Ramirez_Cuevas_Thesis.pdf (7.1 MB)
Characterizing the influence of different factors on acid resistance phenotypes of a panel of Escherichia coli salami validation strains
thesisposted on 2023-05-27, 07:11 authored by Ramirez-Cuevas, S
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 (O157: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 O157: 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 origin. 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.
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