Clostridium

What is it? Morphology, Classification, Characteristics


Overview:  What is Clostridium?

First described in 1880 by Prazmowski, the genus Clostridium is composed of a heterogeneous group of bacteria that are characterized by their rod-like morphology.

Given that the species in this genus are heterogeneous in nature, they display a number of phenotypes ranging from acidophyles to psychrophiles.

These differences have also been identified in their genetic makeup where the G+C content range from about 21 to 54 percent. While some species in this genus may appear to be Gram-negative, Clostridia are generally Gram-positive organisms that are largely described as spore-forming, anaerobic bacteria.

* While a few Clostridium species are pathogenic (causing human and animal disease) a good number of the species are non-pathogenic, some of which have beneficial industrial applications

* Clostridium strains of medical significance are Gram-positive species. However, some may be Gram variable.

Some of the species classified under the genus Clostridium include:

  • C. botulinum
  • C. perfringens
  • C. sporogenes
  • C. bifermentans
  • C. leptum
  • C. difficile


Morphology



The majority of Clostridium strains of medical significance are rod-shaped (straight rods or slightly curved). As such, they resemble cylindrical rods when viewed under the microscope. However, this is not the case with all species/strains of the genus.

Although Gram-positive rods are common, some of the colonies may have a convex shape with a few having a spherical or irregular shape. The ends also vary from rounded to pointed ends depending on the strain.

When viewed under the microscope (after a culture) strains of Clostridium difficile have been shown to vary in shape from short, thick forms to large Gram-positive rods with rounded ends.

With the exception of C. perfringens, Clostridium species have peritrichous flagella that allow the organisms to move from one location to another (swim).

Compared to other flagella structures, peritrichous flagella project from all directions of the cell which allows the bacteria to move rapidly in any direction. Endospores, which may be observed distending from one end of the bacterial cell may be oval or spherical in shape.

* The flagella of C. difficile has also been shown to induce a pro-inflammatory response.

* Sporulation factor and temperature has also been shown to cause morphological changes of such species as C. perfringens in cultures.



Classification


Currently, more than 200 species and about 5 subspecies belonging to genus Clostridium have been identified. A majority of these are free living saprophytes while a few are pathogenic to human beings (e.g, C. botulinum, C. difficile, and C. tetani).


Scientific Classification of Clostridium Species


Kingdom: Bacteria - Like many other types of bacteria, members of the genus Clostridium are single-celled prokaryotes that are characterized by the lack of membrane-bound organelles (nucleus, mitochondria etc). This differentiates them from eukaryotic cells that are well-developed whose nucleus are enclosed within membranes.

Phylum: Firmicutes - Phylum Firmicutes consists of a diverse group of organisms most of which are Gram-positive species. However, some, like members of class Negativicutes, have been shown to be Gram-negative. Together with phylum Bacteroidetes, Firmicutes makes up about 98 percent of gut bacteria.

* This class is also characterized by low G+C Gram-positive bacteria

Class: Clostridia - This is a large class composed of rod or spindle-shaped bacterial cells. Members of class Clostridia are anaerobic in nature and can be found in aquatic environments, soil as well as the human gut. The majority of species are Gram-positive and form spores.

Order: Clostridiales - The order Clostridiales is composed of a diverse (ecologically, phenotypically and physiologically etc) group of organisms. As such, it is composed of both pathogenic and free-living organisms that may be Gram-positive or Gram-negative during staining.

Family: Clostridiaceae - Consisting of more than 30 genera of Firmicutes, members of family Clostridiaceae are anaerobic Gram-positive organisms that are generally characterized by the fact that they form monospores and contain meso-diaminopimelic acid in their peptidoglycan. Members of this class have also been shown to vary in morphology in cultures.

Genus: Clostridium



Characteristics



Ecology/Habitat


Free-Living Clostridium
 


Clostridium species are ubiquitous and thus found in various environments across the world. Oxygen tolerance among these species also varies considerably with some being strict anaerobes. For this reason, oxygen concentration in a given environment will influence the type of species present.

The majority of Clostridium species are saprophytes, which means that they obtain their nutrients from dead plants and animals in soil and various aquatic environments. In these environments, these bacteria break down various organic matters which in turn contributes to humus soil.

Apart from contributing to humus soil, free-living Clostridium found in soil have also been shown to play an important role in nitrogen fixation. For instance, Clostridium pasteurianum, a free-living Clostridium species is able to fix atmospheric nitrogen and convert carbohydrates to other compounds and molecules like carbon-dioxide, acetate, and butyrate. Given that a  majority of these species are anaerobes, they achieve this through fermentation.


Some of the other species of genus Clostridium capable of nitrogen fixation include:

  • C. beijerinkii
  • C. acebutylicum

* Free-living Clostridium species capable of fixing nitrogen have the nif gene. 

* Apart from soil and water (freshwater sediments), free-living Clostridium species can also be found in various moist environments such as certain plant roots.

* Some of the species found in plant's roots cause harm to the plant. For instance, C. puniceum is responsible for potato soft rot.

 
In Human and Animal Bodies 


While some of the Clostridium species can be found in soil and aquatic environments, others can be found in human and animal bodies.

C. perfringens, common in nature, can infect humans and has been found in such organs as the gall bladder. Other species such as C. difficile have been identified in the gastrointestinal tract of mammals where they cause a range of infections.

Such species as C. septicum and C. histolyticum have been shown to infect the body through wounds causing gas gangrene. Many of such species are opportunistic in nature and were particularly prevalent in cases of battlefield injuries.

Gas Gangrene - Also known as clostridial myonecrosis, is a disease that affects muscle tissue. Here, the bacteria (C. perfringens) enters the soft tissue through open wound followed by local growth. However, this only occurs under anaerobic conditions (where blood supply to the affected part is impaired).

This growth and spread of the bacteria into the other cells, however, is limited due to the presence of oxygen. In cases where the bacteria spread to nearby cells and hinder an inflammatory response through phagocytosis, infections of the cells (and tissue) continues allowing gas gangrene to develop. Here, the disease can spread to healthy cells over time thus affecting the entire limb.


Metabolic Properties 


Clostridium species vary considerably when it comes to oxygen tolerance. Whereas some of the species can tolerate some level of oxygen in their environment, others are strict anaerobes and thus thrive in environments with very little oxygen concentration.


Strict Obligate Anaerobes


Whereas species like C. perfringes and C. Clostridium are obligate anaerobes, others like C. Novyi (type A) and C. haemolyticum are very strict obligate anaerobes. As such, they cannot survive in normal atmospheric concentration of oxygen. However, all obligate anaerobes can only survive and thrive in conditions of high reducing intensity with very low oxygen concentration.


Aerotolerant


Although they do not use oxygen for respiration, some Clostridium species can tolerate it in their environment. This includes such species as C. carnis, C. tertium, C. and histolyticum.


Toxins


The genus Clostridium is composed of a number of toxin-producing species. These toxins vary between the species and have different impacts on human beings and animals.


Clostridial toxins include:


Neurotoxins


Neurotoxins are produced by two Clostridium species namely, Clostridium tetani (tetanus toxin) and Clostridium botulinum (botulinum toxin). The two toxins share several features including heavy H and light L chains that are bound by a disulfide bond.


Botulinum Toxin (BoNT)


Produced by C. botulinum, Botulinum toxin (BoNT) is a poisonous substance that works by binding to nerve endings (cholinergic nerve terminals). In doing so, the toxin blocks the release of neurotransmitter acetylcholine thus affecting muscle contraction.

Within the nervous system, the neurotransmitter is the primary neurotransmitter that sends signals to cells thus contributing to the contraction of the smooth muscle as well as the dilation of blood vessels.

By blocking the release of this neurotransmitter, muscle contraction is affected, which results in muscle weakness and paralysis.

* The toxin may be released by intestinal bacteria spores (C. botulinum) or ingested (canned foods etc).

There are several types of toxins (BoNT) that include: A, B, E, F, C, C1, and C2.


Tetanus Toxin (TeNT)


Tetanus toxin is released by Clostridium tetani and is the primary cause of tetanus.

Typically, spores of the bacteria (C. tetani) enter the body through wounds on the epidermal and dermal layers. Once in the body, the spore starts to germinate followed by the release of toxins into the bloodstream as well as the lymphatic system.

From the bloodstream and lymphatic systems, the toxin is taken up through lower motor neurons and transported to the spinal cord or the brainstem.

Although the toxin affects the central nervous system on several points, it significantly affects the neurotransmitter by blocking its release at the central inhibitory interneurons. In turn, this results in the disinhibition of the lower motor neurons which leads to uncontrollable contraction of muscles.

* C. tetani spores germinate in the body under anaerobic conditions

Some of the symptoms of tetanus (Tetanus toxin) include:

  • Rigidity or muscle spasms (hyperactivity of muscles)
  • Lockjaw
  • Respiratory failure (as a result of muscle spasm/rigidity)
  • General muscle ache
  • Neuronal inactivation
  • Tachycardia


Enterotoxin


Enterotoxin, a toxin released by C. perfringens is one of the main causes of gastrointestinal illness.

While the bacteria produces several types of the toxin (A, B, C, D, E), Type A (and Type C in some cases) is the only toxin that is responsible for human illness.

In the body (in vivo) the toxin is released during bacterial sporulation. The toxin is released into the intestinal lumen where it binds to the claudin receptors. This results in the formation of pre-pores and consequently functional pores (CH-1 pore) that cause oncosis through calcium influx. 

Some of the other toxins released by Clostridium bacteria include:

  • Cholesterol-dependent Cytolysins - This toxin is released by some of the Gram-positive Clostridium. Once released, the toxin bind to target cells and cause pore formation.
  • Binary Bacterial Toxins - This toxin is released by a number of Clostridium bacteria including C. perfringens, C. spiroforme  and C. botulinum. Through its mode of action (synergistic binary mechanism) the toxin causes intoxication and destruction of eukaryotic cells. 


Gram Stain


Among Clostridium, a majority are Gram-positive rods. For instance, C. difficile, associated with gastrointestinal illness, is one of the Gram-positive Clostridium that will appear dark blue/violet having retained the primary stain (crystal violet) in their thick peptidoglycan layer.

Although these bacteria may appear Gram-positive, they can lose this appearance under various conditions. In cases of extended incubation, some of the Clostridium species have been shown to lose their Gram-positive appearance. A few species, however, have been shown to be Gram-negative and will stain pink during Gram staining.


Microscopy


Today, well over 200 species belonging to the genus Clostridium have been identified. Of these, at least 30 are of clinical significance.

To observe and identify the species, Gram staining technique can be used.


Requirements


  • A clean glass slide
  • Gram stains (Crystal violet, Gram's iodine, Counterstain (safranin) and a decolorizing agent)
  • Sample - Sample from a patient or culture. (If a sample from a culture is used, it should not have been incubated for long).
  • Water (distilled or tap water)
  • Staining rack
  • Immersion oil
  • Bunsen burner

Procedure

  • Using a cotton swab, make a smear of the sample at the central part of the slide - Use a circular motion to make a good smear
  • Pass the slide over the Bunsen burner flame for about 3 times to heat-fix the smear - avoid overheating
  • Place the slide on the staining rack and carefully flood with crystal violet - Allow to stand for about one minute
  • Tilt the slide and gently rinse with water
  • Flood the slide with Gram's iodine and allow to stand for about 1 minute - Then tilt and rinse with water
  • Tilt the slide and decolorize using acetone (or 95 percent ethyl alcohol) - To do this, apply the decolorizer drop by drop for about 5 seconds in order to avoid excessive decolorization
  • Rinse the slide with water immediately and then flood the slide with the counterstain (safranin) for about 1 minute
  • Tilt the slide and rinse with tap water
  • Rinse excess water using a blotting paper

Place the slide under the microscope and view under immersion oil - compound light microscope or phase-contrast microscope may be used to view the cells.

When viewed under the microscope, one may observe the following, depending on the species:

C. tetani - They will appear purple in color with possible terminal spores. They may also be motile depending on the sample and sample preparation. Students will also notice that they are rod-shaped.


C. perfringens -  Under the microscope, this particular species may appear as either elongated rods or shorter in length. They are also non-motile with no terminal poles. 



See also pages on heterotrophs, and more on unicellular organisms

Return to learning about Bacteria under the Microscope

Return to Prokaryotes Main Page

Return from learning about Clostridium to MicroscopeMaster Home





References


Bjørnar Hassel. (2013). Tetanus: Pathophysiology, Treatment, and the Possibility of Using Botulinum Toxin against Tetanus-Induced Rigidity and Spasms. Toxins 2013.  

Paul A. Lawson  and Fred A. Rainey (2016). Proposal to restrict the genus Clostridium

Prazmowski to Clostridium butyricum and related species. International Journal of Systematic and Evolutionary Microbiology (2016). 

Sabina M. Num and Nicodemus M. Useh. (2014). Clostridium: Pathogenic Roles, Industrial Uses and Medicinal Prospects of Natural Products as Ameliorative Agents against Pathogenic Species. Jordan Journal of Biological Sciences. 

Public Health England. (2015) .UK Standards for Microbiology Investigations Identification of Clostridium species 


Links


https://www.microbiologyresearch.org/docserver/fulltext/micro/151/9/2821.pdf?expires=1547555877&id=id&accname=guest&checksum=A1F593DEA3F649A05B021D1F97572727

https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070878.htm

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