MLAB 2434: M ICROBIOLOGY K ERI B ROPHY -M ARTINEZ Haemophilus and Other Fastidious Gram-Negative...

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.