The role of molecular methods in the detection of pathogens in food

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Title: The role of molecular methods in the detection of pathogens in food
Author: Stevens, Kelly A.
Advisors: Lee-Ann Jaykus, Committee Chair
Wondwossen Gebreyes, Committee Member
Craig Altier, Committee Member
MaryAnne Drake, Committee Member
Abstract: The research described here addresses several issues associated with testing food samples for the presence of pathogens. Based on three different perspectives, the following major objectives are noted: (i) the use of molecular amplification methods to detect Listeria monocytogenes and Salmonella enterica serovar enteritidis directly from food samples, bypassing the need for cultural enrichment; (ii) evaluation of automated ribotyping for the differentiation of Salmonella enterica serovar Typhimurium strains; and (iii) development of a decision model to evaluate a pathogen testing decision in industry. In the first study, we developed and evaluated a method for the direct detection of foodborne pathogens without prior cultural enrichment. Eleven-gram samples of plain nonfat yogurt or mild cheddar cheese were seeded with L. monocytogenes or S. enterica Enteritidis at levels of 10, ², - 10, ⁶, CFU per sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9,700 x g) followed by detection using both cultural and molecular methods. Molecular detection limits of 10, ³,and 10, ¹, CFU per 11 g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. In a second and related study, alternative nucleic acid preparation methods were evaluated to improve the direct detection of Listeria monocytogenes from a frankfurter matrix. Using a combined concentration / extraction sample preparation, 11-g samples were concentrated 100-fold to 100 μl with recovery of target nucleic acids which were further purified by column chromatography and specific bacterial rRNA isolation using two magnetic bead-based technologies, i.e., MICROBEnrich® and MICROBExpress®. PCR detection limits were 10, ⁵, CFU/11g sample and RT-PCR detection limits were 10, ³, CFU/11g. Detection limits were improved an additional 10-fold (to 102 CFU/11g) when extracted RNA was further purified using MICROBExpress®. The third study evaluated the RiboPrinter®l microbial characterization unit for its ability to differentiate thirty-nine isolates of multi-drug resistant Salmonella enterica serovar. Typhimurium. Phenotypically, the isolates varied by phage type and marginally by antibiotic resistance pattern. However, the strains could not be meaningfully differentiated from one another using ribotyping, suggesting that in this case, phenotypic methods may be more discriminatory than this molecular typing method. In the final study, decision analysis tools were used to develop a model addressing the issues encountered when making a testing decision. From a food processors perspective, three potential consequences of foodborne pathogen contamination were considered as elements of the decision, e.g., no consequences, regulatory recall without disease, and disease outbreak. Accordingly, the inputs of the model were (i) costs associated with food-borne contamination (business and health related costs); (ii) reliability of testing; and (iii) prevalence of contamination. In general, the model indicated that testing for highly prevalent pathogens may provide an improvement in food safety but end product testing for pathogens of low prevalence should be carefully considered and may not be justified due to limited return on investment. These efforts represent continued progress in harnessing the power and diversity of molecular methods for the identification and characterization of foodborne pathogens. The development of systematic approaches to making testing decisions is also justified and needed by the industry. Taken together, these studies add to our understanding of the impediments to application of rapid methods for the detection of foodborne pathogens, and provide some solutions to facilitate the practical use of these methods in the future.
Date: 2004-03-18
Degree: PhD
Discipline: Food Science

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