MLAB 2434: MICROBIOLOGYKERI 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 virusesIn some cases of flu, H. influenzae is
secondary infection
HAEMOPHILUS INFLUENZAE: VIRULENCE FACTORS Capsule
Antiphagocytic
IgA Protease Cleaves IgA on mucosal surfaces
Lipid A Effects ciliated respiratory epithelium
Pili Attachment
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
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 MorphologyNo growth on BAP or MACOn 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 Horse/ Rabbit
BAP Hemolysis
H. influenzae + + -
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:
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
o Habitato 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.
CapnophilicFacultative anaerobePart of the normal oralpharygeal
floraCause 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 gram-
negative rods Bipolar staining with
Giemsa or methylene blue
“Safety-pin” appearance
PASTEURELLA MULTOCIDA: IDENTIFICATION
Oxidase positiveIndole positiveNonmotileCatalase positiveGlucose 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-likeFever, headache, malaiseNot 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) 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.
A B
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 childrenStrict human pathogen, spread by
airborne dropletsLives in ciliated epithelium of URTProduces 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 IdentificationDirect fluorescent antibodySlide 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.
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