Download 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.