Final Examination for the Degree of PhD - Abdulhakeem Alzahrani   

Examining Committee  
Dr. Lisa Duizer, Chair  
Dr. Keith Warriner, Advisor  
Dr. Ann Huber, Advisory Committee Member   
Dr. Kari Dunfield, Department Member  
Dr. Pascal Delaquis, AAFC, B.C.,  External Examiner   

TITLE: PERSISTER FORMATION AND REVIVAL IN SHIGA TOXIN-PRODUCING ESCHERICHIA COLI SUBJECTED TO ENVIRONMENTAL STRESS CONDITIONS  
  
ABSTRACT:    
This thesis focused on characterizing the induction and revival of Shiga toxin-producing Escherichia coli (STEC) from the dormant state, along with the contribution of the persister state to survival in soil. The antibiotic selection assay was applied to isolate persisters and it was found that a consistent proportion of the population (0.01%) existed in the dormant state, irrespective of the serotype tested or the effector agents supplemented into media.  STEC persister cells exhibited temporal resistance to ampicillin that was not attributed to mutation. When a growing culture of E. coli O157:H7 was challenged with ampicillin the die-off followed multi-phasic inactivation kinetics with an initial rapid decline in the number of sensitive cells (Phase I) followed by a slower death-rate in tolerant cells (Phase II) that ultimately reached a  plateau (Phase III).  After prolonged exposure to antibiotic, the persister population entered a viable but non-culturable state (VBNC) (Phase IV). While Phase III persisters could be revived by removing the antibiotic pressure, Phase IV persisters could not be cultured despite confirmation of their viability via staining.  STEC introduced into sandy-loam soil formed a subpopulation of VBNC persisters that was distinct from the main population. In loam soil, culturable persisters could be recovered at the end of a 21-day incubation period.  Collectively, the work presented here illustrates the challenges in defining the persister state given the dynamic nature of the phenotype. The experiments in this study also confirm that the persister state contributes to long-term STEC survival in soil, although it remains to be clarified whether the persister state leads to increased virulence.