Bacillus and Anthrax Quiz

Gram Positive Rods

• Bacillus, Clostridia, Listeria, Corynebacterium are gram-positive rods.

Bacillus

• Gram-positive rods, large, arranged singly or in chains.
• Endospore-formers and toxin-producers.
• Aerobic or facultative anaerobic, non-motile/motile, non-hemolytic.
• Ubiquitous in nature (soil, water, or vegetation).
• Involved in the biological cycles of carbon/nitrogen.
• Family Bacillaceae has diverse genera and species.
• Some are commensals of herbivores.
• Some can infect humans.
• Clinically relevant strains for humans include Bacillus anthracis and Bacillus cereus.
• Spore diameter does not exceed the bacterial cell diameter in Bacillus.

Bacillus anthracis

• Gram-positive rod, spore-forming.
• Virulent strains have a polypeptide capsule (poly-D-glutamic acid) encoded by plasmid pXO2.
• Virulent strains carry genes for three toxic proteins (PA, EF, LF) on plasmid pXO1.
• PA (protective antigen)
• EF (edema factor)
• LF (lethal factor).

Anthrax

• Zoonotic disease affecting herbivores (cattle, sheep, goats, deer).
• Humans infected by ingesting contaminated vegetation, water, soil or carcasses/animal products (e.g., meat, wool).
• Transmission routes:
  • Inoculation of spores through exposed skin from contaminated soil or infected animal products.
  • Ingestion of spores (rare in humans, frequent in grass-eating animals).
  • Inhalation of spores (most likely route for biological weapons).
• Type of illness depends on how spores enter the body.
• All types can be fatal if untreated with antibiotics.

Cutaneous anthrax

• Typically starts with a painless papule at the inoculation site.
• Progresses rapidly to an ulcer, then a necrotic eschar.
• Eschar with extensive surrounding edema/inflammation is hallmark.
• Case-fatality: 20% without treatment; <2% with antimicrobial therapy.

Gastrointestinal anthrax

• Ulcers form at invasion site (mouth, esophagus, intestine).
• Leads to regional lymphadenopathy, edema, and sepsis.
• Initial symptoms include nausea, vomiting, and malaise.
• Rapid progression to systemic disease.
• Virtually 100% fatal without treatment.

Inhalation anthrax

• Spores reach lower airways and mediastinal lymph nodes.
• Non-specific initial symptoms after a prolonged latent period (~2 months).
• Symptoms include fever, myalgia, non-productive cough, and malaise.
• The second stage is dramatic with rapidly worsening fever, edema, massive enlargement of mediastinal lymph nodes, respiratory failure, and sepsis.
• Almost all cases progress to shock and death within 3 days of initial symptoms, unless treatment is initiated immediately.
• 100% fatal without treatment.

Laboratory Diagnosis

• Microscopy: Gram stain for observation of rods in clinical specimens.
• Culture: Colonies grow readily on blood agar plates, large, non-pigmented, dry "ground-glass" surface, irregular edges. B. anthracis colonies have a "Medusa Head" appearance.

Treatment of B. anthracis Infection

• Before 2001, penicillin G was the first-line treatment.
• Now ciprofloxacin or doxycycline combined with other antibiotics (rifampin, vancomycin, penicillin, imipenem, clindamycin or clarithromycin) is recommended.
• Treatment course duration is 7 days for topical infections and 60 days for inhalation infections.

Clostridia

• Anaerobic gram-positive rods capable of forming endospores.
• Ubiquitous in soil, water, and sewage.
• Some are part of the normal microbial population in the gastrointestinal tracts of animals and humans.
• Most are harmless saprophytes, but some are well-recognized pathogens.

Clostridium tetani

• Large, motile spore-forming rod.
• Vegetative form is extremely susceptible to oxygen toxicity.
• Sporulates readily and can survive in the environment for a long time.
• Virulence related to two toxins:
  • Tetanolysin (oxygen-labile hemolysin).
  • Tetanospasmin (plasmid-encoded, heat-labile neurotoxin) - causes tetanus.
• Disease characterized by muscle spasm and involving the autonomous nervous system.
• Generalized tetanus is most common.

Tetanospasmin

• Neurotoxin produced during the stationary phase of growth and released when the cell is lysed.
• Responsible for clinical manifestations of tetanus.
• A-B toxin, produced as a single peptide cleaved by endogenous proteases.
• The two parts (A and B) still remain together in the spinal cord.
• A subunit (zinc-endopeptidase) inactivates proteins that regulate the release of glycine and GABA (inhibitory neurotransmitters).
• Leading to unregulated excitatory synaptic activity resulting in spastic paralysis.

Pathogenesis of C. tetani infection

• Spores penetrate through skin wounds, burns, animal bites (splinter wounds are particularly dangerous).
• Spore germination and neurotoxin production >24-36 hours under anaerobic conditions.
• Bacteria are not invasive, staying localized at the entry site without causing suppurative processes.

Tetanus Types

• Generalized tetanus: Pain near wound, trismus (lockjaw), risus sardonicus, contraction at level of cervical musculature and trunk, hyperextension, thoracic and laryngeal spasms; and can lead to respiratory failure and cardiac arrhythmia. Mortality rate is ~30-50%
• Neonatal tetanus: Umbilical stump infection, common in developing countries. Leads to generalized tetanus. Infants typically die. Mortality rate is >90%.

Botulism

• Caused by a heterogeneous collection of fastidious, spore-forming anaerobic rods.
• C. botulinum strains produce various botulinum toxins.
• Spores are common in the environment and can contaminate food (e.g., home-canned food, honey, infant milk powder).
• Spores germinate, produce toxins only after several days of anaerobiosis.

Classic/foodborne botulism

- symptoms can occur from 6h to days after ingestion, severity depends on the dose.
- clinical presentations range from nuanced symptomatology to severe, fatal cases.
- Initial symptoms can include diplopia (difficulty with speech and swallowing), muscle weakness from the shoulders to lower limbs, respiratory paralysis.

Infant botulism

• Associated with consumption of foods (honey, infant milk powder) contaminated with spores.
• Spores germinate and colonize the gastrointestinal tract of the infants, multiplying and producing in vivo toxins. Proliferation is achieved in infants because of the absence of competitors.
• Symptoms are initially non-specific (constipation, weak cry), then flaccid paralysis. Respiratory arrest can occur, with a mortality rate less than 2%.

Wound botulism

• Rare disease, caused by bacteria present in contaminated wounds.
• Outcomes are similar to foodborne botulism, except the incubation period is longer (>4 days).

Inhalation botulism

• Major concern in the era of bioterrorism, caused by aerosolization of the neurotoxin as a biological weapon.
• Rapid onset and potentially high mortality.

Laboratory Diagnosis and Treatment for botulism

• Clinical diagnosis is confirmed by toxin detection in the implicated food, serum, or feces.
• Cultures of heated specimens to kill non-spore forming bacteria are applied on nutritionally enriched anaerobic media.
• Treatment includes adequate ventilatory support, elimination of the organism from the gastrointestinal tract. The use of penicillin or metronidazole, to kill the bacteria and reduce toxin production. Passive immunization with trivalent botulinum antitoxins (A, B, and E) to neutralize unbound toxin.
• Prevention: Spores in food should be destroyed. Germination of spores must be prevented. Preformed toxins in heated foods at temperatures around 60-100C for 10 minutes should be destroyed.

Clostridium perfringens

• Large, rectangular rod with spores that are rarely observed.
• Inhabits the intestinal tracts of humans and animals.
• Widely distributed in nature; particularly in soil and water contaminated with feces.
• Virulence is mediated by toxins (α, β, ε, iota, and enterotoxin).
• α-toxin is a phospholipase C that leads to hemolysis, vascular permeability, and tissue destruction.
• Enterotoxin causes food poisoning (produced during the transition phase of vegetative cells to spores in the small intestine).

Soft tissue infections caused by C. perfringens:

• Clostridial spores or vegetative organisms can be introduced into tissue during surgery or traumatic injury.
• Initial symptoms of intense pain and discolored, bullae skin develop and subcutaneous gas are present.
• Followed by muscle necrosis, shock, and possible renal failure, culminating in death within 2 days (mortality range 40-100%).

Laboratory diagnosis and Treatment for C. perfringens soft tissue infections

• Microscopy: Identification of the bacteria with characteristic morphology (gram-positive rectangular rods) in clinical specimens. Cultures can be grown on agar media or blood culture broths.
• Treatment: Surgical debridement, high doses of penicillin, and hyperbaric oxygen treatment may be used (inconclusive results).

Clostridium difficile

• Large, anaerobic rod that forms spores in vivo and in culture.
• Part of the normal intestinal flora in healthy individuals.
• Agent of endogenous infections and is associated with 15-25% of antibiotic-associated diarrhea (AAD) cases.
• The occurrence of this disease is a complication of antibacterial therapy.
• Symptoms are mild watery diarrhea progressing to severe diarrhea (bloody mucous diarrhea) and pseudomembranous colitis.
• Pathogenesis mediated by toxins A (cholera-like) and B (potent cytotoxin inhibiting protein synthesis).

Laboratory diagnosis and Treatment for C. difficile

• Diagnosis: confirm disease via toxin detection in stool or by detecting the C. difficile toxin genes via nucleic acid amplification techniques (molecular tests) in clinical specimens.
• Treatment: Discontinuation of the implicated antibiotic may alleviate mild disease. Metronidazole or vancomycin is necessary for severe diarrhea and colitis.

Listeria monocytogenes

• Facultatively anaerobic gram-positive rod with flagella.
• Short rod (coccobacillus), sometimes in pairs or short chains.
• Grows in a wide range of temperatures, pH, and high salt concentration.
• Facultative intracellular pathogen.
• Ubiquitous in nature (soil, vegetation, water) and in the gastrointestinal tract of animals (but not in humans).
• Human disease is uncommon, usually restricted to pregnant women/neonates, elderly, patients with defective cellular immunity.
• Primary source of infection → consumption of contaminated food (undercooked/processed meats, unpasteurized/contaminated milk, soft cheese, unwashed vegetables).

Listeria monocytogenes Pathogenesis

• Enters gastrointestinal tract after food ingestion.
• Adheres to epithelial cells via internalin A protein that triggers phagocytosis.
• Inside infected cells, produces listeriolysin O and two phospholipase C enzymes.
• Released into cytosol and replicates within infected cells.
• Replicates into epithelial cells and moves from cell to cell without immune system exposure.
• Enters macrophages after passing through intestinal lining. Dissemination ensues throughout the body(liver, spleen, etc.) triggering a disseminated disease.

Listeria monocytogenes Life Cycle

• Bacteria are phagocytized by host cells.
• Lysing of phagosomes enables bacteria to escape.
• Proliferation occurs within host cells.
• Cell to cell spread (actin based locomotion).
• Formation of filopods to invade adjacent cells.

Listeriosis Risk Factors

• Pregnant women (nonspecific influenza-like symptoms).
• Newborns/infants (early-onset or late-onset disease).
• Early-onset listeriosis is acquired during pregnancy and leads to abortion, stillbirth, or premature birth.
• Granulomas form in multiple organs. High mortality without treatment.
• Late-onset listeriosis acquired soon after birth triggers meningitis or meningoencephalitis with septicemia.
• Elderly and immunocompromised patients (invasive infections, such as sepsis, meningitis, and meningoencephalitis).
• Healthy adults may experience mild, self-limited influenza-like illness.

Listeria monocytogenes Laboratory Diagnosis and Therapy

• Microscopy: Gram stain for viewing short rods (coccobacillus).
• Culture: Small colonies on conventional media, with beta-hemolysis on blood agar (differentiation from streptococci); cold enrichment for culture from food.
• Treatment: Gentamicin with penicillin or ampicillin for serious infections (i.e., septicemia and meningitis).

Corynebacterium spp.

• Includes >100 species.
• Gram-positive bacteria, irregular shape, arranged in clumps or short chains.
• Aerobic or facultatively anaerobic, nonmotile, non-spore forming, club-shaped.
• Widely distributed in nature, especially soil, water, and human skin/respiratory tract.
• C. diphtheriae (most important), the etiologic agent of diphtheria.
• Humans are only known reservoir for this bacteria.
• Often have metachromatic granules (containing phosphates, lipids, RNA) at ends of bacterial cells.

Corynebacterium diphtheriae

• Major virulence factor: diphtheria toxin. Tox gene is introduced by a lysogenic bacteriophage, β-phage.
• Diphtheria toxin inhibits protein synthesis.
• Toxin receptor: is heparin-binding epidermal growth factor, present in many cells, particularly on heart and nerve cells.

Diphtheria (Respiratory Infection)

• Bacteria enters respiratory tract.
• Localizes on epithelial cells (pharynx).
• Causes localized damage, malaise, sore throat, exudative pharyngitis, low-grade fever.
• Exudate evolves into a thick pseudomembrane (bacteria, lymphocytes, plasma cells, fibrin, dead cells).
• Toxin spreads via blood to non-contiguous sites.
• Severe disease involves the heart (myocarditis) and nervous system (neuropathy, dysphagia, paralysis).

Diphtheria (Cutaneous Infection)

• Organisms invade subcutaneous tissue, leading to a papule, that develops a grayish membrane.
• Toxin elicits a systemic response with fever.
• Can affect the heart and nervous system.

Diphtheria Laboratory Diagnosis and Therapy

• Microscopy: Gram stain of pseudomembrane, respiratory swabs, or cutaneous lesions.
• Cultures: Corynebacterium spp. grown on Loffler medium (horse serum) and selective agar plates (e.g., cysteine tellurite blood agar, colistin-nalidixic agar).
• Treatment: Penicillin or erythromycin for eliminating C. diphtheriae and reducing toxin production. Early administration of diphtheria antitoxin is used to neutralize exotoxin before binding to epithelial cells.