Faculty of Medicine

Hashemite University
Dr Mohammad Al-Tamimi, MD, Master Biomed, PhD
Objectives
Understand the morphology, epidemiology,
pathogenesis, and laboratory diagnosis of Bacillus
anthracis
Understand the morphology, epidemiology,
pathogenesis, and laboratory diagnosis of Bacillus
cereus
Understand the morphology, epidemiology,
pathogenesis, and laboratory diagnosis of Clostridium
perfringens, Clostridium tetani, Clostridium difficili
and Clostridium botulinum
 Medically Important Gram-Positive
Bacilli
Three general groups:
1. Endospore-formers
Bacillus, Clostridium
2. Non-endospore-formers
Listeria, Erysipelothrix
3. Irregular shaped and staining properties
Corynebacterium, Proprionibacterium,
Mycobacterium, Actinomyces, Nocardia

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Classification

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Bacillus Bacillus anthracis
Bacillus cereus
General Characteristics of the Genus
Bacillus
Gram-positive, endospore-forming, mostly
motile rods
Aerobic and catalase positive
Versatile in degrading complex macromolecules
Primary habitat is soil
Thermophilic (< 75C) and psychrophilic (5-
8C)
Can flourish at extremes of acidity & alkalinity
(pH 2 to 10)
2 species of medical importance:
Bacillus anthracis
Bacillus cereus 6
Bacillus anthracis

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Introduction
Louis pasture and Robert Kock experiments
Anthrax disease:
1. Farmers disease
2. Former soviet union accidental attack 1979 (50
deaths)
3. USA attach by mailing envelop 2001 (5 deaths)
4. Cloud of death? Biological weapon
 Epidemiology
B. anthracis is the most highly virulent species for humans
and is the causative agent of anthrax
Anthrax is a devastating diseases that rarely encountered in
developed countries but still evident in some foreign
countries (Turkey, Iran, Pakistan, Sudan)
The spores usually survive in soil. Then transmitted to
herbivorous animals, infection transmitted to humans by
1. Direct contact with animal products (e.g., wool and hair)
2. Inhalation & ingestion of spores
3. Breaks in the skin or mucous membranes
4. Encountered as an occupational disease (Veterinarians,
agricultural workers)
Once enter human body the spores turn to vegetative form
and induce virulent infection
Virulent Factors
Polypeptide capsule: antiphagocytosis
Exotoxins: including edema toxin and lethal toxin, that
mediates cell and tissue destruction responsible for
virulent and lethal effect
Clinical Presentation of Anthrax
3 types of anthrax:
Cutaneous (95% of infections)
Spores enter through skin
Small, pruritic, non-painful papule at inoculation site.
Papule develops into hemorrhagic vesicle & ruptures, Slow-
healing painless ulcer. Infection may spread to lymphatic's
or blood
20% mortality in untreated cutaneous anthrax
Pulmonary (5%)
Inhalation of spores
Induce sever pneumonia
Virtually 100% fatal
Gastrointestinal (rare)
Ingested spores
Abdominal pain and Hemorrhagic ascites
Virtually 100% fatal
Laboratory Diagnosis
Specimens (skin, blood, sputum, food)
No special requirements for collection and transport
No blood culture isolate of a large gram-positive
rods should ever be discarded without first ruling
out anthrax
1. Gram Stain
Gram-positive rods that become gram-variable or
gram-negative with age, usually in long chains
Spores remain clear because they do not retain the
stain
Spores can be stained with specific dyes (malachite
green)
2. Cultivation
Media of choice:
5% sheep blood agar, chocolate agar, blood culture media
Special media:
1. PLET media (polymixn-lysozyme-EDTA-thallous acetate)
2. Bicarbonate agar that induce B. anthracis capsule
formation
Incubation conditions:
Detectable growth apear after 24 hours incubation at 35 C in
ambient air or 5% CO2, bicarbonate agar must be
incubated in CO2
Colonial appearance:
Medium-large, gray, flat, irregular, non hemolytic, ground
glass appearance, Medusa head
3. Immunological and
Molecular assays
Indirect haemagglutination test and ELISA test for
detection of B. anthrax antibodies are available but not
widely used
PCR technique for detection of B. anthrax DNA is
sensitive but highly technical
 4. Confirmation
1. Susceptible to penicillin
2. Produces a wide zone of lecithinase on egg yolk agar
3. Gamma bacteriophage testing
 Treatment and Prevention
Treated with penicillin, tetracycline, or
ciprofloxacin
Vaccines
live spores and toxoid
purified toxoid; for high risk occupations and
military personnel; toxoid 6X over 1.5 years; annual
boosters

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Bacillus cereus
General Characteristics
Gram-positive rods, facultative
anaerobe, with endospores
Grows in foods, spores survive cooking and reheating
Ingestion of toxin-containing food causes nausea,
vomiting, abdominal cramps and diarrhea; 24 hour
duration
Transmitted mainly by ingestion of contaminated food
(mainly meat and rice)
Increasingly reported in immunosuppressed
Virulence Factor
Heat stable enterotoxin
Heat labile enterotoxin
Pyogenic toxin

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Clinical Presentation
Food poisoning of 2 types:
1. Diarrheal type: abdominal pain and watery diarrhea
2. Emetic type: profuse vomiting
Laboratory Diagnosis
1. Gram stain
2. Culture
3. Stool analysis
Treatment and Prevention
Symptomatic treatment, no need for specific
treatment (generally self-limited disease)
Prevention by proper cooking of food, proper
preparation, direct consumption of food or
refrigeration
 Faculty of Medicine
Hashemite University
Dr Mohammad Al-Tamimi, MD, Master Biomed, PhD
Introduction
The most important pathogens are:
1. Clostridium perfringens
2. Clostridium tetani
3. Clostridium difficili
4. Clostridium botulinum
Clostridium perfringens
 General Characters
Large rectangular gram-positive bacilli
Obligate anaerobic
Non-motile
Spore forming but rarely seen in clinical samples or
culture. Spores found in soil, human skin, intestine, and
vagina
Replicate and grow rapidly
Involved mainly in soft tissue and wound infections
Virulence Factors
Toxins
Collagenase
Hyaluronidase
Dnase
Licithenase
Pathology
Not highly invasive; requires damaged and dead
tissue and anaerobic conditions
Conditions stimulate spore germination, vegetative
growth and release of exotoxins, and other
virulence factors
Fermentation of muscle carbohydrates results in
the formation of gas and further destruction of
tissue
Clinical Presentations
Clostridial cellulites
Gas Gangrene
Laboratory Diagnosis for C. perfringens
Gram stain and microscopy:
Gram-positive large rods
Culture: anaerobic media/anaerobic condition
Culture media: blood agar
Culture conditions: The optimum growth occurs at 7.3 pH,
37C temperature, and obligatory anaerobic conditions
Colony: smooth, large, regular, convex, and slightly opaque
colonies, and zone of complete hemolysis (inner zone due to
beta hemolysis) surrounded by wider zone of incomplete
hemolysis (alpha hemolysis)
Lecithinase production
Lecithinase (-toxin; phospholipase) hydrolyzes
phospholipids in egg-yolk agar around streak
Biochemical tests:
lactose and glucose fermentation positive
 Treatment and Prevention
Immediate cleansing of dirty wounds, deep wounds,
Debridement of disease tissue
Large doses of cephalosporin or penicillin
Hyperbaric oxygen therapy
Revascularization to improve blood supply
No vaccines available
Clostridium tetani
General Characteristics
Gram-positive long thin bacilli, with prominent
terminal spores (drumstick appearance)
Strictly anaerobe, difficult to isolate from clinical
samples
Motile by peritrichous flagella
Spores resist boiling for 20 minutes
Virulent Factors
Spore formation
Tetanospasmin: very poisonous and responsible for
clinical picture (spastic paralysis)
Tetanolysin: its action is not well known
 Pathology
Spores usually enter through accidental puncture
wounds, burns, umbilical stumps, frostbite, and
crushed body parts.
Anaerobic environment is ideal for vegetative cells to
grow and release toxin.
Tetanospasmin neurotoxin causes paralysis by
binding to motor nerve endings; blocking the release
of neurotransmitter for muscular contraction
inhibition; muscles contract uncontrollably
Death most often due to paralysis of respiratory
muscles
Clinical Presentations
Tetanus
Tetanus affects skeletal muscle only
In recent years, approximately 11% of reported tetanus
cases have been fatal
The highest mortality rates are in unvaccinated
persons and persons over 60 years of age
C. tetani, the bacteria that causes tetanus, is recovered
from the initial wound in only about 30% of cases
Opisthotonos in tetanus patient Locked jaw
Laboratory Diagnosis
Gram stain, microscopy and culture of poor sensitivity
Culture: Grow on blood agar or cooked meat medium
Beta-hemolysis exhibited by isolated colonies
Tetanus toxin and antibodies detection also not
reliable
Diagnosis is based mainly on classical clinical picture
 Treatment and Prevention
Antitoxin therapy with human tetanus immune
globulin; inactivates circulating toxin but does not
counteract that which is already bound
Control infection with penicillin or tetracycline
Muscle relaxants
Vaccine available; booster needed every 10 years
Thank you....