Bacillus cereus acid stress responses

J.M. Mols

Research output: Thesisinternal PhD, WU

Abstract

Bacillus cereus is a ubiquitous Gram-positive organism, which frequently causes foodborne
illnesses. The widespread prevalence of B. cereus makes it a common contaminant in
fresh foods where it also can cause spoilage. To prevent food-borne diseases and food
spoilage, foods are often processed and/or preserved. In recent years, consumers’
preferences have directed to fresher and tastier foods and this has acted as a driver for food
industry to use milder processing and preservation techniques. Examples of hurdles that can
be applied to preserve foods are low pH and the addition of organic acids. B. cereus may
overcome these adverse conditions by displaying an adaptive stress response. The response
of B. cereus upon exposure to these hurdles was investigated using two model strains,
ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific
genes and differences in metabolic capacities, including a urease encoding gene cluster in
ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC
10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e.,
the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates.
However, the activity appeared to be too low to contribute to acid resistance in the strains
tested. To search for other acid resistance mechanisms, comparative phenotype and
transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic
and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the
addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of
the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed.
Furthermore, a major oxidative response was displayed, which included the induction of
several oxidative stress related genes and the production of inactivation-associated reactive
oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were
detected using fluorescent probes in combination with flow cytometry, including a newly
developed method using a specific probe that enables superoxide detection in Grampositive
and Gram-negative bacteria. The formation of ROS was also shown upon exposure
to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus
stress survival capacity revealed increased heat- and acid-resistance with cells grown and
exposed to stresses in the absence of oxygen. The excess ROS may originate from stressinduced
dysfunction of the aerobic electron transfer chain, which was indicated by the
induction of alternative electron transfer chain components upon exposure to organic and
inorganic acid shocks. Upon exposure to stress, superoxide is generated through the
premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated
rates. Subsequently, superoxide may promote the formation of other ROS, which can cause
cellular damage leading to cell death. The induction of oxidative stress related genes has
been reported in numerous other studies involving a wide range of bacteria exposed to
different adverse conditions. However, a clear relation between the formation of ROS and
the applied environmental stress was up to now not established. Secondary oxidative responses, including the formation of ROS, are possibly common bacterial responses to
severe stresses under aerobic conditions. This thesis describes genomic differences between
B. cereus strains and the acid stress response of these strains on transcriptome and
phenotype levels, including measurements of intracellular ROS. The findings in this study
can contribute to further understanding of bacterial stress responses and secondary
oxidative responses. Furthermore, the results obtained may aid to optimize and select
(combinations of) stresses to apply in hurdle technology, thus enabling design of safe,
milder food processing and preservation techniques.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Abee, Tjakko, Promotor
  • Zwietering, Marcel, Promotor
  • Moezelaar, Roy, Co-promotor
Award date4 Nov 2009
Place of Publication[S.l.
Print ISBNs9789085854944
Publication statusPublished - 2009

Keywords

  • bacillus cereus
  • stress response
  • food preservation

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