Bacillus Genus and Anthrax Quiz

Gram Positive Rods: Genus Bacillus

• Bacillus is a genus of gram-positive bacteria, often rod-shaped.
• Young/fresh cultures exhibit square-ended bacilli.
• Some cultures may appear gram-negative in older stages.
• Aerobic or facultative anaerobic bacteria.
• Form spores.
• Mainly harmless saprophytes (decomposers).
• Found in soil and water, and sometimes in surgical environments or dressings.

Bacillus anthracis

• Causing anthrax—a significant disease in herbivores.
• Distinguishing Feature: unique capsule (poly-D-glutamate) type & several exotoxins.
• Capsule prevents phagocytosis.
• Potential biowarfare agent.
• Reservoir: a wide range of animals

Bacillus anthracis: Virulence Factors

• Capsule—plasmid encoded.
• Anthrax toxin (AB toxin): consists of 3 proteins (edema factor [EF], lethal factor [LF], protective antigen [PA]).
• EF: an adenylate cyclase.
• LF: kills cells.
• PA: mediates the entry of LF or EF into eukaryotic cells.

Bacillus anthracis: Pathogenesis

• Anthrax is primarily a disease affecting herbivores.
• Humans become infected through contact with infected animals or their materials (e.g., hides, hair, animal products).
• Human infection potentially from handling infected animal material (e.g. skins, hides).
• Other sources include animal hair, bones, and bedding from infected animals.

Bacillus anthracis: Diseases

• Cutaneous anthrax (most common): bacilli enter damaged skin, create a blister ("malignant pustule"), and eventually a scab with edema..
• Pulmonary anthrax ("wool sorter's disease"): a life-threatening pneumonia caused by inhaling spores.
• Enteric anthrax (rare): severe gastrointestinal inflammation, vomiting, and bloody diarrhea due to infected meat.
• Septicemia and Meningoencephalitis: might follow other types of anthrax

Bacillus anthracis: Laboratory Diagnosis

• Specimen: fluid/pus from a lesion, blood, or sputum.
• Microscopic: stained smears from lesions/blood from dead animals display gram-positive rod chains.
• Microscopic: Loeffler's polychrome stain highlights the gram-positive bacilli, showing square ends and a pink/purple capsule.
• Other techniques utilize immunofluorescence for identification on dried smears.
• Culture: usually grows non-hemolytic colonies on blood agar; colonies are gray to white with a rough texture.
• Culture: Gram-positive spores may be exhibited.
• Biochemical tests: ferments glucose, maltose, and sucrose with acid production, but no gas. Positive for Nitrate reduction test, Catalase test, and starch hydrolysis tests.

Bacillus anthracis: Prevention & Control

• Treatment: antibiotics such as ciprofloxacin or doxycycline are effective.
• Prevention: Disposal of animal carcasses by burning or deep burial in lime pits, decontamination of animal products, and handling infected materials, while wearing appropriate clothing and gloves.

Bacillus cereus: Distinguishing Features

• Reservoir: naturally found in the environment—soil, water.
• Distinguishing feature: spores.
• Transmission: food-borne intoxication.
• Associated with fried rice from Chinese restaurants.

Bacillus cereus: Pathogenesis

• Produces enterotoxins, causing food poisoning.
• Emetic type: associated with fried rice and preformed foods, is fast onset (1–6 hours) similar to S. aureus.
• Diarrheal type: associated with meat dishes and sauces, later onset (18–24 hours).

Bacillus cereus: Diseases

• Gastrointestinal illness (e.g., gastroenteritis) resulting from toxin production in food.

Bacillus cereus: Laboratory Diagnosis

• Specimen: suspected food, vomiting, or patient material (e.g., rice, meat).
• Microscopic examination typically shows large Gram-positive rods.
• Distinctive cultures are displayed on blood agar plates; exhibiting non-hemolytic, gray-white, colonies with rough textures.
• B. cereus is often motile, non-capsulated, and displays hemolytic colonies on blood agar; produces colorless colonies on MacConkey Agar.
• A lecithinase test and similar tests can distinguish this species.

Bacillus cereus: Prevention & Control

• Treatment: generally self-limiting. Antibiotics sometimes used—gentamicin, erythromycin, vancomycin, clindamycin.
• Prevention: proper food handling practices, avoiding keeping rice warm for extended durations.

Genus Clostridium

• Gram-positive, rod-shaped anaerobic bacteria (often forming spores).
• Many are motile (certain exceptions).
• Form spores.
• Ferment organic compounds; often producing acid and gas.
• Often produce foul-smelling or offensive-smelling products.
• Commonly found in soil, water, and animal or human digestive tracts.
• Produce extracellular enzymes, degrading biological molecules.
• Play significant roles in biodegradation and carbon cycling.
• Some are medically important, causing disease (e.g. tetanus, botulism).

Clostridium perfringens: Distinguishing Features

• Large Gram-positive, often spore-forming rods (spores are less common in tissue samples).
• Anaerobic; exhibits "stormy fermentation" in laboratory media (e.g., in milk).
• Double zones of hemolysis, as observed in laboratory media.
• Oval, sub-terminal, often non-bulging spores.
• Non-motile.

Clostridium perfringens: Virulence Factors

• Exotoxin, enterotoxin, hydrolytic enzymes.
• Based on surface proteins and lethal toxins; Five primary types (A-E) have been identified.

Clostridium perfringens: Pathogenesis

• Spores germinate in tissues under anaerobic conditions.
• Vegetative cells produce toxins; some destroy tissues, others cause illness such as enterotoxins (emetic toxin).
• Alpha toxin: a phospholipase C damaging red blood cells (RBCs), platelets, and white blood cells (WBCs), causing tissue damage.
• Enterotoxin is produced in the gut and causes watery diarrhea (within a 24hr period post-ingestion).

Clostridium perfringens: Diseases

• Human illness is sometimes associated with type A or C.
• Food poisoning (e.g., from contaminated food); most common clinical presentations; type A.
• Gas gangrene; type A1.
• Other similar forms of illness are also possible related to different types.

Clostridium perfringens: Laboratory Diagnosis

• Specimen: material from wounds, pus, and tissue.
• Microscopic examination displays large Gram-positive rods.
• Culturing in specific mediums such as chopped meat glucose medium, thioglycolate medium, and blood agar (with anaerobic incubation).
• Milk cultures sometimes used.
• Biochemical tests include catalase, oxidase, lecithinase, lipase, and lactose fermentation.

Clostridium perfringens: Prevention & Control

• Treatment of gangrene: typically involves debridement (removal of damaged tissue), medications (e.g. clindamycin, penicillin) or hyperbaric oxygen therapy (HBO).
• Treatment of food poisoning: usually self-limiting.
• Prevention: proper food handling, including avoiding contamination, and cooking food adequately can often prevent illness

Clostridium botulinum: General Features

• Anaerobic.
• Gram-positive spore-forming rods.
• Oval, sub-terminal endospores.
• Motile.
• Grows best in neutral or low-acid environments.
• Reservoir: soil/dust.
• Transmission: foodborne/traumatic implantation.

Clostridium botulinum: Virulence Factors

• Botulism toxin (neurotoxin)—heat labile.
• Seven toxin types recognised.
• Types A, B and E commonly related to human disease.
• Type E usually associated with fish, and Type D with mammals.

Clostridium botulinum: Pathogenesis

• Botulism is an intoxication, not an infection.
• Resulting from ingesting food containing preformed toxin.
• Preformed botulinum toxin produces symptoms within a day or two post-exposure, and is usually fatal.
• Proteolytic cleavage activates the toxins.

Clostridium botulinum: Pathogenesis—Infant Botulism

• Primarily results from colonization of C. botulinum in infants.
• Absorption of toxin generated in the infant gut.

Clostridium botulinum: Pathogenesis—Wound Botulism

• Enters necrotic tissue.
• Causes disease in those areas.

Clostridium botulinum: Laboratory Diagnosis

• Specimens: Include suspected food/samples and patient excretions/serum/faeces.
• Cultures/Biochemical tests: Robertson's cooked meat medium (RCMM).
• Growth media/incubation usually required—usually for 3 to 5 days.
• Specific tests can be performed on agar or subcultured blood agar cultures.

Clostridium botulinum: Prevention & Control

• Treatment: trivalent antitoxin (A, B, E) which should be administered intravenously.
• Prevention: Strict regulation/observation of canning or food safety processes and procedures.

Clostridium tetani: General Features

• Terminal spores within a swollen sporangium (drumstick/tennis racquet appearance).
• A potent toxin-neurotoxin—potentially fatal toxin.
• Most strains are motile (peritrichous flagella)..
• Horses and humans are susceptible, often causing tetanus.
• Reservoir: soil.

Clostridium tetani: Pathogenesis

• Tetanus results from tissue contamination often secondary to puncture-type wounds.
• A non-invasive disease generated by the toxin.
• C. tetani produces a spasmogenic toxin (tetanospasmin).
• Tetanospasmin is a protein that prevents release of glycine (a neurotransmitter).
• Without glycine, muscle spasms and convulsions occur.

Clostridium tetani: Diseases

• Tetanus ("lockjaw"): characterized by muscle spasms, often starting in the jaw.

Clostridium tetani: Laboratory Diagnosis

• Specimen: wound swabs or excised bits of necrotic tissue.
• Microscopic examination typically displays the characteristic gram positive rods with terminal spores. (drumstick/tennis racquet appearance).
• Specific isolation from culturing in a variety of media.

Clostridium tetani: Prevention & Control

• Treatment: Hyper-immune globulin (TIG) & medications (antibiotics e.g., metronidazole or penicillin), to neutralise the toxin and debride (remove damaged tissue).
• Prevention: Active immunization, passive immunization, and combined immunization procedures are used.

Clostridium difficile: General Features

• Slender bacilli with large, oval, subterminal spores.
• Members of the intestinal flora.
• Reservoirs: human colon or gastrointestinal tract.
• Mode of transmission: endogenous.

Clostridium difficile: Virulence Factors

• Produces two toxins: toxin A (enterotoxin), toxin B (cytotoxin).

Clostridium difficile: Pathogenesis

• Toxin A damages the intestinal mucosa, and increases fluid accumulation.
• Toxin B is cytopathic and cytotoxic.

Clostridium difficile: Diseases

• Antibiotic-associated diarrhea and colitis—caused by antibiotic therapy; sometimes referred to as pseudomembranous colitis.

Clostridium difficile: Laboratory Diagnosis

• Culture not diagnostic due to presence in normal flora, (however, sometimes microscopic visualisation of the species is used to confirm diagnosis).
• Other diagnostic tests often used are examination of patient's stool, and endoscopic assessments may also be used.

Clostridium difficile: Prevention & Control

• Treatment: use metronidazole; vancomycin if needed (used for severe cases only—if metronidazole is not effective).
• Prevention: Avoiding unnecessary antibiotic use and appropriate or enhanced hygiene practice.

Genus Corynebacterium

• Non-spore-forming gram-positive bacilli.
• Often club-shaped or irregular in shape.
• High guanosine and cytosine content.
• Found (normally) in or on human skin and/or mucous membranes.
• Some species are found in other animals and plants—often producing exotoxins.
• Includes medically significant species, such as Corynebacterium diphtheriae, which causes diphtheria.

Corynebacterium diphtheriae: Distinguishing Features

• Gray-to-black colonies from club-shaped/irregularly shaped gram positive rods.
• Frequently visualized in V or L arrangements in gram stained samples.
• Granules (volutin) are detectable using specific staining mechanisms (e.g. Loeffler's medium).
• Non-capsulated, and non-motile.
• Aerobic.
• Do not form spores.

Corynebacterium diphtheriae: Biovars

• Four biovars identified: gravis, intermedius, mitis, and belfanti.
• Originally severity classifications, less frequently used.
• Toxigenic and non-toxigenic strains occur in each.

Corynebacterium diphtheriae: Reservoir

• Throat and nasopharynx.

Corynebacterium diphtheriae: Transmission

• Spread by respiratory droplets from affected individuals.

Corynebacterium diphtheriae: Virulence Factors

• Produces a potent diphtheria toxin (an ADP ribose).

Corynebacterium diphtheriae: Pathogenesis

• Non-invasive; colonises mucosal surfaces (e.g., throat).
• Produces diphtheria toxin (A-B component) which inhibits protein synthesis, by adding ADP-ribose to EF-2.
• Often results in a pseudomembrane formation on the throat (from dead cells, and fibrin exudate, etc)
• Can cause obstruction in larynx and trachea.
• Also potential systemic effect on heart and nerve tissue—inducing damage.

Corynebacterium diphtheriae: Diseases

• Diphtheria: characterized by a pseudomembrane formation in the throat.

Corynebacterium diphtheriae: Laboratory Diagnosis

• Specimens: throat or nasopharyngeal swabs (or skin swabs if cutaneous diphtheria suspected).
• Microscopic: gram positive; short rods; clubbed/enlarged at one end.
• Culture: growth observed on specific media such as tellurite blood agar or Tinsdale medium—producing characteristic colonies such as grey-black colonies.
• Biochemical tests and toxigenicity tests.

Corynebacterium diphtheriae: Prevention & Control

• Treatment: rapid administration of antitoxin, as well as antibiotics such as penicillin or erythromycin are important.
• Prevention: active and passive immunisations (or a combined approach).

Genus Listeria

• Gram-positive bacilli; non-spore-forming
• Facultative intracellular bacteria.
• Tumbling motility (and often display actin motility in cells).
• Frequent cold-growth.

Listeria monocytogenes: Distinguishing Features

• Small gram-positive rods.
• Beta-hemolytic.
• Nonspore-forming.
• Camp factor is often positive
• Tumbling motility (in broth)
• Facultative intracellular parasite.
• Cold growth characteristic is often observed.

Listeria monocytogenes: Reservoir

• Widely distributed (animals, unpasteurized milk, plants, etc.).
• Often found in the gastrointestinal and genital tracts of animals.
• Frequent contamination of some foods (e.g. refrigerated foods, cold cuts, soft cheeses, etc.).

Listeria monocytogenes: Pathogenesis

• Listerolysin O is a beta-hemolysin involved in overcoming phagocytosis.
• Facilitates rapid egress from phagosomes and enters the cytoplasm.
• Immunological immaturity in certain groups can increase the risk of infection.

Listeria monocytogenes: Diseases

• Listeriosis (human disease)
• A systemic disease, sometimes with focal infection, often in immunocompromised individuals (or pregnant women especially).
• Various clinical presentations, sometimes with septicaemia, and meningitis.

Listeria monocytogenes: Laboratory Diagnosis

• Specimens: typically blood or CSF.
• Microscopic: typically small Gram-positive rods displayed.
• Culture: in selective mediums with appropriate incubations.
• Other tests help assess specific aspects of the bacteria.

Listeria monocytogenes: Prevention & Control

• Prevention methods include cooking and handling of refrigerated foods appropriately.
• Pregnant and immunocompromised individuals should avoid foods from which this species could be isolated.
• Antibiotics are frequently used.

Genus Mycobacterium

• Acid-fast rods with a waxy cell wall.
• Obligate aerobes (and often have slow growth characteristics).
• Cell walls contain a high proportion of lipids (especially mycolic acids), which contributes to their resistance to many chemicals and detergents—frequently used in bacterial isolation.

Mycobacterium tuberculosis: Distinguishing Features

• Auramine-rhodamine staining—the resulting color is often described as fluorescent apple green.
• Acid-fast staining—displayed in appropriate mediums.
• Aerobic and grows slowly in Lowenstein-Jensen mediums.
• Produces niacin.

Mycobacterium tuberculosis: Reservoir

• Human lungs.

Mycobacterium tuberculosis: Transmission

• Respiratory droplets.

Mycobacterium tuberculosis: Pathogenesis

• Sulfatides and Cord factor are involved in intracellular survival.
• Tuberculin is involved in delayed-hypersensitivity and cell-mediated immunity.
• Primary pulmonary tuberculosis develops in individuals exposed to the bacteria; may then later reactivate under conditions of reduced cellular immunity (T-cell mediated).

Mycobacterium tuberculosis: Disease

• Primarily pulmonary tuberculosis, but potentially systemic or disseminated such as miliary TB.

Mycobacterium tuberculosis: Laboratory Diagnosis

• Microscopy of sputum sample often used for visualisation.
• Acid-fast staining is often used and visualization of bacilli in specific samples is a key diagnostic process.
• PPD skin test (Mantoux).
• Quantiferon-TB Gold Test—measuring interferon-gamma production.
• Culture in Lowenstein-Jensen medium.
• Biochemical tests such as catalase and niacin production.

Mycobacterium tuberculosis: Prevention & Control

• Control involves multi-method approaches and specific antibiotics.