Gram positive spore –forming Bacilli

Gram positive spore –forming Bacilli
The genera Bacillus and Clostridium belong to the Bacillaceae family of sporing bacteria. There are numerous species in the genus Bacillus (e.g., B. cereus, B. subtilis, etc.) that normally live in the soil. The organism in the group that is of veterinary and human medical interest is Bacillus anthracis

Bacillus anthracis (Anthrax)
Occurrence. Anthrax occurs primarily in animals, especially herbivores. The pathogens are ingested with feed and cause a severe clinical sepsis that is often lethal.
Morphology and culturing. The rods are 1 lm wide and 2–4 lm long, nonflagellated, with a capsule made of a glutamic acid polypeptide. The bacterium is readily grown in an aerobic milieu. Non-motile. Capsule (poly d-glutamate-protein formed in 10-25% CO2 bicarbonate, serum albumin charcoal, and starch
Spores characters
? Aerobic bacilli forming heat-resistant
spores
? Formed in culture/soil
? survive in soil for decades
? Never formed in animal body
? Occurs under un-favourable conditions
? Encouraged by DW, 2% NaCl, oxalated agar
? Inhibited by calcium chloride
? central or subterminal, oval
? Of same width as the bacillary body-no bulging
? resistant to drying/UV/gamma rays/heat
Bacillus anthracis.
Gram positive bacilli. The cells have characteristic squared ends. The endospores are ellipsoidal shaped and located centrally in the cell. The spores are highly retractile to light and resistant to staining.

Cultural characteristics
They grow at 35-37° C Optimum for sporulation 25-30°C Good growth occurs on ordinary media colony are grey white on sheep blood agar Irregular, non-haemolyticn rough and form Medusa head colony Edge of a colony is composed of long, interlacing chains of bacilli resembling locks of matted hair
Virulence factors
The two principal virulence factors of B. anthracis are the toxin complex and the polypeptide capsule. In general terms, its role has long been viewed as protecting the bacterium from phagocytosis, but the details of how this occurs and how it influences the pathogenesis of the disease have not been elucidated.
Anthrax Toxin – it is a Complex of proteins ( all the components thermolabile)
• Edema factor (Factor I)
• Protective factor (Factor II)
• Lethal Factor (Factor III) Anthrax Toxin
Protective antigen : Binds plasma membrane of target cells then Cleaved to 2 fragments ( cellular trypsin or proteases) Larger fragment is attached to cell surface – binding domain for LF & EF Specific receptor mediated endocytosis of LF & EF
The three component interact between them to produce the clinical picture of pathogen
( Edema Factor + Protective Ag = Edema toxin)which can exert
Calmodulin dependent adenyl cyclase Increased cellular cAMP Edema Impaired Neutrophil function Depletes ATP from Macrophages
( Lethal Factor + Protective Ag = Lethal toxin)
Zinc metallo proteases that inactivates protein kinases then stimulates Macrophages –TNF alpha and IL – 1 beta finally hock & death. Death due to oxygen depletion, secondary shock, increased vascular permeability, respiratory failure and cardiac failure.
Clinically three forms of Human anthrax occur
A-Cutaneous anthrax-Hide-porters disease
B. Pulmonary anthrax-Wool sorters disease
C. Intestinal anthraxare
Differentiated based on the pathogen’s portal of entry. In dermal anthrax, which accounts for 90–95% of human B. anthracis infections) the pathogens enter through injuries in the skin. A local infection focus similar to a carbuncle complicated to Necrotic ulcer . These may develops to sepsis with
. Inhalational anthrax Wool sorter’s disease (bioterrorist anthrax), with its unfavorable prognosis, results from inhalation of dust containing the pathogen. Ingestion of contaminated foods can result in intestinal anthrax with vomiting and bloody diarrheas.

Bacillus cereus
is a Gram-positive, spore-forming microorganism capable of causing foodborne disease At present three enterotoxins, able to cause the diarrheal syndrome, have been described: hemolysin BL (HBL), nonhemolytic enterotoxin (NHE) and cytotoxin K. HBL and NHE are
three-component proteins, whereas cytotoxin K is a single protein toxin. Symptoms caused by the latter toxin are more severe and may even involve necrosis. In general, the onset of symptoms is within 6 to 24 h after consumption of the incriminated food. B. cereus food poisoning is underestimated probably because of the short duration of the illness (~24
h).
Name of Illness Caused by B. cereus
B. cereus food poisoning is the general name used for the illness. However, B. cereus has two recognized types of food borne illness: diarrheal and emetic. The emetic syndrome is caused by a heat- and pH-stable peptide toxin. Consumption of food contaminated with this toxin
may lead to emesis between 30 min and 5 h after ingestion. The diarrhoeal syndrome is caused by enterotoxins that are produced during growth of B. cereus in the small intestine

Clostridium
Most Clostridium species decompose proteins or form toxins and some do both. Their natural habitat is the soil or intestinal tract as saprophytes. The important pathogenic species are:
? Clostridium botulinum: Causes botulism
? Clostridium tetani: Causes tetanus
? Clostridium perfringens: Causes gas gangren

Morphology
Large anaerobic gram positive motile rods. The spore is usually wider than the rods. Spores are placed centrally, terminally, or subterminally according to the genus

Colony forms
Clostridium perfringens: Large raised colonies with entire margins.
Clostridium tetani : Smaller colonies with fine filaments.
Most Clostridia produce a zone of hemolysis on blood agar.
Growth characteristics of anaerobic microorganisms are:
1. Unable to utilize O2 as the final oxygen acceptor.
2. Lack of cytochrome and cytochrome oxidase.
3. Unable to break down hydrogen peroxide H2O2 because they lack catalase of peroxidase so H2O2 will accumulate to toxic conc. in the presence of O2.

Clostridium botulinum
It causes botulism, infant’s botulism, and rarely wound infection. It is found in soil and animal feces. The spores are subterminal highly resistant to heat. They resist boiling 3-5 hours. This resistance is diminished at acidic pH and salt. It produces toxin during life and autolysis of bacteria.


Toxin …

there are eight Types of Clostridium botulinum: They are from A – H according to the type of toxin produced.

Types A, B, and E are the most commonly associated with illness
Toxins of types A, B, and E have the following characteristics:
1. They are among the most highly toxic substances known.
2. They are neurotoxic proteins (MW = 150 000)
3. Lethal dose for human is 1 – 2 mg
4. They are destroyed by heating for 20 minutes at 100 oC
5. Toxin production is under the control of a viral gene (Bacteriophage yielded from toxigenic strain and it may infect non-toxigenic strain and convert it to toxigenic).

… Action of botulism toxin …
It is a neurotoxic protein. All of its types (A, B, and E) are made of heavy and light chains linked by disulfide bonds. The heavy chain is thought to bind the toxin to the motor nerve end plate. The light chain blocks the calcium-mediated release of acetyl choline. The toxin acts by blocking the release of acetyl choline at synapses and neuromuscular junctions causing flaccid paralysis.
… Pathogenesis …
Botulism is intoxication. It results from ingestion of food in which Clostridium botulinum spores germinate and produce toxins under anaerobic conditions. These foods are spiced, smoked, vacuum-packed, or canned alkaline foods (if eaten without smoking). The toxin acts by blocking the release of acetyl choline at synapses and neuromuscular junctions causing flaccid paralysis. Patients who recover don’t develop an antitoxin in the blood. Patients develop Symptoms … [ within 18 – 24 hours ] which are

? Visual disturbances
? Inability to swallow
? Speech difficulty
? Respiratory paralysis or cardiac arrest
Lab diagnosis …
Toxin can be detected in the patient serum and left over food.
1. Mice are injected with the specimen and then neutralized by injections of antitoxin.
2. Culture of food remains of its growth test for toxin production.
3. Toxin is tested by hemoagglutination or radioimmunoassay (RIS).