Control and Detection of Enterohaemorrhagic Escherichia coli

• Author / Creator

  Laidlaw, Anna M

• Enterohaemorrhagic Escherichia coli (EHEC) is a pathogen that causes severe disease in humans and has a low infectious dose. Since foodborne EHEC outbreaks continue to be a problem worldwide, improved control and detection methods for EHEC on at-risk foods, such as spinach and beef, are essential. While new methods of control and detection may prove to be effective in broth or buffer, it is important that they are also tested in a food matrix since food systems are more complex and may lead to different results. A novel intervention method was developed to control EHEC on spinach and lettuce with a volatile antimicrobial from mustard, allyl isothiocyanate (AITC). AITC released from its precursor sinigrin in mustard meal was limited by the activity of mustard’s endogenous enzyme myrosinase at 4°C. While the requirement of endogenous myrosinase in mustard meal to catalyze this reaction was known, decreased activity at refrigeration temperature was not, and made this antimicrobial intervention on produce impractical. Secondly, this work explored improving EHEC detection methods. Enrichment remains a necessary but lengthy step in pathogen detection methods and a major impediment to rapid detection. Therefore, decreases in the current validated enrichment times for EHEC detection were investigated with aim to reduce pathogen detection times. Individual lag phase of heat injured E. coli O157:H7 cells were measured to determine necessary enrichment times. However, decreasing enrichment times increased the probability of not detecting sub-lethally injured cells since sub-lethal cell injury significantly increased lag phases and therefore overall time to detection. Lastly, a major challenge of qPCR detection is the inability to discriminate between live and dead cells within a sample and this limitation could lead to false positive result. This is especially of importance when using this method for pathogen detection in food. The current work investigated five different detection methods to determine the concentration of viable EHEC cells on beef steaks after interventions of lactic acid, peroxyacetic acid and hot water and included 1) use of a DNA binding dye propidium monoazide (PMA) to prevent dead cell DNA amplification when used in conjunction with qPCR 2) PMA in addition to membrane emulsifying deoxycholate treatment to increase penetration of the dye and therefore increase accuracy of viable cell DNA amplification with qPCR quantification 3) mRNA and 4) rRNA qPCR quantification and 5) conventional plating. Treatment of samples with PMA and deoxycholate was reported in the literature to be successful in broth in preventing dead cell amplification in qPCR and within this research, the same treatment used within a food system confirmed these findings; however, it proved to be more complicated than in broth. While the combination treatment of PMA and deoxycholate prevented amplification of all DNA from dead cells, it also killed the sub-lethally injured cell population within samples and subsequently this rendered their DNA inaccessible for quantification in qPCR. This could lead to false negative result if the sub-lethally injured cells would have otherwise recovered and remained viable on the beef. Therefore, PMA with deoxycholate treatment requires further optimization before being considered for viable pathogen detection in qPCR. While mRNA and rRNA provided viable cell quantification in qPCR, it was more time and labor intensive and results had higher variability. Overall, conventional plating provided the most robust and reliable result for quantification of live EHEC cells on beef steaks after intervention.

• Subjects / Keywords

  • Detection
  • Controls
  • Enterohaemorrhagic Escherichia coli

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3JM23M8Z

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.