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Section I– Microbiology By Dr. Mohammed Ayad

Animals acquire anthrax through ingestion of spores and spread of the organisms from the intestinal tract. This

is rare in humans, and gastrointestinal anthrax is extremely uncommon. Abdominal pain, vomiting, and bloody

diarrhea are clinical signs.

Injection anthrax is characterized by extensive, painless, subcutaneous edema and the notable absence of the

eschar characteristic of cutaneous anthrax. Patients may progress to hemodynamic instability due to septicemia.

Laboratory Diagnosis

Specimens to be examined are fluid or pus from a local lesion, blood, pleural fluid, and cerebrospinal fluid in

inhalational anthrax associated with sepsis and stool or other intestinal contents in the case of gastrointestinal

anthrax. Stained smears from the local lesion or of blood from dead animals often show chains of large grampositive rods. Anthrax can be identified in dried smears by immunofluorescence staining techniques.

When grown on blood agar plates, the organisms produce nonhemolytic gray to white, tenacious colonies with

a rough texture and a ground-glass appearance. Comma-shaped outgrowths (Medusa head, “curled hair”) may

project from the colony. Demonstration of capsule requires growth on bicarbonate- containing medium in 5–7%

carbon dioxide. Gram stain shows large gram-positive rods. Carbohydrate fermentation is not useful. In

semisolid medium, anthrax bacilli are always nonmotile, but related organisms (e.g., B cereus) exhibit motility

by “swarming.”

Definitive identification requires lysis by a specific anthrax γ-bacteriophage, detection of the capsule by

fluorescent antibody, or identification of toxin genes by polymerase chain reaction (PCR). These tests are

available in most public health laboratories. A rapid enzyme-linked immunoassay (ELISA) that measures total

antibody to PA, but the test result is not positive early in disease.

Bacillus cereus

Food poisoning caused by B cereus has two distinct forms;

1. The emetic type; which is associated with fried rice, milk, and pasta

2. The diarrheal type; which is associated with meat dishes and sauces

B cereus produces toxins that cause disease that is more of intoxication than a food-borne infection. The emetic

form is manifested by nausea, vomiting, abdominal cramps, and occasionally diarrhea and is self-limiting, with

recovery occurring within 24 hours. It begins 1-5 hours after ingestion of a plasmid-encoded preformed cyclic

peptide (emetic toxin) in the contaminated food products.

B cereus is a soil organism that commonly contaminates rice. When large amounts of rice are cooked and

allowed to cool slowly, the B cereus spores germinate, and the vegetative cells produce the toxin during logphase growth or during sporulation. The diarrheal form has an incubation period of 1-24 hours and is

manifested by profuse diarrhea with abdominal pain and cramps; fever and vomiting are uncommon. In this

syndrome, ingested spores that develop into vegetative cells of B cereus secrete one of three possible

enterotoxin which induce fluid accumulation in the small intestine.

The presence of B cereus in a patient’s stool is not sufficient to make a diagnosis because the bacteria may

be present in normal stool specimens; a concentration of 105

bacteria or more / gram of food is considered

diagnostic.

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Clostridia species

The Clostridia are large anaerobic, gram-positive, motile rods. Many decompose proteins or form toxins, and

some do both.

Their natural habitat is the soil, marine sediments, sewage, or the intestinal tract of animals and humans,

where they live as saprophytes. Among the pathogens are the organisms causing botulism, tetanus, gas

gangrene, and pseudomembranous colitis.

Clostridia are usually wider than the diameter of the rods in which they are formed; the spore is placed

centrally, subterminally, or terminally.

Most species of Clostridia are motile and possess peritrichous flagella. They are anaerobes and grow under

anaerobic conditions ((grow well on the blood-enriched media)).

Clostridia produce large raised colonies (e.g., C perfringens); others produce smaller colonies (e.g., C tetani).

Some Clostridia form colonies that spread or swarm on the agar surface (Clostridium septicum). Many

Clostridia produce a zone of β-hemolysis on blood agar. C perfringens characteristically produces a double

zone of β-hemolysis around colonies.

Clostridia can ferment a variety of sugars (saccharolytic) and many can digest proteins (proteolytic); some

species do both. These metabolic characteristics are used to divide the Clostridia into groups. Milk is turned

acid by some and digested by others and undergoes “stormy fermentation” (clot torn by gas).

Clostridium botulinum

C botulinum which causes the disease botulism is worldwide in distribution; it is found in soil and occasionally

in animal feces. Types of C botulinum are distinguished by the antigenic type of toxin they produce. Spores of

the organism are highly resistant to heat, withstanding 100°C for several hours. Heat resistance is diminished at

acid pH or high salt concentration.

During the growth of C botulinum and during autolysis of the bacteria, toxin is liberated into the environment.

Seven antigenic varieties of toxin (serotypes A-G) are known. Types A, B, E, and F are the principal causes of

human illness.

Botulinum toxins have three domains:

1- Two of the domains facilitate binding to and entry of toxin into the nerve cell

2-The third domain is the toxin which is protein that is cleaved into a heavy chain and a light chain that are

linked by a disulfide bond.

Botulinum toxin is absorbed from the gut, enters the blood circulation, and binds to receptors of motor neurons

of the peripheral nervous system and cranial nerves. The toxin does not cross the blood brain barrier or affect

the central nervous system. It inhibits the release of acetylcholine at the neurons synapse, resulting in lack of

muscle contraction and paralysis.

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Figure shows C botulinum toxins action pattern

Pathogenesis

Most cases of botulism represent an intoxication resulting from the ingestion of food in which C botulinum has

grown and produced toxin. The most common offenders are spiced, smoked, vacuum packed or canned

alkaline foods that are eaten without cooking. In such foods, spores of C botulinum germinate; that is, under

anaerobic conditions, vegetative forms grow and produce toxin. In infant botulism, honey is the most frequent

vehicle of infection.

The infant ingests the spores of C botulinum, and the spores germinate within the intestinal tract. The vegetative

cells produce toxin as they multiply; the neurotoxin then gets absorbed into the bloodstream.

The toxin acts by blocking release of acetylcholine at synapses and neuromuscular junctions; the result is

flaccid paralysis.

Figure shows C botulinum gram stained film

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The clinical picture begin 18-24 hours after ingestion of the toxic food, with visual disturbances (incoordination

of eye muscles, double vision), inability to swallow, and speech difficulty, and death occurs from respiratory

paralysis or cardiac arrest.

The infants in the first months of life develop poor feeding, weakness, and signs of paralysis (floppy baby).

Infant botulism may be one of the causes of sudden infant death syndrome. C botulinum and botulinum toxin

are found in feces but not in serum.

Laboratory diagnosis carried out by detection of toxin and not the organism. Toxin can often be demonstrated

in serum, gastric secretions, or stool from the patient, and toxin may be found in leftover food.

Clinical swabs or other specimens obtained from patients should be transported using anaerobe containers.

Mice injected intraperitoneally with such specimens from these patients die rapidly. The antigenic type of toxin

is identified by neutralization with specific antitoxin in mice. This mouse bioassay is the test of choice for the

confirmation of botulism. In infant botulism, C botulinum and toxin can be demonstrated in bowel contents but

not in serum. Other methods used to detect toxin include Elisa and PCR.

Clostridium tetani

C tetani which causes tetanus is worldwide in distribution in the soil and in the feces of horses and other

animals. Several types of C tetani can be distinguished by specific flagellar antigens. All share a common O

(somatic) antigen, which may be masked, and all produce the same antigenic type of neurotoxin

((tetanospasmin)) which composed of two peptides linked by a disulfide bond. The larger peptide initially

binds to receptors on the presynaptic membranes of motor neurons; it then migrates by the retrograde axonal

transport system to the cell bodies of these neurons to the spinal cord and brainstem. The release of the

inhibitory glycine and GABA (γ-aminobutyric acid) is blocked, and the motor neurons are not inhibited; so

hyper-reflexia, muscle spasms, and spastic paralysis result.

Pathogenesis

C tetani is not an invasive organism, the infection remains strictly localized in the area of devitalized tissue

(wound, burn, injury, umbilical stump, surgical suture) into which the spores have been introduced.

Figure shows C tetani gram film (tennis rackets or drumsticks)

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The volume of infected tissue is small, and the disease is almost entirely a toxemia. Germination of the spore

and development of vegetative organisms that produce toxin are aided by:

1- Necrotic tissue

2- Calcium salts

3- Associated pyogenic infections all of which aid establishment of low oxidation-reduction potential

The toxin released from vegetative cells reaches the central nervous system and rapidly becomes fixed to

receptors in the spinal cord and brainstem and exerts the actions

The incubation period may range from 4-5 days up to 3 weeks. The disease is characterized by tonic contraction

of voluntary muscles. Muscular spasms often involve first the area of injury and infection and then the muscles

of the jaw (trismus, lockjaw), which contract so that the mouth cannot be opened. Gradually, other voluntary

muscles become involved, resulting in tonic spasms. The patient is fully conscious, and pain may be intense.

Death usually results from interference with the mechanics of respiration. The mortality rate in generalized

tetanus is very high.

Diagnosis and tetanus prevention

Anaerobic culture of tissues from contaminated wounds may yield C tetani, but neither preventive nor

therapeutic use of antitoxin should ever be withheld pending such demonstration. Proof of isolation of C tetani

must rest on production of toxin and its neutralization by specific antitoxin.

Prevention of tetanus depends on:

1- Active immunization with toxoids

2- Aggressive wound care

3- Prophylactic use of antitoxin

4- Administration of penicillin

Active immunization with tetanus toxoid should accompany antitoxin prophylaxis; they are given very large

doses of antitoxin (3000-10,000 units of tetanus immune globulin) intravenously in an effort to neutralize toxin

that has not yet been bound to nervous tissue. Surgical debridement is vitally important because it removes the

necrotic tissue that is essential for proliferation of the organisms. Penicillin strongly inhibits the growth of C

tetani and stops further toxin production.

Tetanus toxoid is produced by detoxifying the toxin with formalin and then concentrating it. Aluminum salt

adsorbed toxoids are used. Three injections comprise the initial course of immunization followed by another

dose about 1 year later. Initial immunization should be carried out in all children during the first year of life.

“booster” injection of toxoid is given upon entry into school. Thereafter, “boosters” can be spaced 10 years

apart to maintain serum levels In young children; tetanus toxoid is often combined with diphtheria toxoid and

a cellular pertussis vaccine (DPT).

Clostridia with tissue invasion

Many different toxin-producing Clostridia (C perfringens and related Clostridia) can produce invasive

infection (including myonecrosis and gas gangrene) if introduced into damaged tissue.

About 30 species of Clostridia may produce such an effect, but the most common in invasive disease is C

perfringens (90%). An enterotoxin of C perfringens is a common cause of food poisoning. The invasive

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Clostridia produce a large variety of toxins and enzymes that result in a spreading infection. Many of these

toxins have lethal, necrotizing, and hemolytic properties.

The alpha toxin of C perfringens type A is a lecithinase, and its lethal action is proportionate to the rate at

which it splits lecithin (an important constituent of cell membranes). Alpha toxin also aggregates platelets,

thereby leading to formation of thrombi in small blood vessels and adding to poor tissue profusion and

extending the consequences of anaerobiosis, namely, destruction of viable tissue (gas gangrene).

The theta toxin has similar hemolytic and necrotizing effects but is not a lecithinase that act by forming pores

in cell membranes.

Epsilon toxin is a protein that causes edema, and hemorrhage is very potent. DNase and hyaluronidase, a

collagenase that digests collagen of subcutaneous tissue and muscle, are also produced.

Some strains of C perfringens produce a powerful enterotoxin especially when grown in meat dishes. When

more than 108

vegetative cells are ingested and sporulate in the gut. It induces intense diarrhea in 7-30 hours.

The action of C perfringens enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of

fluids and electrolytes in diarrhea; this illness is similar to that produced by B cereus and tends to be selflimited.

Pathogenesis

In invasive Clostridia infections, spores reach tissue either by contamination of traumatized areas (soil, feces)

or from the intestinal tract. The spores germinate at low oxidation-reduction potential; vegetative cells multiply,

ferment carbohydrates present in tissue, and produce gas.

The distention of tissue and interference with blood supply, together with the secretion of necrotizing toxins

and hyaluronidase, favor the spread of infection. Tissue necrosis extends, providing an opportunity for

increased bacterial growth, hemolytic anemia, and, ultimately, severe toxemia and death.

Figure shows C septicum gram stained slide from infected wound

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In gas gangrene (Clostridial myonecrosis), a mixed infection is the rule. In addition to the toxigenic Clostridia,

proteolytic Clostridia and various cocci and gram-negative organisms are also usually present. C perfringens

occurs in the genital tracts of 5% of women.

In contaminated wound (e.g., a compound fracture, postpartum uterus), the infection spreads in 1-3 days to

produce crepitation in the subcutaneous tissue and muscle, foul-smelling discharge, rapidly progressing

necrosis, fever, hemolysis, toxemia, shock, and death.

In the laboratory specimens consist of material from wounds, pus, and tissue. The presence of large grampositive rods in Gram-stained smears suggests gas gangrene Clostridia; spores are not regularly present.

Material is inoculated into chopped meat glucose medium and thioglycolate medium and onto blood agar

plates incubated anaerobically.

After pure cultures have been obtained by selecting colonies from anaerobically incubated blood plates, they

are identified by biochemical reactions (various sugars in thioglycolate, action on milk); hemolysis, and

colony morphology, lecithinase activity is evaluated by the precipitate formed around colonies on egg yolk

media.

Clostridium difficile

Pseudomembranous colitis is diagnosed by detection of one or both C difficile toxins in stool and by

endoscopic observation of pseudomembrane or micro-abscesses in patients who have diarrhea and have been

given antibiotics.

Plaques and micro-abscesses may be localized to one area of the bowel. The diarrhea may be watery or bloody,

and the patient frequently has associated abdominal cramps, leukocytosis, and fever.

Many antibiotics have been associated with pseudomembranous colitis; the most common are ampicillin and

clindamycin and, more recently, the flouroquinilones. The disease is treated by discontinuing administration of

the offending antibiotic and orally giving metronidazole, vancomycin.

Fecal transplantation has become a successful and routine method for recurrent and refractory disease. This

usually involves administration of the feces of a healthy related donor by way of colonoscopy or less commonly

via a nasogastric tube into the gastrointestinal tract of the patient.

Administration of antibiotics results in proliferation of drug-resistant C difficile that produces two toxins:

1- Toxin A is a potent enterotoxin that also has some cytotoxic activity, binds to the brush border

membranes of the gut at receptor sites

2- Toxin B is a potent cytotoxin

Both toxins are usually found in the stools of patients with pseudomembranous colitis. Not all strains of C

difficile produce the toxins, and the toxin genes are found on a large, chromosomal pathogenicity island along

with three other genes that regulate toxin expression.

Diagnosis is made clinically and supported by demonstration of toxin in the stool by a variety of methods that

includes anaerobic toxigenic culture, enzyme immunoassay, and molecular tests that detect the genes that

encode toxins A or Antibiotic associated diarrhea

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The administration of antibiotics frequently leads to a mild to moderate form of diarrhea, termed antibioticassociated diarrhea. This disease is generally less severe than the classic form of pseudomembranous colitis;

25% of cases of antibiotic-associated diarrhea are caused by C difficile infection, other Clostridium species such

as C perfringens and C sordellii have also been implicated; but they are not associated with pseudomembranous

colitis.

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Lecture 12

Spore-forming gram-positive Bacilli: Bacillus and Clostridium Species

These bacilli are ubiquitous, and because they form spores, they can survive in the environment for many years.

Bacillus species are aerobes and the Clostridium species are anaerobes.

Many species of Bacillus and related genera, most do not cause disease and are not well characterized. There

are a few species, however, that cause important diseases in humans. Anthrax, a classical disease in the history

of microbiology, is caused by Bacillus anthracis.

Anthrax remains an important disease of animals and occasionally of humans. Because of its potent toxins, B

anthracis is a major potential agent of bioterrorism and biologic warfare.

Bacillus cereus and Bacillus thuringiensis cause food poisoning and occasionally eye or other localized

infections.

The genus Clostridium is extremely heterogeneous and more than 200 species have been described.

Clostridia cause several important toxin mediated diseases, including tetanus (Clostridium tetani), botulism

(Clostridium botulinum), and gas gangrene (Clostridium perfringens), and antibiotic-associated diarrhea and

pseudomembranous colitis (Clostridium difficile).

Bacillus species

The genus Bacillus includes large aerobic, gram-positive rods occurring in chains. The members of this genus

are closely related but differ both phenotypically and in terms of pathogenesis.

Pathogenic species possess virulence plasmids. Most members of this genus are saprophytic organisms

prevalent in soil, water, and air, and on vegetation (e.g., Bacillus subtilis).

Some are insect pathogens, such as B thuringiensis. This organism is also capable of causing disease in

humans. B cereus can grow in foods and cause food poisoning by producing either an enterotoxin (diarrhea) or

an emetic toxin (vomiting). Both B cereus and B thuringiensis may occasionally produce disease in

immunocompromised humans (e.g., meningitis, endocarditis, endophthalmitis, conjunctivitis, or acute

gastroenteritis). B anthracis, which causes anthrax, is the principal pathogen of the genus.

The typical cells, measuring 3–4 μm, have square ends and are arranged in long chains; spores are located in

the center of the bacilli