef, apple juice and baby powder respectively after 22 h incubation at 37°C. Ambiguous results were obtained in the broccoli, mushroom and sausage sample where positive ECL signals were obtained in unspiked samples, indicating cross-reactivity with other organisms. Enterobacter agglomerans, Enterobacter cloacae, Citrobacter freundii, Psuedomonas spp. and generic E. coli were isolated using the antibody-coated beads used with the ORIGENTM Analyser. In total, 43 competing strains were biochemically and serologically identified. DNA fingerprinting and ribotyping of all isolates by PCR was completed and comparative analysis identified 18 clusters of micro-organisms competing with the spike strain. Of this study population 6 strains were identified harbouring plasmids and a multiplex PCR indicated no E. coli 0157 virulence markers present.

Access Level

info:eu-repo/semantics/openAccess

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