3. Background of the Study
Quorum sensing is the regulation of gene expression in response to
fluctuations in cell-population density. Quorum-sensing bacteria
produce and release chemical signal molecules called autoinducers
that increase concentration as a function of cell density. Their roles
include population density detection, virulence, biofilm formation,
and the maintenance of the stress response. Quorum signaling
inhibitors (QSIs) have been extensively studied for medical
applications and food safety. However, recent reports have opened
questions on potential advantages and implied a need to investigate
the unveiled characteristics of QSIs.
4. The expected anti-pathogenicity of QSIs can be
incapacitated by multiple quorum sensing systems, mutations,
efflux systems, or environmental conditions. Quorum-sensing
interference can affect bacterial viability. Recent research has
unveiled novel roles for quorum sensing systems to impact
collective signaling and global regulation of bacterial physiology.
Studies on the involvement of AI-2 quorum sensing in stress
response have provided evidence for a significant impact on
bacterial growth, survival, metabolism, adaptation, and
colonization.
5. Enterohemorrhagic Escherichia coli (EHEC) is a foodborne
pathogen responsible for outbreaks of bloody diarrhea and
hemolytic-uremic syndrome. EHEC has a LuxS/AI-2 signaling
system for the expression of its virulence factors, but there is
limited information on its impact on EHEC growth or vital
mechanisms under gastrointestinal conditions.
6. Statement of the Problem
This study aims to determine the impact of osmotic stress on the
qourum sensing mechanism of Enterohemorrghic Escherichia coli
(EHEC) and its bacterial communication and virulence.
7. Objectives
Specifically, this study aims to:
To investigate the AI-2-mediated differences in the expression
of stress response and virulence factors in EHEC under various
stress conditions and its impact.
8. Significance of the Study
This study will be relevant and valuable to the researchers,
healthcare professionals, public health organizations, food safety
regulators, and industries involved in biotechnology and
pharmaceuticals. The findings can contribute to improved disease
management, public health strategies, and the development of
novel interventions.
10. MATERIALS AND METHODS
First, we will prepare a culture of EHEC in LB broth with 20%
glycerol added and grown at 37 oC in the LB broth. After that, osmotic
stress will be introduced by adding an osmotic stress-inducing agent
which is the pH, will be adjusted by using HCl (5M) and NaOH (10N).
Anaerobic experiments will be performed in an anaerobic chamber with
an atmosphere consisting of 5% CO2, 10% H2, and 85 % N2 to the culture.
Fourth, the culture under osmotic stress conditions will be incubated.
Fifth, we will regularly measure the optical density of the culture to
monitor bacterial growth. Sixth, we will use an osmometer to confirm
the presence of osmotic stress.
11. Seventh, we will plate samples on agar plates to assess viability
under osmotic stress. Eight, we will compare the growth curve and
colony-forming units (CFUs) of EHEC with and without AI-2 to
understand the impact of quorum sensing. Finally, we will then
analyze the data to determine the influence of AI-2 quorum sensing
on EHEC viability under osmotic stress conditions.
12. STATISTICAL METHOD
Statistical Analysis
The experimental data will be analyzed by a one-way analysis
of variance (ANOVA) and t-test using GraphPad Prism 6. the
comparative analysis between test sample and control sample will be
carried out fold change. For a DEG set, hierarchical cluster analysis
will be performed using complete linkage and Euclidean distance as a
measure of similarity. Gene-Enrichment and Functional Annotation
analysis for a significant probe list will be performed using DAVID.
