Kinetics and mechanisms of the low pH-induced inactivitation of Escherichia coli

Newly emerged foodborne pathogens with low infectious doses and remarkable capacities to tolerate environmental stress have prompted the use of food processing strategies that not only restrict the growth of contaminating microorganisms but also effect their inactivation. The rational design of such processing technologies depends on the accurate prediction of microbial responses to lethal constraints and hence on an appreciation of the mechanism(s) of microbial inactivation. This thesis describes the process of the low pH-induced inactivation of Escherichia coli populations by low pH and attempts to provide a mechanistic interpretation that is consistent with the kinetic data presented. To present an =ambiguous account of the low pH-induced inactivation of E. coli those uncertainties associated with defining and reporting microbial death were addressed. An operational definition of microbial viability, and a lack thereof, was established, and the methods employed to enumerate viable cells (traditional culture-based methods) were optimised for the recovery of low pH treated E. coil. In addition precautions were taken to eliminate kinetic artefacts. Initial experiments provided evidence of two distinct phases of inactivation in low pH-treated exponential phase populations of E. coli - an initial phase of rapid inactivation whose rate is influenced both by the stringency of the low pH treatment imposed and by temperature, and a protracted phase of much slower inactivation whose rate is independent of the severity of the lethal agent employed, and of temperatures 25°C. Subsequent studies illustrated that a third phase of inactivation, characterized by the rapid and complete loss of population viability, is observed if population viability is monitored over a sufficiently long period of time. A considerable degree of 'day to day' kinetic variability was observed among exponential phase populations of E. coil prepared from stationary phase inocula. That variability was attributed to small differences in the initial viable counts and the number of residual stationary phase cells in individual populations, and precautions were taken to minimise that variability in subsequent experiments. Those experiments indicate that the low pH tolerance of E. coli decreases with increasing cell density in purely exponential phase populations, that it increases with the physiological age of populations whose initial viable counts exceed 1 x 10 8 cfu.ml-1 reaching a maximum when purely exponential phase population are cultivated for thirty-two generation time equivalents at 25°C, and that it declines conspicuously in populations cultivated for longer periods of time. Hypotheses concerned with the basis of the shape of non-linear inactivation curves were introduced and critically evaluated in light of published data, kinetic data obtained in initial experiments, and data obtained from new experiments designed specifically to test alternative hypotheses. A case for the inherent differential resistance of individuals within a population is argued but not promoted as a comprehensive interpretation of the kinetic data presented. Finally, an E. coli mutant carrying an unmarked deletion in cfa, the gene encoding cyclopropane fatty acid (CFA) synthase was constructed, and employed with its parental strain to evaluate the role of CFAs as molecular mediators of low pH tolerance in E. coli. The results obtained indicate that CFAs play no role in mediating the intrinsic or inducible low pH tolerance of E. coli.