Assessment of molecular virulence gene profiling and antibodies for rapid detection of pathogenic Escherichia coli isolates
In recent years, there have been several fatal Escherichia coli O157:H7 outbreaks in the United States. Therefore, precise detection and identification of virulent foodborne pathogens such as E. coli O157:H7 are now a high priority for food companies and environmental agencies alike. Methods used for this purpose should ideally utilize molecular biological tools that are precise, selective, convenient, rapid and automated. The objectives of this study were: (1) to utilize the TagMan® PCR system (Applied Biosystems) to assess the presence of virulence genes in both foodborne and environmental E. coli isolates, (2) to assess the presence of E. coli toxin genes (encoding STI, LTI and LTII) in the environment, and (3) to characterize monoclonal antibodies (3C4A11 and 9C9C4) to E. coli O157:H7. TagMan ® pathogen detection kits (O157:H7, stxI and stxII) correctly identified 54 foodborne E. coli O157:H7 isolates. Ninety (15%) out of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TagMan® PCR amplification products from these ninety environmental isolates were analyzed by agarose gel electrophoresis, and 90% (81/90) of these samples contained the expected PCR product. Serotyping and ribotyping data indicate TagMan® pathogen detection kits were useful for identification of pre-screened foodborne E. coli O157:H7 isolates but may give false positive results with environmental isolates. These 90 isolates also were tested for the presence of ETEC-specific toxin genes STI, LTI and LTII. These genes were present in the environmental isolates at a frequency of 1.1% (STI and LTII) and 54.4% (LTI). Neither 3C4A11 nor 9C9C4 antibodies reacted preferentially with E. coli strains by ELISA. In Western blot experiments, 3C4A11 reacted with two protein bands of approximately 45 and 60 kDa in both 204P and non-pathogenic E. coli K12. The N-terminal sequence data from the 45kDa band indicated that this protein was either a putative E. coli flippase or BetU, a glycine betaine transporter. The molecular biological methods used in this study were indeed rapid and sensitive regarding their abilities to detect E. coli O157:H7 isolates, but should be used in conjunction with each other before a presumptive positive result is reported. ^
Major Professor: Arun K. Bhunia, Purdue University.
Biology, Molecular|Agriculture, Food Science and Technology|Biology, Microbiology