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MLAB 2434: MICROBIOLOGY KERI BROPHY-MARTINEZ Chapter 1

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MLAB 2434: MICROBIOLOGY KERI BROPHY-MARTINEZ Chapter 18 Haemophilus and Other Fastidious Gram-Negative Rods ... – PowerPoint PPT presentation

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Title: MLAB 2434: MICROBIOLOGY KERI BROPHY-MARTINEZ Chapter 1


1
MLAB 2434 MicrobiologyKeri Brophy-Martinez
  • Chapter 18Haemophilus 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

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 epiglottis 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
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
  • Clinical Significance
  • Infective endocarditis

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
  • Legionnaires 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 pneumophila
A
B
(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
  • Carrions 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.
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