Effect of Carbon Dioxide on

Neurotoxin Gene Expression

in Nonproteolytic Clostridium
botulinum Type E
(2007)

Authors: Ingrid Artin, Andrew T. Carter,

Elisabet Holst, Maria Lovenklev, David R.
Mason, Michael W. Peck, and Peter Radstrom
Presenter: Hamza Shah

 Terminology:

Terminology
and
Abbreviatio
ns:

Clostridium botulinum: spore forming, anaerobic, toxin

producing bacterium
Modified atmospheric packaging: packaging that
contains a selective gas inside for antimicrobial
applications
Cnt: one of the genes responsible for toxin production in
clostridium botulinum

Abbreviations:

MAP: modified atmospheric packaging

BoNT: Botulinum neurotoxin
RTE: ready to eat foods
OD: optical density
RE: relative expression

 Related to Food Industry:

Consumption of pre-formed neurotoxins results in
bacterial intoxication
Modified-Atmospheric Packaging (MAP) is used to resist
microbial issues

Introduction
:

MAP has been very useful against aerobic microbes, but not
as effective against anaerobic microbes (e.g. C. botulinum)
CO2 is often issued as the primary gas in MAP

C. Botulinum thrives by degrading sugars and is able to

produce toxins in temperatures as low as 3C.
The mild heat treatment exposed to foods is often enough
to kill vegetative cells, but not spores
In some cases the mild heat can cause the initiation of
germination

Introduction
Continue:

A previous study done on C. botulinum type Bs ability

to express the cntB gene and the production of BoNT/B
showed:
70% CO2 atmosphere 5 folds greater expression of the
gene and production of the toxin than in a 10% CO2
atmosphere
35% CO2 atmosphere showed an intermediate result

Bacterial
Growth
Curve:

Purpose:

How does the concentration of carbon dioxide effect

the growth of C. botulinum type E (strain ATCC 9564)
and the expression of the cntE gene used to produce
BoNT/E.

 Bacterial strains and culture conditions:

Tryptone-peptone-yeast extract (TPY) broth was used in
the study for bacterial growth
Bacterial growth was measured using
spectrophotometer with an optical density of 620
nm.

Methods
Overview:

qRT-PCR was used to quantify the cntE gene

Enzyme-linked immunosorbent assay (ELISA) was
used to quantify the neurotoxin production

Results:

Growth and
expression under
Ideal Conditions

Results:

Rate of Growth
Influence by CO2
Levels

Results:

CO2 and Growth

Phase Relation on
cntE and
Neurotoxin
Production

Conclusion:

Growth of C. botulinum is slowed with the increase in

carbon dioxide concentrations
The expression of cntE by nonproteolytic C. botulinum
type E strains ATCC 9564 showed a relationship with
the growth phase of the bacterium and the amount of
CO2 present
The expression of cntE was at its highest between late
exponential and stationary phase, and then it
decreased rapidly
The steady decrease and no arrest to the expression of
the cntE gene may be due to the continued
transcription of the cntE gene, and/or the bacterial cells
entering the stationary phase as a different time
Even though there is a slow in growth, an increase in
the C. botulinum toxin is observed with and increase in
CO2 concentrations

 From the results produced from this study it can be

concluded that the use of CO2 can be responsible for an
increase in production of neurotoxins in nonproteolytic
C. botulinum type E

Discussion:

These result should lead to less use of CO2 in packaging

for foods that are prone to C. botulinum contaminations
Since CO2 is a one of the major gases used in MAP for its
antimicrobial effects the results published by this study
will effect the use in food/environments prone to C.
botulinum.

Some previous studies examined the behavior of C.

botulinum in the presence of sorbic acid and sodium
nitrite, and both showed a reduction in growth rate and
expression of the cntE
Carbon dioxide has induced similar results in different
types of bacterium (e.g. Staphylococcus aureus, Vibrio
cholera, and Bacillus anthracis)

 Future studies:

Future
Potential:

Explore the cellular mechanisms how CO2 is actually

having an cellular effects that is resulting in macrochanges (e.g. rate of growth and toxin production)
If specific food compositions, preservatives, or
temperature have an effect on growth and/or toxin
production with the presence of high concentration of
CO2

Literature and online:

Centers for Disease Control and Prevention. Centers for
Disease Control and Prevention, 25 Apr. 2014. Web. 30
Jan. 2015.
<http://www.cdc.gov/nczved/divisions/dfbmd/diseases/bot
ulism/>.

References:

"Effect of Carbon Dioxide on Neurotoxin Gene Expression in

Nonproteolytic Clostridium Botulinum Type E." Applied
and
Environmental Microbiology 74.8 (2008): 2391-2397.
Print.
"U.S. Food and Drug Administration." BBB. Web. 30 Jan.
2015.
<http://www.fda.gov/Food/FoodborneIllnessContaminants/
CausesOfIllnessBadBugBook/ucm070000.htm>.
Books:
Hauschild, Andreas H. W. Clostridium Botulinum: Ecology
and Control in
Foods. New York: M. Dekker, 1993. Print.

References
(Images):

Thank you,