Library Open Repository

Characterizing the influence of different factors on acid resistance phenotypes of a panel of Escherichia coli salami validation strains

Downloads

Downloads per month over past year

Ramirez-Cuevas, S (2010) Characterizing the influence of different factors on acid resistance phenotypes of a panel of Escherichia coli salami validation strains. PhD thesis, University of Tasmania.

[img]
Preview
PDF (Front matter)
front-_Ramirez_...pdf | Download (354kB)
Available under University of Tasmania Standard License.

[img] PDF (Whole thesis)
Ramirez_Cuevas_...pdf | Request a copy
Full text restricted
Available under University of Tasmania Standard License.

Abstract

Acid resistance of Escherichia coli (E. coli) is relevant to food safety, as certain
foods rely on acidity for preservation and pathogen inactivation. In Australia, food
borne disease outbreaks associated with pathogenic E. coli and involving salami and
other fermented food products have been well documented. The ability of pathogenic
E. coli to survive through the food chain, together with its low infectious dose, is
hypothesized to be partly due to its ability to resist acid challenge. In the last two
decades numerous laboratories worldwide have worked to understand and
characterize E. coli acid resistance. These laboratories have reported that the
mechanisms used by E. coli against acid challenge are yet to be completely
understood and many knowledge gaps exist. Careful analysis of the current
published data on the E. coli acid resistance reveals substantial differences in the
way experiments are conducted. These include differences in media, acid type and
pH values. The hypothesis and central argument of this thesis is that slight variations
in the parameters used for in vitro acid challenges might have a significant effect on
the acid resistance profiles of E. coli cells. More specifically this project focuses on
demonstrating that slight variations in the parameters for an acid challenge
experiment can vary its outcome and ultimately the interpretation and application of
the experiment results. The document's central argument is the contribution of
knowledge regarding the response of E. coli cells to in vitro acid challenge.
The acid response of a panel of 5 E. coli salami validation strains and two controls
was studied using a proteomic gel-based approach. Proteomic analysis revealed
time-dependent protein expression differences following acid challenge. It was concluded however that such approach was too coarse to reveal differences in
protein expression profiles that may be associated with differences in the acid
resistance phenotypes amongst E. coli strains studied.
In order to obtain a better understanding of the phenotypes of the salami validation
strains, a series of small experiments were performed. These experiments
investigated the effects that storage, acid adaptation, type of acid used; salt and
temperature have on the ability of E. coli strains to tolerate acid challenge. An
interesting finding from these experiments was the fact that a significant percentage
of cells in cultures challenged with acid may enter a viable but non-culturable state
following acid challenge. Using fluorescent microscopy, it was demonstrated that
following acid exposure the number ofviable cells using plate counts as an indicator
was considerably less than when assessed using fluorescent microscopy.
In addition, it was also demonstrated that the inclusion of 1% glucose to the growth
media, increased the capacity of cultures to withstand acid challenge when compared
with cultures grown without the addition of glucose.
Subsequently, two E. coli strains (0 157:H7 and an Australian salami validation E.
coli strain) were tested for their ability to tolerate acidic conditions, in broth and in
semi-solid agar. In this study a blend of carrageenans was developed to assess
survival of E. coli in an acidic semi-solid environment. The novel blend allowed for
a homogeneous semi-solid-agar to remain stable at pH 4.5 following the addition of
acid. Studies revealed that survival of both strains were affected by the physical state
of the challenge media. Bacterial cells, regardless of pathogenicity, appeared to have a higher acid resistance when challenged m the semi-solid media than when
challenged in broth.
A panel of 12 E. coli strains (pathogenic and non-pathogenic) was challenged
utilizing four different media (BHI, TSB, NB and MM), four different salt
concentrations (0.5%, 3.5%, 8.5% and 12.5%) and three different acid treatments
varying in pH values (2.4 to 4.6) and L-lactic acid concentrations (0.42 to 3.7 g/L).
Results demonstrated that the acid resistance profiles within the panel of strains
significantly varied across the treatments. Of the components that altered acid
resistance, NaCl and media type had the largest effect. In particular, BHI broth
appeared to have the greatest protective effects across all treatments. Buffer
composition altered the amount of acid required to reach a set pH level and
ultimately the amount of free acid in the solution. In some cases individual strains
that appeared to be acid resistant under a certain set of conditions displayed no
ability to resist acid under another.
The acid resistance of the pathogenic E. coli 0 157: H7 (Sakai, ATCC BAA-460) was
characterized on a genetic level using microarray analysis. Genetic expressiOn
profiles of exponential phase cells (acid sensitive), stationary phase cells (acid
resistant) and stationary phase cells challenged with acid in the presence or absence
of 3.5% NaCl were analyzed. Transcriptome analysis revealed the presence of
previously identified acid-resistance-associated genes for stationary phase cultures
prior to and following acid challenge. Transcripts for the challenged cultures
revealed the presence of a large quantity of up-regulated genes of bacteriophage
ongm. These results suggest that the given challenge conditions were capable of triggering the activation of the bacteriophage inserted genome. Such activation might
lead to toxin production or the promotion of genes that might be advantageous for
intestinal colonization.
The hypothesis of this thesis was supported by the results. The outcome of various
acid challenges was altered with changes of the experimental parameters. The
experimental data demonstrated that the parameters of in vitro acid challenge
experiments have an independent effect on the acid resistant phenotypes of
Escherichia coli cells. E. coli strains that might appear resistant to acid challenge
within a panel of strains under one condition, might appear to be acid-sensitive if the
conditions of the challenge are changed. The data also demonstrated that changes of
the physical state of the challenge media (liquid versus semi-solid) can have a direct
effect on acid survival of. While at a genetic level, there significant changes were
observed when NaCl is introduced in an acid challenge.
The results suggest that the interaction of environmental factors play an important
role in setting acid resistance levels of E. coli strains. These observations argue the
closer attention must be paid to the parameters used in acid challenge experiments as
variations on experimental outcomes may arise due to the possible secondary effects
of secondary variables.

Item Type: Thesis (PhD)
Keywords: Ramirez, CS
Date Deposited: 23 Mar 2014 23:13
Last Modified: 11 Mar 2016 05:53
Item Statistics: View statistics for this item

Actions (login required)

Item Control Page Item Control Page