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

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Brown, Janelle L. (Janelle Louise) (2003) Kinetics and mechanisms of the low pH-induced inactivitation of Escherichia coli. PhD thesis, University of Tasmania.

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Abstract

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.

Item Type: Thesis (PhD)
Copyright Holders: The Author
Copyright Information:

Copyright 2002 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 2003. Library has additional copy on microfiche.

Date Deposited: 25 Nov 2014 01:00
Last Modified: 21 Jun 2016 02:12
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