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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 RadstromPresenter: Hamza Shah

The study was conducted at the Lund University, located in Sweden.

Proteolytic are able to break proteins 1

Terminology and Abbreviations: Terminology: Clostridium botulinum: spore forming, anaerobic, toxin producing bacteriumModified atmospheric packaging: packaging that contains a selective gas inside for antimicrobial applicationsCnt: one of the genes responsible for toxin production in clostridium botulinum

Abbreviations: MAP: modified atmospheric packagingBoNT: Botulinum neurotoxin RTE: ready to eat foodsOD: optical density RE: relative expression

BoNT the toxin blocks the acetylcholine release prevents muscle contraction causing paralysis

Endotoxin: a toxin that is present inside a bacterial cell and is released when the cell disintegrates.

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Introduction: Related to Food Industry:Consumption of pre-formed neurotoxins results in bacterial intoxicationModified-Atmospheric Packaging (MAP) is used to resist microbial issuesMAP 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 sporesIn some cases the mild heat can cause the initiation of germination

Consumption of neurotoxins, botulinum toxinModified atmospheric packaging is used to resist microbial growthGood against aerobic but not that good against anaerobicC. botulinum lives by degrading sugars and can survive in environment as low as 3C.C. botulinum are spore forms and for that reason mild heat treatment is able to kill vegetative cells but the spores.

CO2 is used due to its antimicrobial quality

Food-borne botulism is caused by consuming preforming neurotoxins in food. As little as 30ng of the toxin can be lethal

Preservatives such as salts, nitrites, sugars

Many times if a food company is responsible for deaths there are penalties, imprisonment, fines, and many times the company ends up shutting down

Often times the vegateatied cells are killed some small doses of thermal heat, but the spores survive and can cause issues

There are about 50 cases of C. bot that are related to foods3

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

A previous study done with C. botulinum B showed that higher the levels of CO2 higher the gene expression and the production of the toxinBy 5 foldsThis should be kept in mind4

Bacterial Growth Curve:

Important to recreate this curve in your mind since this curve since a lot of the terms here are used throughout the study. 5

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.

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Methods Overview:

Bacterial strains and culture conditions: Tryptone-peptone-yeast extract (TPY) broth was used in the study for bacterial growthBacterial growth was measured using spectrophotometer with an optical density of 620 nm. qRT-PCR was used to quantify the cntE gene Enzyme-linked immunosorbent assay (ELISA) was used to quantify the neurotoxin production

Quantitative reverse transcriptase PCR (QRT-PCR or qRT-PCR) is a PCR technique used to determine the amount of cDNA in a sample. It is the most commonly used form of quantitative PCR (qPCR). This technique is also called real-time reverse transcriptase PCR7

Results:Growth and expression under Ideal Conditions

Figure 1: This was done at 30 degrees Celsius, with 10% H2, 10% CO2, and 80% N2 (anaerobic environment)

Shows the growth of the bacteria (measured by optical density), with its relation to time Shows the C. bot toxin present over time It also shows the amount of expression of the cntE gene

So what do we see: (1) The bacteria grows for about 10 hrs and then hits its stationary phase(2) The peek in the cntE expression happens between growth and stationary phase then it decreases rapidly(3) the toxin production increases rapidly with the cntE gene expression (also with the growth phase), and once the gene expression halts the amount of toxin present starts to increase at a slower rate

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Results: cntE mRNA Half-life

Here we can observe the relation between the mRNA accumulation an its rate of gene expression

The rate of cntE mRNA breakdown was measured to conform the close relation between mRNA accumulation and its rate of gene expression.

This graph helps us figure out the half-life of the mRNA product for the toxin, and it has a half-life of about 9 minutes.

We can see that the mRNA for the cntE gene reaches less than 1% within 1 hour.

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Results: Rate of Growth Influence by CO2 Levels

This shows the proliferation of C. bot within an environment with certain carbon dioxide levels

We can see that the higher the concentration of CO2 slower the growth of the bacterium - 10% CO2 reached its peak at about 9 hrs- 35% CO2 reached its peak at about 14 hrs- 70% CO2 reached its peak at about 17 hrs

We can also see that overall the CO2 is having an antimicrobial effect.

They all hit the transition been the growth and the stationary phase later in the ideal anaerobic environment, this clarifies that as CO2 concentration increases the rate of growth decreases - 10

Results: Expression of cntE According to Growth Stage and CO2 levels

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Results: CO2 and Growth Phase Relation on cntE and Neurotoxin Production

Here it can be seen that the expression of cntE is affected by both the stage of growth and the concentration of CO2 present in the bacteria environment 12

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 timeEven though there is a slow in growth, an increase in the C. botulinum toxin is observed with and increase in CO2 concentrations

The increase in the toxin production is what the food industry cares about13

Discussion: 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 EThese result should lead to less use of CO2 in packaging for foods that are prone to C. botulinum contaminationsSince 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 cntECarbon dioxide has induced similar results in different types of bacterium (e.g. Staphylococcus aureus, Vibrio cholera, and Bacillus anthracis)

The increase in the toxin production is what the food industry cares about14

Future Potential: Future studies: Explore the cellular mechanisms how CO2 is actually having an cellular effects that is resulting in macro-changes (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

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References: Literature and online:Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 25 Apr. 2014. Web. 30 Jan. 2015. ."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. .Books:Hauschild, Andreas H. W. Clostridium Botulinum: Ecology and Control in Foods. New York: M. Dekker, 1993. Print.

References (Images):

Thank you,

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