Haemophilus and other Fastidious Gram
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Transcript Haemophilus and other Fastidious Gram
MLAB 2434: MICROBIOLOGY
KERI BROPHY-MARTINEZ
Haemophilus and Other
Fastidious Gram-Negative Rods
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
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
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
HAEMOPHILUS INFLUENZAE:
VIRULENCE FACTORS
Capsule
IgA Protease
Cleaves IgA on mucosal surfaces
Lipid A
Antiphagocytic
Effects ciliated respiratory epithelium
Pili
Attachment
HAEMOPHILUS INFLUENZAE:
CLINICAL INFECTIONS: TYPABLE STRAINS
Acute epiglottitis or laryngotracheal infection in small
children
Cellulitis/arthritis
Children under 6 years
Contagious, vaccine has decreased incidence
Pneumonia/septicemia
cheek and upper extremities
Meningitis
Can cause airway obstruction needing immediate tracheostomy
In children
Conjunctivitis “pink eye”
very contagious
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
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
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.
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
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.
HAEMOPHILUS SPECIES
Colony Morphology
No growth on BAP or MAC
On CA:
semi-opaque, gray-white, convex, mucoid.
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
HAEMOPHILUS SPECIES: IDENTIFICATION
This organism would be identified as H. influenzae
because it is using both X and V factors.
HAEMOPHILUS SPECIES: IDENTIFICATION
This organism would be identified as H.
parainfluenzae because it is using V factor only.
HAEMOPHILUS SPECIES:
IDENTIFICATION
Quad plates
Contain X and V
factors & sheep blood
agar
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
HAEMOPHILUS SPECIES:
IDENTIFICATION
Haemophilus sp.
X
V
H. influenzae
+
+
Horse/Rabbit
BAP
Hemolysis
-
H. haemolyticus
+
+
-
H. parainfluenzae
+
V
-
H. parahaemolyticus
+
V
-
H. ducreyi
+
-
+/-
H. aphrophilus
+/-
-
-
V=variable
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
TAKE 5!
HACEK GROUP
HACEK is an acronym of the first initial of each genus that belong in
the group:
Haemophilus aphrophilus:
o
Actinobacillus actinomycetemcomitans
Cardiobacterium hominis
Eikenella corrodens
Kingella species
Habitat
o
NAME ALERT: Now called Aggregatibacter aphrophilus
Not a true Haemophilus because does not need X nor V
Commensals of oral cavity
Clinical Significance
Infective endocarditis
Peridontal disease
Dental caries
Infections following dental procedures
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
CAPNOCYTOPHAGA SP.
Capnophilic
Facultative anaerobe
Part of the normal oralpharygeal flora
Cause periodontal disease, sepsis
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
PASTEURELLA MULTOCIDA
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
PASTEURELLA MULTOCIDA
Microscopic
examination
Very small gramnegative rods
Bipolar staining with
Giemsa or methylene
blue
“Safety-pin”
appearance
PASTEURELLA MULTOCIDA:
IDENTIFICATION
Oxidase positive
Indole positive
Nonmotile
Catalase positive
Glucose fermenter
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.
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
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
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
LEGIONELLA SPECIES
General characteristics
Habitat
Aquatic sources
Cooling towers, condensers
Ubiquitous gram-negative rods
Acquired by humans primarily through inhalation of
aerosols
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)
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)
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.
LEGIONELLA SPECIES: IDENTIFICATION
Oxidase positive
Catalase Positive
Motile by polar flagella
Short, thin GNR, may be faint staining
LEGIONELLA PNEUMOPHILA
Misc. Identification methods
Rapid Methods for Identification
Urine Antigen test
Direct Fluorescent Antibody test (DFA)
DNA Detection
Serological tests (IFA)
LEGIONELLA SPP.:
TREATMENT
Susceptibility testing not routinely performed
Erythromycin alone or Rifampin used to treat
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)
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
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
BORDETELLA SPP.:
MISC. IDENTIFICATION METHODS
Serologic Identification
Direct fluorescent antibody
Slide agglutination tests
Nucleic Acid Detection by PCR
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
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.