BACTERIOLOGY

• Corynebacterium


  Gram positive rod-shaped bacteria or bacilli, swollen or club-shaped at the ends, slight variation in shape and size, stain irregularly, non-spore formers, non-motile, facultative anaerobes
• Corynebacterium species


  • Corynebacterium diphtheriae (main pathogenic species)
  • Diphtheroids or coryneform organisms (other species)
• Corynebacterium diphtheriae


  • Gram positive bacilli with variation in shape and size, stain irregularly and are easily decolorized in Gram staining, contain metachromatic or volutin granules in the cytoplasm, tend to be placed at the ends of the bacillus, form pairs attached at one end or parallel rows (palisade arrangement)
• Significant physical properties of C. diphtheriae

  • Can remain viable for weeks in dust or on dry articles, susceptible to heat and regularly used chemical disinfectants
• Main pathogenicity properties of C. diphtheriae

  • Invasion of tissues locally at the infected site, exotoxin (diphtheria toxin)
• Diphtheria toxin


  Responsible for the severity of the disease diphtheria, not released by all C. diphtheriae, ability to produce the toxin is developed as a result of genetic material acquired via bacteriophages, species is further classified into toxigenic and non-toxigenic strains
• Severity of diphtheria caused by toxigenic strains

  • Severe
  • Intermediate
  • Mild
• Clinical significance of C. diphtheriae


  • Causes diphtheria as the major illness, occasionally causes wound infection
• Diphtheria


  Acute disease which occurs in outbreaks, affects children more than adults, transmitted via secretions or droplets from the upper respiratory tract, initial manifestations are confined to the oropharynx due to local invasion of the mucosal surface by bacilli, effects of diphtheria toxin at the site, and inflammatory response
• Manifestations of diphtheria


  • Fever
  • Inflammatory process in upper respiratory tract observed as greyish membrane-like covering (pseudomembrane) in the throat and externally a swelling around the neck due to oedema
  • Difficulty in breathing
• Complications and other effects of the diphtheria toxin

  • Airway obstruction, damage to the heart muscle, degeneration of peripheral nerves, damage to the adrenal glands
• Laboratory investigation of diphtheria

  Specimen can be fluid or infected tissue or part of the pseudomembrane from oropharynx, procedures include Gram stain of smear for Gram positive bacilli, isolation by culture, and identification
• Isolation of C. diphtheriae

  Culture media to consider include SBA (serum or whole blood provides necessary nutrients for growth) and selective media like Potassium tellurite blood agar (potassium tellurite suppresses the growth of other organisms)
• Loeffler's serum agar for C. diphtheriae

  Used after primary isolation to facilitate rapid growth and marked volutin granules formation, volutin granules are demonstrated as part of identification process by Albert's stain or Neisser's stain
• Other factors applied in identification of C. diphtheriae

  • Characteristics of the colonies
  • Gram's stain and microscopic morphology
  • Biochemical tests where appropriate
  • Tests to demonstrate toxin production by the isolated organism
• Management methods of diphtheria

  Specific treatment includes diphtheria antitoxin and antimicrobial treatment (penicillin and erythromycin), supportive care
• Prevention and control of diphtheria

  Detection and treatment of carriers and patients, management of patients in separate rooms from others, active immunization (DPT in childhood immunization)
• Diphtheroids


  Microscopically similar to C. diphtheriae, occur as saprophytes or normal flora on the skin and mucous membranes, commonly encountered as contaminants of specimens and cultures, uncommon causative agents of opportunistic infections
• Bacillus


  Gram positive rods, arranged singly or in chains, spore-formers, most species are motile, majority of the species form colonies with complete zones of haemolysis on blood agar, aerobic or facultative anaerobes
• Bacillus species


  • B. anthracis (main pathogen)
  • B. cereus
  • B. subtilis
  • B. pumilis
  • B. stearothermophilus
• Bacillus anthracis


  • Gram or rods observable as single cells or one cell attached to another to form chains, capsulated in , , forms outside animal host
• Significant physical properties of B. anthracis

  • Bacilli are destroyed by heat at a temperature of 60 degrees centigrade for 30 minutes, spores are relatively resistant to chemical disinfectants and heat, resistant to physical changes in the environment, survive in the environment or in animal products longer than other organisms
• Pathogenicity properties of B. anthracis

  • Ability to form spores, capsule which inhibits phagocytosis, exotoxin (a complex which consists of three parts, each part has its function in pathogenesis)
• Clinical implication of B. anthracis

  • Causative agent of anthrax, primarily a disease of animals including cattle, sheep, and goats, human infections are accidental and occur in outbreaks
• Transmission of B. anthracis

  Transmitted agent in most cases is the spore, spores originate from bacilli shed from infected animals through various openings, soil and pastures become contaminated with spores, humans and other animals are accidentally exposed from the contaminated sites, spores can also be acquired through handling contaminated animal products
• Entry of B. anthracis into humans

  • Contamination of damaged skin or mucous membranes
  • Inhalation of spores
  • Ingestion
• Manifestations of human anthrax

  Cutaneous anthrax (infection occurs through broken skin, develops a dark-colored eschar), inhalational anthrax or Woolsorter's disease (severe signs and symptoms due to infection and inflammation of the lungs), gastrointestinal anthrax (acquired mainly by eating meat from infected animals, includes abdominal pain, vomiting, fever, diarrhoea), septicaemic anthrax (may or may not be as a complication of the above initial manifestations, predisposes to spread of infection to other internal tissues)
• Laboratory investigation of anthrax in humans

  Specimens include fluid or pus from cutaneous lesions, blood, sputum, highly infectious specimens collected, transported, and processed with recommended protection methods, examination procedures include staining and microscopy of smears, culture of B. anthracis
• Anthrax


  Acquired mainly by eating meat from infected animals
• Manifestations of anthrax


  • Abdominal pain
  • Vomiting
  • Fever
  • Diarrhoea which may be bloody
• Septicaemic anthrax


  May or may not be as a complication of the initial manifestations, predisposes to spread of infection to other internal tissues
• Specimens for laboratory investigation of anthrax in humans

  • Fluid or pus from cutaneous lesions
  • Blood
  • Sputum
• Anthrax specimens


  • Highly infectious
  • Collected, transported and processed as higher risk specimen
  • Recommended protection methods are applied
• Examination procedures for anthrax

  1. Staining and microscopy of smears

  2. Culture of B. anthracis
• Culture media for B. anthracis

  S.B.A., Nutrient agar
• Incubation conditions for B. anthracis

  In air, 35 to 37 degrees C, 18 to 24 hours
• Colonies of B. anthracis

  • Greyish opaque with a wavy margin and small projections (Medusa head), 2 to 5mm diameter, rough surface, non-haemolytic for most strains
• Gram's stain of B. anthracis

  Gram positive bacilli
• Identification of Bacillus anthracis

  1. Growth requirements

  2. Appearance of colonies on various media

  3. Gram's stain morphology

  4. Motility test (non-motile)

  5. Biochemical tests including a positive gelatin liquefaction test
• Antimicrobial agents B. anthracis is susceptible to

  • Penicillin
  • Ciprofloxacin