1. Gram-Positive Bacilli
Part-7
Gram-Positive Bacilli
The Most Medically Important Species That Belong to the Genera: Bacillus, Clostridium and Corynebacterium
((First Semester ))

2. Gram-Positive Bacilli
There are four medically important genera of Gram-positive bacilli:
The genus Bacillus
The genus Clostridium
The genus Corynebacterium
The genus Listeria

3. I- The Genus Bacillus: Gram-positive bacilli Aerobic, Spore-forming
Cellular arrangement: occurs in chains (Streptobacilli)
Medically important species:
Bacillus anthracis
Bacillus cereus
A- Bacillus anthracis: A Gram-positive capsulated bacilli that occurs in chains (Streptobacilli). It has a capsule that is made of amino acids (D-glutamate) (Remember: most bacteria have capsules that are made of polysaccharides)
Disease: Anthrax, which a zoonotic disease of animals such as sheep and goats.
Transmission: In most cases, humans are infected with Bacillus anthracis endospores from animals. Infection with Bacillus anthracis endospores occurs while dealing with infected animals. Humans are infected with endospores:
Through skin wounds, cuts and micro-cuts: upon contact with infected animals that may have the endospores on their wool and hair.
Through the respiratory tract by inhalation of endospores
Through the digestive tact by ingestion of contaminated meat
Once in human’s body (in skin, in lung or in the digestive tract), endospores of Bacillus anthracis germinate and vegetative bacterial cells start to replicate.

4. According to where infection starts, Anthrax can be classified into:
Cutaneous anthrax
Pulmonary anthrax
Gastrointestinal anthrax
Note:
Humans are infected from animals. However, the infection is not transmitted from human to human.
Pathogenesis of Bacillus anthracis:
Pathogenesis of Bacillus anthracis is mainly based primarily on:
Its capsule: which is made of D-glutamate (anti-phagocytic)
Its exotoxin: which is an AB toxin known as Anthrax toxin
Anthrax toxin: it is an AB exotoxin, however, unlike other AB toxin, anthrax toxin is consist of two kinds of A subunits, which are known as the Edema factor (EF) an the Lethal factor (LF).

5. The edema factor is an adenylate cyclase that causes an increase in the intracellular concentration of cyclic AMP (c-AMP). This causes an outpouring of fluid from the cell into the extracellular space, resulting in tissue edema.
The lethal factor is a protease that specifically cleaves mitogen-activated protein kinase kinase (MAPKK), resulting in its inactivation. MAPKK is involved in cell a signaling pathway that that delivers survival signal for human cells. Inactivation of MAPKK, implies absence of this survival signal, and thus initiation of programmed cell death (apoptosis).
Note: the B subunit is known as protective antigen (PA), since it can be used as a vaccine

6. Clinical Findings:
1-Cutaneous anthrax:
It is characterized by a painless black ulcer known as the black eschar.
Local edema around the black eschar.
The lesion is also called a malignant pustule.
At this stage the infection may heal leaving a skin scar.
However, some cases if left untreated may progress to bacteremia, hemorrhagic mediastinitis, hemorrhagic meningitis, septic shock, edema and death.
2-Pulmonary (inhalation) anthrax:
It is known as "wool sorter's disease
The disease starts in the lungs with nonspecific respiratory tract symptoms resembling influenza, especially a dry cough and substernal pressure.
This rapidly progresses to hemorrhagic mediastinitis, hemorrhagic meningitis, septic shock, and death.
3- Gastrointestinal Anthrax:
The symptoms of gastrointestinal anthrax include, vomiting, abdominal pain, and bloody diarrhea.

7. During cutaneous anthrax, pulmonary anthrax or gastrointestinal anthrax, the bacterium may reach the blood stream, septicemia, septic shock, sepsis, hemorrhagic meningitis, generalized edema and death

8. The Genus Clostridium:
Clostridia are anaerobic, spore-forming, Gram-positive rods.
There are four medically important species of this genus:
Clostridium tetani
Clostridium botulinum
Clostridium perfringens
Clostridium difficile.
Clostridium tetani:
Disease: Tetanus (lockjaw)
Transmission: the endospores of Clostridium tetani are found in soil world-wide. Humans are infected with the endospores of this bacterium through skin wounds (most commonly, puncture wounds), and deep skin cuts that get contaminated with soil.
Why deep wounds and cuts?

9. Pathogenesis:
Once in the wound, the endospores germinates into vegetative cells and start replicating and producing its exotoxin (an AB toxin) at the wound site.
Note: Damages tissue of the wound has a low Oxygen tension that is suitable for the growth of this anaerobic bacterium.
Clostridium tetani is not highly invasive and by it self, it does not cause major tissue damage. However, its AB exotoxin (Tetanus toxin / Tetanospasmin) is a highly potent neurotoxin toxin.
Toxin molecules bind to the local neurons at wound site through its B subunit, which in turns, facilitate the entry of the A subunit into the neurons.
By axonal retrograde transport, the A subunit is carried into the CNS and then reaches the motor endplate of motor neurons (junction of motor neurons with skeletal muscles). At the motor end plates, the A subunit functions as a protease that specifically cleaves the proteins involved in the exocytosis of the vesicles which contain the inhibitory neurotransmitter Glycine.
Accordingly, skeletal muscles receive only the signals for their contraction (mediated by the neurotransmitter acetylcholine), but not the relaxation signal (mediated by Glycine).
The net results: descending sustained muscle contraction (spasmic paralysis).
Note: the term paralysis means that the muscle has stopped its normal function (contraction and relaxation). In case of tetanus, affected muscles contract and remain contracted (locked on contracted status ) (spasmic paralysis).

11. Clinical Symptoms:
Tetanus is characterized by strong muscle spasms (Spastic paralysis).
A specific initial clinical feature is lockjaw (trismus), due to rigid contraction of the jaw muscles.
This is followed by descending spasmic paralysis of skeletal muscle
Death is mainly due to respiratory failure because of spasmic paralysis of the diaphragm (skeletal muscle).
Treatment includes administration of both:
Pre-formed antitoxin antibodies
Antibiotics
Prevention: vaccination by using the tetanus TOXOID, which is part of the DPT vaccine.
Note: Immunization with DPT consists of three doses given at 2, 4, and 6 months of age, with boosters at 1 and 6 years of age. Because immunity wanes, a booster every 10 years is recommended

12. Clostridium botulinum
The endospores of Clostridium botulinum are found in soil world wide. Accordingly, these endospores can easily contaminate our food such as fruits, vegetables and meet.
It is important to know that Clostridium tetani in its vegetative status, does not replicate in the digestive tract of adults, however, it can survive and replicate in the digestive tract of infants.
Diseases:
1- Food Botulism: it is a potentially-fatal food poisoning that occur as a result of ingestion of improperly-prepared, expired canned food (such as canned meat and vegetables).
Improperly prepared: means that it was not washed well or it was contaminated with soiled before being canned. This implies that it may be contaminated with the endospores of this bacterium since these endospores are found in soil.
Expired: This implies the the substances used in canned food to suppress bacterial growth is not longer effective.
Canned: provide an anaerobic environment suitable for the growth of Clostridium botulinum, because it is an anaerobic bacterium.
Under the above mentioned conditions, the endospores of this bacterium germinate to generate vegetative bacterial cells, which start to replicating and producing its exotoxin , which is known as Botulinum toxin (food cans are usually kept at room temperature).

13. Upon ingestion of this contaminated canned food (without being heated), the bacterial exotoxin is absorbed from the intestinal tract to reach CNS though the blood stream.
Botulinum toxin is an AB toxin. The B subunit mediate the internalization of the A subunit into nerve cells.
The A subunit is a protease . So, when the A subunit reaches the motor end plate of motor neurons, it specifically cleaves the proteins involved in the exocytosis of the vesicles which contain acetylcholine. Thus a acetylcholine is will not be released.
This implies that skeletal muscles will receive only the inhibitory signals mediated by the neurotransmitter glycine, which cause muscle relaxation.
In the absence of acetylcholine, there will be no activation signals that induce muscle contraction.
In this case, skeletal muscles stay locked in its relaxation status and this is known as flaccid paralysis.
Clinical symptoms:
Blurred vision
Descending muscle weakness and paralysis
Respiratory failure due to paralysis of the diaphragm, which can results in death.
Treatment:
Respiratory aid
Antitoxin
Why there is no need to give antibiotics?

14. .

15. 2- Infant Botulism:
It is mainly occur as a consequence of feeding infants honey that is contaminated with the endospores of Clostridium botulinum.
Unlike in adults, Clostridium botulinum can survive and replicate in the digestive tract of infants.
Accordingly, once an infant is fed honey that is contaminated with the endospores of this bacterium, the endospores germinate in the digestive tract of infants to generate vegetative cells that start replicating and producing the botulinum exotoxin.
This exotoxin is then absorbed from digestive tract to reach CNS through the blood stream.
Once the A subunit of this toxin reached the motor end plate of motor, it specifically cleaves the protein involved in the exocytosis of the vesicles that contain acetylcholine, resulting in sustained muscle relaxation (flaccid paralysis).
Death may occur die reparatory arrest as a result of flaccid paralyses of the diaphragm.
This exotoxin.
Treatment:
Antitoxin along with respiratory support.
Note: In case of infant botulism, antibiotics are not recommended since antibiotics may increase the release of the toxin from the bacterial cells being killed by antibiotics (lysis of bacterial cells in the digestive tract will release all the toxin found in their cytoplasm, resulting in exacerbating the infant’s clinical situation).
Prevention:
Infants under 1 yea of age, are not recommended to be fed honey.

16. Wound Botulism:
Wound Botulism occurs when the spores of Clostridium botulinum that is found in soil contaminate a wound that has damaged tissue with low oxygen tension
In the wound, the spores of germinate to give vegetative Clostridium botulinum cells, which start to replicate and produce the toxin.
When the toxin reaches the blood stream, the A subunit of the toxin enter motor neurons to inhibit the release of acetylcholine at the neuromuscular junction. This will result in descending flaccid paralysis as mentioned before.
If the paralysis reaches the diaphragm, death may occur due to respiratory arrest.
Note: Wound botulism has increased in recent decades in people who inject heroin, which is contaminated with the endospores of the bacteria. In fact, this type of botulism is most common in people who inject black tar heroin.
Treatment:
Surgical debridement of infected tissue
Administration of antibiotics and anti-toxin antibodies
Respiratory aid

17. Clostridium perfringens: causes gas gangrene (also known as Myonecrosis)
Epidemiology:
Clostridium perfringens is widely distributed in nature. Its endospores are found almost everywhere in dry soil. In addition, Clostridium perfringens vegetative cells are members of the normal flora of the colon and vagina.
One of the most important diseases that is caused by Clostridium perfringens is Gas gangrene (also known as Myonecrosis)
Gas gangrene may results due to the introduction of Clostridium perfringens endospores that are found in soil into major wounds that are contaminated with soil.
When these soil-contaminated wounds (which contain damaged tissue with low oxygen tension) are not cleaned properly, the endospores of the endospores of Clostridium perfringens germinate in the damaged tissue and start replicating.
Clostridium perfringens produces many exotoxins that have the ability to destroy connective tissue (connective tissue degrading enzymes) and cells (by a membrane distrusting enzyme, known as alpha toxin, which is a phospholipase (also knows as Lecithenase).

18. This will result in massive tissue necrosis (Myonecrosis) and development of gangrene. Clostridium perfringens is an anaerobic bacteria that during infection ferments the destroyed tissue to produce gas that Crepitation (hence the name Gas gangrene). The bacteria then may invades the blood stream to results in septicemia and septic shock can result in death.
Clinical symptoms of Gas gangrene:
Pain, edema, and cellulitis occur in the wound area
Crepitation indicates the presence of gas in tissues (the gas is foully smelling)
Discoloration of the infected tissue occurs
Hemolysis and jaundice are common
Mortality rates are high.
Treatment:
Surgical detriment of the infected necrotic tissue
Administration of antibiotics and anti-toxins antibodies
Hyperbaric oxygen therapy
If this treatment strategy fails, amputation of the affected limp must be carried out.
If failed…death is imminent

19. Clostridial Endometritis:
Clostridium perfringens colonizes the genital tract of about 5% of women.
Clostridial Endometritis is a life-threatening condition that can be associated with incomplete abortion, or the use of inadequately-sterilized instruments.
The introduction of Clostridium perfringens vegetative cells or endospores into the uterus after incomplete abortion that may be found in the vagina or into the uterus after incomplete abortion can result in a potentially-fatal gangrenous infection of uterine tissue is followed by toxemia and bacteremia that may lead to death.

20. Clostridium difficile
Epidemiology and transmission:
Clostridium difficile is carried in the gastrointestinal tract in approximately 3% of the general population and up to 30% of hospitalized patients.
Clostridium difficile can be transmitted among people fecal–oral route
Pathogenesis:
In healthy individuals, Clostridium difficile (if present) occupies a small percentage of the total population of the intestinal microbiota (other members of the intestinal microbiota compete with Clostridium difficile so that they won’t allow it to replicate in high numbers).
It is known that Clostridium difficile produces two potentially-harmful exotoxins that are known as Exotoxin A and Exotoxin B.
However, and as it was mentioned above that in healthy individuals, this bacterium (if present…it was mentioned above that about 3% of healthy individuals have this bacterium as a member of their intestinal microbiota) is found in small numbers, accordingly, the amounts of the two toxins produced by this bacterium are so small so that they will NOT have a significant effect on the function as well the survival of the mucosal cells of large intestine (colon).

21. However, prolong administration of certain antibiotics (which usually occurs at hospitals) such as clindamycin, third-generation cephalosporins, ampicillin and fluoroquinolone; can will suppress the replication of many of intestinal microbiota that are sensitive to the above- mentioned antibiotics.
Clostridium difficile is not affected by the above mentioned antibiotics, so that suppression of other members of the intestinal microbiota, allows Clostridium difficile to replicate in high numbers and thus to produce large amounts of its toxins.
The presence of large amounts of these two toxins (Exotoxin A and Exotoxin B) will have a significant effect on the function as well the survival of the mucosal cells of large intestine (colon).

22. Clinical conditions that are associated with the production of large amounts of Clostridium difficile exotoxins:
A- Antibiotic Associated Diarrhea (which is mediated by Exotoxin A)
B- Pseudomembranous colitis (which is mediated by exotoxin B)
Initially, the produced large amounts of Exotoxin A affects the function of the colon mucosa so that the mucosal cells secrete electrolytes and water, (instead of absorbing electrolytes and water) resulting in a watery diarrhea that can be associated with fever and abdominal cramps.
B- Pseudomembranous colitis (which is mediated by exotoxin B):
Later on, Exotoxin B causing the death of the colon mucosal cells, resulting in mucosal ulceration so that the diarrhea progresses into bloody diarrhea….. the intestinal ulcerations may become more severe and may result in a potentially-fatal massive colon bleeding.

23. Corynebacterium diphtheriae:
Corynebacterium diphtheriae is a Gram-positive aerobic, club-shaped bacilli that may form V-shaped, L-shaped and Chinese letter arrangements.
The cytoplasm of exhibit a beaded appearance due to the presence of cytoplasmic inclusions (accumulation of nutrients)
Pathogenic strains Corynebacterium diphtheria produce an AB exotoxin, known as Diphtheria toxin. This toxin is encoded by a gene that is found on a prophage (a provirus integrated with the bacterial chromosome). Accordingly, pathogenic Corynebacterium diphtheria is called Lysogenic Corynebacterium diphtheria.
Disease: Diphtheria
Diphtheria is characterized by a severe throat inflammation (local infection), that is associated with the formation of a gray pseudomemrane at the upper part of the respiratory tract (Throat infection).
Transmission: Humans are the only host for this bacterial pathogen. It is transmitted from person to person by respiratory aerosols .

24. This will inhibit the translation process translation.
Pathogenesis:
Diphtheria toxin inhibits translation in mucosal cells at infection site.
Once the A subunit enters the mucosal cells at infection site, it adds ADP-ribose to a translation factor known as Elongation factor-2 (EF-2).
This will inhibit the translation process translation.
Inhibition of translation (protein synthesis) in eukaryotic cells results in apoptosis
Note: Corynebacterium diphtheriae that is not lysogenic DOES NOT produce the Diphtheria toxin and thus does cause the disease Diphtheria
Clinical symptoms:
The most characteristic symptom of Diphtheria is the formation of a thick, gray, adherent pseudo-membrane that extends over the over the tonsils and throat.
Other symptoms: fever, sore throat, and cervical adenopathy

25. Complications: Treatment: Prevention:
Extension of the membrane into the larynx and trachea, causing airway obstruction
((لهذا يسمى المرض بالعربيه الخانوق
The Diphtheria toxin may reach the blood stream (toxemia) to cause:
Paralysis of the muscles of the soft palate and pharynx: this can lead to regurgitation of fluids through the nose.
Myocarditis accompanied by arrhythmias and circulatory collapse (this can be fatal)
Peripheral neuritis: this may temporarily affect the muscles of the extremities (weakness or paralysis)
Treatment:
The patient must be given:
Anti-toxin
Antibiotics
Prevention:
DPT vaccine, which contains the TOXOID of Diphtheria toxin