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Contents
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1 Introduction
2 Cell Structure
3 Genome Structure
4 Metabolism
5 Formation of Endospores
6 Pathology
8 Conclusion
9 References
Introduction
By Rebecca Dann
Clostridium botulinum is a gram-positive, rod-shaped bacterium and pathogen that is
prevalent in marine and soil environments around the world (Figure 1).
Cell Structure
Clostridium botulinum is a spore-forming, gram-positive firmicute. These bacteria have the
ability to form spores even when in a thriving environment. The formation of spores does not
serve the sole purpose of protecting and storing the cell, but it also provides the means for the
bacteria to release neurotoxins when they begin germinating. The factors to initiate
germination greatly vary and depend on where the spore is located (Webb et al. 2011). In
many food products, spores are activated by salt and pH concentrations, or by the addition of
chilled or heated temperatures for either storage or preparation. A study by Webb et al.
demonstrated that the rate and amount to which spores germinate is directly related to the
proximity of spores to each other. The larger the density of spores in the same location, the
more germination will occur (Webb et al. 2011). Through designed experiments, a model
could be generated to predict the growth rate of the bacterium and determine what conditions
initiate the activation of a spore (Webb et al. 2011). This could lead to the discovery of
improved prevention techniques to ensure that food is safe and free of Clostridium botulinum.
Genome Structure
This species can be categorized into four genetically and physiologically distinct groups that
all produce different types of neurotoxins. The Clostridium botulinum in Group I produce the
toxin types A, B, and F. They are proteolytic and have an optimal growth temperature of 37
degrees Celsius but can thrive in temperatures ranging from 12.8-48 degrees Celsius (Peck et
al. 2010). This group of toxins most commonly causes infant botulism and contaminates
food. Group II consists of Clostridium botulinum that produces type B, E, and F toxins and is
also mostly found in contaminated food products. They are nonproteolytic and develop at a
lower optimal temperature of 25 degrees Celsius. Group III contains toxins type C and D and
are most commonly associated with botulism in animals. Group IV produces toxin type G.
The genome for the group I bacteria has been fully sequenced revealing that this bacteria
contains chromosomal DNA and a bacteriocin-encoding plasmid (Figure 2).
Metabolism
Clostridium botulinum can fully metabolize amino acids and chitin, and can partially
metabolize several other polysaccharides. Proteases secreted by the cell can cleave
surrounding polypeptides so the bacterium can digest smaller molecules (Sebaihia et al.
2007). There is evidence in the genome that this bacteria uses fermentation pathways that
utilize a series of coupled oxidation-reduction reactions to acquire energy, where the
oxidation of one amino acid is directly coupled to the reduction of another amino acid.
Studies have compiled evidence that the amino acid glycine is first reduced by a glycine
reductase complex and then oxidized by a glycine cleavage system (Sebaihia et al. 2007). It
has been shown that this bacteria ferments glycine, proline, phenylaline, and leucine. The
remaining energy is obtained by sugar metabolism. Clostridium botulinum uses the second
most abundant sugar, Chitin, as its second main source of energy (Sebaihia et al. 2007).
Chitin is commonly found in the exoskeletons of arthropods and insects, such as lobsters and
mollusks, and in the cell walls of fungi, both of which are prevalent in the marine and soil
environments in which this bacteria resides (Figure 3).
Formation of Endospores
A key characteristic of Clostridium botulinum is their ability to form dormant spores that can
withstand extreme conditions, such as high pressure, UV light, and heat treatments.
Sporulation is the reason for why this bacterium is such a food hazard and poses such a threat
to those who are exposed to the species. To ensure that food is free of proteolytic botulinum,
it must be treated with a temperature of at least 121 degrees Celsius for a minimum of three
minutes; this process has been termed the botulinum cook. Spore germination in proteolytic
Clostridium botulinum is initiated by the presence of the amino acid L-alanine, which
activates germinant receptor proteins located in the inner membrane of the spore. These
proteins are encoded by three germinant receptor operons that are expressed during
germination (Peck et al. 2010).
Spores of the non-proteolytic Clostridium botulinum are less heat resistant in comparison
with the proteolytic bacteria. A temperature range of only 80-85 degrees Celsius for several
minutes prevents spore germination. Unlike the proteolytic bacteria, this strain of botulinum
can be activated by the presence of a lysozyme, an enzyme that is prevalent in common
foods. The lysozyme can enter the protective spore coat and hydrolyze the peptidoglycan
layer of the cortex (Peck et al. 2010). This allows the core, which contains the bacterias
DNA, to be exposed to its surroundings, which activates germination. Spore germination in
non-proteolytic botulinum can also be initiated by the presence of two amino acids, such as
L-alanine and L-lactate. The germinant receptor proteins that allow the amino acids to initiate
a response are only encoded by one germinant receptor operon instead of three as in the
proteolytic botulinum (Peck et al. 2010).
Pathology
If an adult individual is already intoxicated by the botulinum neurotoxin, equine antitoxin can
be administered to help rid of the symptoms. The drug inhibits the neurotoxins that have yet
to bind to the nerve receptors from doing so, decreasing the toxins ability to cease muscle
movement. Some cases of botulism can take months to years for a full recovery, and fatality
occurs in approximately 5-10% of the cases reported (Peck et al. 2010). The cost to treat this
devastating disease can be very expensive as it can be a long process. Cases of infant
botulism can be treated with Botulism Immune Globulin Intravenous, which provides
essential fluids and nutrients.
Conclusion
While there are currently successful methods to rid of Clostridium botulinum from
contaminated food sources, further research is still needed to ensure that this bacteria presents
no threat, as botulism is one of the most devastating diseases. By gaining a more
comprehensive understanding of the structure of the Clostridium botulinum neurotoxins and
the mechanisms for why and how they are synthesized by the bacteria can help generate ideas
as to how to cure this disease and prevent it from even occurring.
References
1) Webb, M., Stringer, S., Le Marc, Y., Baranyi, J., and Peck, M. 2011. Does proximity to
neighbors affect germination of spores of non-proteolytic Clostridium botulinum? Food
Microbiology: 32, 104-109
2) Malakar, P.K., Barker, G. C., and Peck, M. W. 2010. Qualitative risk assessment for
hazards that arise from non-proteolytic clostridium botulinum in minimally processes chilled
dairy-based food. Food Microbiology: 28, 321-330
3) Nakamura, K., Kohda T., Seto, Y., Mukamoto, M., and Kozaki, S. 2013. Improved
detection methods by genetic and immunological techniques for botulinum C/D and D/C
mosaic neurotoxins. Veterinary Microbiology: 162, 881-890
4) Sebaihia, M., Peck, M., Minton, N., Thomson, N., Holden, M., Mitchell, W., Carter, A.,
Bentley, S., Mason, D., Crossman, L., Paul, C., Ivens, A., Wells-Bennik, M., Davis, I.,
Cerdeno-Tarraga, A., Churcher, C., Quail, M., Chillingworth, T., Feltwell, T., Fraser, A.,
Goodhead, I., Hance, Z., Jagels, K., Larke, N., Maddison, M., Moule, S., Mungall, K.,
Norbertczak, H., Rabbinowitsch, E., Sanders, M., Simmonds, M., White, B., Whithead, S.,
and Parkhill, J. 2007. Genome sequence of proteolytic Clostridium botulinum strain Hall A
and comparative analysis of the clostridial genomes. Genome Research: 7, 1082-1092
5) Verderio, C., Rossetto, O., Grumelli, C., Frassoni, C., Montecucco, C., and Matteoli, M.
2006. Entering neurons: botulinum toxins and synaptic vesicle recycling. EMBO Reports: 10,
995-999
6) Todar, K. Lectures in Microbiology by Kenneth Todar, University of Madison-Wisconsin
Department of Bacteriology. 2009
7) Nevas, M., Lindstorm, M., Hautamaki, K., Puoskari, S., and Korkeala, H. 2005.
Prevalence and diversity of Clostridium botulinum types A, B, E, and F in honey produced in
the Nordic countries. Science Direct: 105, 145-151
8) Smelt, J., Stringer, S.C., Brul, S. 2013. Behavior of individual spores of non proteolytic
Clostridium botulinum as an element in quantitative risk assessment. Food Control: 29, 358363
9) Pirazzini, M., Rossetto, O., Bertasio, C., Bordin, F., Shone, C., Binz, T., and Montecucco,
C. 2013. Time course and temperature dependence of the membrane translocation of tetanus
and botulinum neurotoxins C and D in neurons. Biochemical and biophysical research
communications: 430, 38-42
10) Rajkovic, A., El Moualij, B., Fikri, Y., Dierick, K., Zorzi, W., Heinen, E., Uner, A., and
Uyttendaele, M. 2011. Detection of Clostridium botulinum neurotoxins A and B in milk by
ELISA and immuno-PCR at higher sensitivy than mouse bio-assay. Food Analysis Methods:
5, 319-326
11) Peng, L., Tepp, W., Johnson, E., and Dong, M. 2010. Botulinum neurotoxin D uses
synaptic vesicle protein SV2 and gangliosides as Receptors. PLOS: 7, 1371
12) Sudhof, T.C., and Rizo, J. 2011 Synaptic vesicle exocytosis. Cold Spring Harbor Perspect
Biology: 12, 1101.
13) Chen, S., Karalewitz, A., and Barbieri, J. 2012. Insights into the different catalytic
activities of Clostridium neurotoxins. Biochemistry: 51, 3941-3947
14) Connan, C., Bruggemann, H., Mazuet, C., Raffestin, S., Cayet, N., and Popoff, M. 2011.
Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in
Clostridium botulinum type A strain hall. PLOS: 10, 1371
15) Peck, M., Stringer, S., and Carter A. (2010). Clostridium botulinum in the post-genomic
era. Food Microbiology 28, 183-191
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