Miscellaneous Gram-Negative Bacilli

• Capnocytophaga


  • Normal microbiota of the oral cavity of humans
  • Causes septicemia in patients with neutropenia
• Capnocytophaga


  • Common sites of clinical isolation: blood cultures from patients who have neutropenia with oral ulcers (source of the Capnocytophaga), soft tissue infections, peritonitis, and endocarditis
• Capnocytophaga species

  • C. ochracea- most common clinical isolate
  • C. canimorsus and C. cynodegmi- normal inhabitants of the oral cavity of dogs and cats
• Capnocytophaga

  • Fastidious, facultative anaerobe
  • Thin and often fusiform (pointed ends) resembling Fusobacterium spp.
  • No flagella but can produce gliding motility on solid surfaces
  • Ferment sucrose, glucose, maltose, and lactose
  • Indole negative
• Legionella pneumophila causes Legionnaire's disease
• Legionnaire's disease first recognized during an epidemic of pneumonia that occurred among members of the Pennsylvania American Legion who had gathered in Philadelphia to celebrate the bicentennial


  1976
• Legionella


  • Non–spore-forming, faintly staining, thin, gram-negative bacilli
  • Found in the environment especially water
  • Tolerate chlorine concentrations of 3 mg/L
• Transmission of Legionella

  1. Exposure to contaminated water (faucets, shower heads, public fountains)

  2. Hot water systems, cooling towers, and evaporative condensers are major reservoirs
• Legionella virulence factors

  • Ability to exist as intracellular pathogens (amebae and mammalian cells)
  • Survive inside phagosomes, prevent the formation of phagolysosome (can't survive by phagocytosis)
  • Ability to multiply over the temperature range of 20° to 43° C and survive for varying periods at 40° to 60° C
  • Capacity to adhere to pipes, rubber, plastics, and sediment and persist in piped water systems, even when flushed
• Legionnaire's disease

  Febrile disease with pneumonia
• Legionnaire's disease

  • Pneumonia is the predominant manifestation
  • Community acquired bacterial pneumonia
  • Incubation period: 2-10 days
  • Symptoms: nonproductive cough, fever, headache, and myalgia
  • Sputum may be bloody or purulent
  • May lead to extrapulmonary infection
• Pontiac fever

  • Influenza-like febrile disease
  • Non-pneumonic form of legionellosis
• Pontiac fever


  • Incubation period of 2 days
  • Caused by inhalation of bacterial toxins or an acute allergic reaction to the bacteria
  • Affects previously healthy individuals who complain of flulike symptoms of fever, headache, and myalgia that last 2 to 5 days and then subside without medical intervention
• Asymptomatic infection can occur with Legionella
• Laboratory diagnosis of Legionnaire's disease

  Combination of culture and urine antigen detection
• Specimen for Legionella culture and direct examination

  • Sputum, bronchoalveolar lavage, and bronchial washings
  • Other tissues or fluids, such as pleural fluid, are generally acceptable when suspicion is high
  • Freeze specimens at −70° C if processing will be delayed for several days
• Microscopic examination of Legionella

  • Pleomorphic, weakly staining, gram-negative bacilli found outside of and within macrophages and segmented neutrophils
  • Other stains that can be used: Diff-Quik and Giemsa
• Isolation of Legionella

  1. Fastidious, aerobic bacteria that do not grow on SBA (sheep blood agar) and require L-cysteine for growth

  2. Acid treatment of specimen before inoculation to dilute microbial inhibitors such as complement antibodies and antimicrobial agents

  3. BCYE (buffered charcoal yeast extract) Agar with L-cysteine is best for Legionella isolation

  4. Colonies appear grayish-white or blue-green, convex, and glistening, measuring approx. 2 – 4 mm in diameter

  5. Central portion of young colonies has a "ground-glass" appearance, light gray and granular

  6. Periphery has pink or light blue or bottle green bands with a furrowed appearance

  7. Incubate at 35° C in air; increased CO2 can enhance the growth of some of the more fastidious species

  8. Selective BCYE agar contains polymyxin B, anisomysin, and either vancomycin or cefamandole
• Bordetella pertussis and Bordetella parapertussis cause whooping cough or pertussis
• Bordetella pertussis and Bordetella parapertussis

  Cause mild illness and asymptomatic infection, primarily in household contacts and in a number of unvaccinated and previously vaccinated children
• Transmission of whooping cough or pertussis

  Occurs person to person through inhalation of respiratory droplets
• Whooping cough or pertussis

  • Highly contagious, acute infection of the upper respiratory tract
  • Caused by Bordetella pertussis
• Stages of whooping cough or pertussis

  1. Catarrhal stage: symptoms are the same as for a mild cold with a runny nose and mild cough, lasts several weeks

  2. Paroxysmal stage: severe and violent coughing, 15 to 25 paroxysmal coughing episodes can occur in 24 hours, vomiting and "whooping" (the result of air rapidly inspired into the lungs past the swollen glottis), lasts 1-4 weeks

  3. Convalescent phase: begins within 4 weeks of onset with a decrease in frequency and severity of the coughing spells
• Major virulence determinants of Bordetella pertussis

  • Fimbriae types 2 and 3: serotype-specific agglutinins for colonization of respiratory mucosa
  • Filamentous hemagglutinin (FHA): mediates adhesion to the ciliated upper respiratory tract
  • Pertactin (PRN): mediates Eukaryotic cell binding and is highly immunogenic
  • Tracheal colonization factor Brk A
  • Pertussis toxin: encoded by ptx gene which is an A/B toxin related to cholera toxin, induces lymphocytosis and suppresses chemotaxis and oxidative responses in neutrophils and macrophages
  • Adenylate cyclase toxin: hemolyzes red cells and activates cAMP, thereby inactivating several types of host immune cells
  • Dermonecrotic toxin: exact role unknown
  • Tracheal cytotoxin: ciliary dysfunction and damage
  • Endotoxin: lipopolysaccharide
  • Type III secretion: to transport proteins directly to host cells
  • Outer membrane: inhibits host lysozyme
  • Siderophore production: prevents host lactoferrin and transferrin from limiting iron
• Specimen of choice for Bordetella diagnosis

  • Nasopharyngeal aspirates or a nasopharyngeal swab (calcium-alginate or Dacron on a wire handle)
  • Specimens obtained from throat, sputum, and anterior nose are unacceptable since these sites are not lined with ciliated epithelium
• Transport media for Bordetella specimens

  Half-strength Regan-Lowe agar, cold casein hydrolysate medium, casamino acid broth
• Selective media for Bordetella pertussis and parapertussis

  • Bordet-Gengou: potato infusion agar with glycerol and sheep blood with methicillin or cephalexin
  • Modified Jones-Kendrick charcoal: charcoal agar with yeast extract, starch, and 40 μg cephalexin
  • Regan-Lowe: charcoal agar with 10% horse blood and cephalexin, best for recovery of B. pertussis
  • Stainer-Scholte: synthetic agar lacking blood products
• Cephalexin is superior to methicillin and penicillin for inhibiting normal respiratory flora in Bordetella isolation
• Bordetella pertussis and parapertussis colonies

  • Young colonies have a "mercury drops" appearance, becoming whitish gray with age
• Erythromycin


  Drug of choice for pertussis
• Differential characteristics of Bordetella species

  • Catalase: B. pertussis, B. parapertussis, B. bronchiseptica are all positive
  • Oxidase: B. pertussis and B. bronchiseptica are positive, B. parapertussis is negative
  • Motility: B. pertussis and B. parapertussis are non-motile, B. bronchiseptica is motile
  • Nitrate: B. pertussis, B. parapertussis are negative, B. bronchiseptica is positive
  • Urease: B. pertussis is negative, B. parapertussis is positive in 24 hours, B. bronchiseptica is positive in 4 hours
  • Growth on media: B. pertussis takes 3-6 days on Regan-Lowe agar, B. parapertussis takes 2-3 days, B. bronchiseptica takes 1-2 days
  • Growth on blood agar: B. parapertussis and B. bronchiseptica are positive, B. pertussis is negative
  • Growth on MacConkey agar: B. parapertussis is variable, B. bronchiseptica is positive, B. pertussis is negative
• Pasteurella is a zoonosis, a disease that humans acquire from exposure to infected animals
• Pasteurella is normal flora of the oral cavity in birds and mammals
• Charcoal agar with 10% horse blood and cephalexin


  1. to 8-week shelf life
• Stainer-Scholte


  Synthetic agar lacking blood products
• Cephalexin


  Superior to methicillin and penicillin for inhibiting normal respiratory flora
• Incubation of plates


  Incubated at 35°C in a humidified atmosphere without elevated carbon dioxide for up to 12 days
• Young colonies of B. pertussis and B. parapertussis

  • "MERCURY DROPS"
• Colonies


  • Become whitish gray with age
• Differential characteristics of Bordetella species

  • Catalase (+)
  • Oxidase (B. pertussis +, B. parapertussis -, B. bronchiseptica +)
  • Motility (B. pertussis -, B. parapertussis -, B. bronchiseptica +)
  • Nitrate (B. pertussis -, B. parapertussis -, B. bronchiseptica +)
  • Urease (B. pertussis -, B. parapertussis + (24 hours), B. bronchiseptica + (4 hours))
  • Growth on Reagan-Lowe agar (B. pertussis 3-6 days, B. parapertussis 2-3 days, B. bronchiseptica 1-2 days)
  • Growth on Blood Agar (B. pertussis -, B. parapertussis +, B. bronchiseptica +)
  • Growth on MacConkey agar (B. pertussis -, B. parapertussis +/-, B. bronchiseptica +)