1. MLAB 2434: Microbiology Keri Brophy-Martinez
Haemophilus and Other Fastidious Gram-Negative Rods

2. Haemophilus and Other Fastidious Gram-negative Rods
The fastidious group of gram-negative bacilli include:
Haemophilus
HACEK( Haemophilus, Actinobacillus, Cardiobacteria, Eikenella & Kingella)
Legionella
Bordetella
Pasteurella
Brucella
Francisella
Bartonella

3. Haemophilus Species Haemophilus = “blood loving”
Require either heme (X factor) or NAD (V factor)
Haemophilus is facultative and can grow anaerobically
Organism is sensitive to drying and extremes in temperature
Distinctive “mousy” or “bleach-like” odor

4. Haemophilus Influenzae
Misnamed – originally thought to cause the “flu”
Now know that flu is caused by viruses
In some cases of flu, H. influenzae is secondary infection

5. Haemophilus Influenzae: Virulence Factors
Capsule
Antiphagocytic
IgA Protease
Cleaves IgA on mucosal surfaces
Lipid A
Effects ciliated respiratory epithelium
Pili
Attachment

6. Haemophilus Influenzae: Clinical Infections: Typable strains
Acute epiglottitis or laryngotracheal infection in small children
Can cause airway obstruction needing immediate tracheostomy
Cellulitis/arthritis
cheek and upper extremities
Meningitis
Children under 6 years
Contagious, vaccine has decreased incidence
Pneumonia/septicemia
In children
Conjunctivitis “pink eye”
very contagious

7. Haemophilus Influenzae: Clinical Infections: nontypable strains
Otitis media
Children 6 months- 2 years
Sinusitis
Pneumonia, bronchitis
In adults
These sites are all in proximity to respiratory tract

8. Haemophilus Species Haemophilus species require growth factors:
X-factor ( hemin)
Heat-stable substance
Present in RBC and released with degradation of hemoglobin
V-factor (NAD: nicotinamide adenine dinucleotide)
Heat- labile
Found in blood or secreted by certain organisms

9. Haemophilus Species
H. influenzae satellitism around and between the large, white, hemolytic staphylococci.
This occurs when another organism produces V factor as a bi-product.

10. Haemophilus Species Gram Stain Morphology
Usually very small pleomorphic gram negative cb or rod
May be able to observe a halo around the organism
Gram stain can be enhanced by extending time for safranin to 2 minutes OR substitute carbolfuschin for safranin

11. Haemophilus Species
Direct smear of H. influenzae in CSF in a case of meningitis. Note the TINY intracellular and extracellular pleomorphic gram-negative bacilli.
Remember to look for capsules surrounding the rod.

12. Haemophilus Species Colony Morphology No growth on BAP or MAC On CA:
semi-opaque, gray-white, convex, mucoid.

13. Haemophilus Species: Identification
Gram stain
Gram negative cocco-baccillus
Catalase +
Oxidase +
X and V factor strips or disks
Quad plates
Rapid ID Panels
NHI cards- automated

14. Haemophilus Species: Identification
This organism would be identified as H. influenzae because it is using both X and V factors.

15. Haemophilus Species: Identification
This organism would be identified as H. parainfluenzae because it is using V factor only.

16. Haemophilus Species: Identification
Quad plates
Contain X and V
factors & sheep blood
agar

17. Haemophilus ducreyi
Causative agent of chancroid or soft chancre (STD), highly contagious
Specimens should be collected from base of lesion, inoculated directly to enriched media and held for 5 days
Gram stain appears as groups of coccbacilli that resemble a ‘school of fish” or “railroad tracks”
Requires only X factor to grow

18. Haemophilus Species: Identification
V=variable

19. Haemophilus Antibiotic therapy
Historically ampicillin was the drug of choice. However, resistance has developed due to production of beta-lactamase or altered penicillin binding proteins and cell wall permeability
Susceptibility testing can be performed by disk diffusion, broth dilution or E-test
Primary antibiotics include cefotaxime or ceftriaxone

20. Take 5!

21. HACEK Group
HACEK is an acronym of the first initial of each genus that belong in the group:
Haemophilus aphrophilus:
NAME ALERT: Now called Aggregatibacter aphrophilus
Not a true Haemophilus because does not need X nor V
Actinobacillus actinomycetemcomitans
Cardiobacterium hominis
Eikenella corrodens
Kingella species
Habitat
Commensals of oral cavity
Clinical Significance
Infective endocarditis
Peridontal disease
Dental caries
Infections following dental procedures

22. HACEK Group: General Characteristics
Gram-negative bacilli
Require an increased CO2 (5%-10%) environment
Slow/poor growers
Usual flora of the oralpharyngeal cavity
Opportunists in immunocompromised hosts

23. Capnocytophaga sp. Capnophilic Facultative anaerobe
Part of the normal oralpharygeal flora
Cause periodontal disease, sepsis

24. Pasteurella species General characteristics
Colonizes mucous membranes of the upper respiratory tract and gastrointestinal tracts of mammals and birds
Human infections occur from bites and scratches inflicted by animals, primarily felines
Results in a localized, pus- producing infection
Can cause life-threatening systemic disease
Most common isolated species is Pasteurella multocida

25. Pasteurella multocida

26. Pasteurella multocida
Culture characteristics
Growth on 5% blood or chocolate shows small, smooth, grayish,convex colonies
Non-hemolytic
“Musty” or earthy odor
No growth on MacConkey agar

27. Pasteurella multocida
Microscopic examination
Very small gram-negative rods
Bipolar staining with Giemsa or methylene blue
“Safety-pin” appearance

28. Pasteurella multocida: Identification
Oxidase positive
Indole positive
Nonmotile
Catalase positive
Glucose fermenter

29. Brucella species Causes infection in cattle (zoonosis)
Acquired through aerosol, percutaneous and oral routes of exposure
Brucellosis
Primarily seen with animal handlers and those who handle animal products
Also known as Malta or undulant fever
Type 3 biohazard – can be transmitted through unbroken skin
Category B Biological agent- easy to disseminate and cause moderate morbidity, but low mortality.

30. Brucella species: Identification
Colony Morphology
Small, smooth, convex, nonhemolytic
May require holding culture for 21 days
Gram Stain Morphology
Small gram-negative coccobaccilli
Nonmotile
Aerobic
Oxidase positive
Catalase positive
Urease positive

31. Francisella tularensis
Highly infectious Type 3 biohazard – can be transmitted through unbroken skin, bite from an insect, direct contact with infected animals or inhalation of aerosols
Category A Biological agent-it can be spread from person to person or disseminated, high mortality rates
Infection in rabbits, sheep, squirrels and ticks
Zoonotic infection in humans
Tularemia

32. Francisella tularensis: Identification
Colony Morphology
BAP = No growth
MAC = No growth
Choc = Small, smooth, gray gncb at 2-5 days
Requires special media (BCYE or MTM)
Oxidase: negative
Catalase: negative- weak positive
Ferments glucose
X and V negative
NOTE: Usually identified by DFA or direct agglutination tests due to risk of lab acquired infection

33. Legionella Species General characteristics Habitat
Aquatic sources
Cooling towers, condensers
Ubiquitous gram-negative rods
Acquired by humans primarily through inhalation of aerosols

34. Legionella Species: Clinical Infections
Legionnaire’s disease
Disease with pneumonia and extrapulmonary involvement
Malaise, rapid onset of dry cough and fever
Illness is fatal in 15-30% of cases not treated
Pontiac fever
Influenza-like
Fever, headache, malaise
Not fatal- short lived (2-5 days)

35. Legionella Species Specimen Handling & Processing
BAL, bronchial washings, lung biopsy and pleural fluid are appropriate specimens
Avoid aerosolization & transport ambient temperature
Buffered Charcoal Yeast Extract (BCYE) most widely used
Organism requires cysteine & iron salts for growth
Incubate at 35o C in 5-10% CO2 with increased humidity for 10 days
Slow growth (2-4 days)

36. Legionella pneumophilaB
(A) Nonselective buffered charcoal yeast extract (BCYE) plate inoculated with sputum specimen. Colonies appear blue-green or gray-white and glistening
(B) Selective BCYE ( has added antibiotics) inoculated with the same specimen but treated before inoculation. Legionella colonies are the smallest visible colonies. Colonies are grayish-white and glistening at 2-4 days.

37. Legionella Species: Identification
Oxidase positive
Catalase Positive
Motile by polar flagella
Short, thin GNR, may be faint staining

38. Legionella pneumophila
Misc. Identification methods
Rapid Methods for Identification
Urine Antigen test
Direct Fluorescent Antibody test (DFA)
DNA Detection
Serological tests (IFA)

39. Legionella spp.: Treatment
Susceptibility testing not routinely performed
Erythromycin alone or Rifampin used to treat

40. Bordetella spp. B. pertussis and B. parapertussis Cause pertussis
“Whooping cough”
Highly communicable disease of children
Strict human pathogen, spread by airborne droplets
Lives in ciliated epithelium of URT
Produces toxins and virulence factors
Required vaccination (DTaP)

41. bordetella spp: Specimen collection, transport and processing
Nasopharyngeal swab or aspirate is the specimen of choice.
Swabs should be calcium alginate or dacron polyester
Specimen should be plated at the bedside and a smear made OR placed in casamino acid for transport
Regan-Lowe is recommended for transport

42. Bordetella spp: Identification
Requires Bordet-Gengou agar
Cough plate
Appears slightly beta hemolytic smooth, shiny, resembling a mercury droplet
Regan-Lowe agar
Domed and shiny with a white mother of pearl opalescence
BAP & MAC: no growth
Organism is a fastidious obligate aerobe
Gram stain: small faint staining GN coccobacilli
Can increase counterstain of safranin to 2 minutes for improved visibility
Oxidase positive
Nonmotile

43. Bordetella spp.: Misc. Identification methods
Serologic Identification
Direct fluorescent antibody
Slide agglutination tests
Nucleic Acid Detection by PCR

44. Bartonella Spp. Facultative Intracellular gram negative cocco-bacillus
Transmitted by direct contact or blood-sucking arthropods
Infect RBCs and vascular endothelial cells in the host leading to circulatory system infections
Clinical Infections
Cat Scratch disease
Others
Carrion’s disease
Trench fever

45. References
Engelkirk, P. G., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams & Willkins.
Kiser, K. M., Payne, W. C., & Taff, T. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education, Inc.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.