Fastidious gram-negative rods
General Information
Fastidious Gram-negative Rods
Haemophilus, Bordetella, Legionella
CLS 511 Medical Microbiology
Karen Honeycutt, M.Ed, MT(ASCP)SM
Fastidious gram-negative rods
Haemophilus influenzae
• Haemophilus means blood loving,
requires nutrients in blood for growth
• Normal flora of respiratory tract
• Encapsulated strains
– 6 antigen types (a-f) based on capsular
polysaccharides
– Type b causes most human disease
– Systemic and life threatening diseases
Haemophilus influenzae
Clinical Significance
• Respiratory tract infections
– Usually determined as pathogenic by
predominance or isolation of moderate to
p
many organisms from sputum culture
assocatiated with PMN’s
CLS 511 Medical Microbiology
Unit 13 Fastidious Gram-negative rods
• Gram stain =
gram-negative coccobacilli
(very small gram-negative rods)
• Fastidious = require
special
nutrient/atmosphere/temperature conditions
during specimen transport and for growth
• Haemophilus sp., Bordetella sp. and
Legionella sp.
Haemophilus influenzae
Clinical Significance
• Respiratory tract infections (often
secondary to viral infections)
• Meningitis
– Dramatic decrease (Hib vaccine)
• Otitis media
• Conjunctivitis (pinkeye) - contagious
(H. influenzae biogroup aegypticus)
Haemophilus influenzae
Virulence factors
• Capsule – most significant role
– Most invasive infections caused by
encapsulated
p
strains
– antiphagocytic
1
Haemophilus influenzae
Prevention – Hib Vaccine
• Prevents most systemic disease
• See for current vaccine guidelines
http://www cdc gov/vaccines/vpd
http://www.cdc.gov/vaccines/vpdvac/hib/default.htm#recs
Haemophilus influenzae
Laboratory Diagnosis
Haemophilus influenzae
Current treatment
• Life threatening disease (meningitis)
– Cefotaxime or ceftriaxone
– Alternatives: trimethoprimsulfamethoxazole imipenem,
sulfamethoxazole,
imipenem ciprofloxacin
• Non-life threatening disease
– Amoxicillin-clavulanate, oral second or third
generation cephalosporins or trimethoprimsulfamethoxazole
Haemophilus influenzae
Susceptibility Testing
• Culture appropriate specimen
– Organism will grow on chocolate agar,
which is usually part of routine cultures where
Haemophilus sp.
sp is often recovered (respiratory,
(respiratory
csf, eye)
• Direct specimen Gram stain =
gram-negative coccobacilli (sometimes
reported as gram-negative rods)
• Growth within 24-48 hours
Fastidious gram-negative rods
Bordetella pertussis
• Etiologic agent of pertussis
• Only human-to-human transfer
• Children <1 year of age at risk for most
severe disease (nonvaccinated at
greatest risk)
• Infection initiated when infectious
aerosols are inhaled
• Bacteria attach and proliferate on
ciliated epithelial cells
CLS 511 Medical Microbiology
Unit 13 Fastidious Gram-negative rods
• Non-life threatening disease
– Provide beta-lactmase test result
• If positive = organism ampicillin/amoxacillin (R)
• Life-threatening disease
– Provide full susceptibility test results
Bordetella pertussis
Clinical Disease
• 7-10 day incubation
• 1-2 wks Catarrhal stage: common
cold, very infectious
• 2-4 wks Paroxysmal state: repetitive
cough, inspiratory whoops, vomiting
• 3-4 wks Convalescence/secondary
complications
2
Bordetella pertussis
Laboratory Diagnosis
• Direct specimen detection (results
within 2-24 hours)
– Antigen testing: Direct fluorescent antibody
testing - organism detected directly in specimen
• Organism does not need to remain
viable for accurate
test results
Positive fluorescing
bacilli in
nasopharyngeal
specimen
Bordetella pertussis
Laboratory Diagnosis
• Culture – specifically order B. pertussis
culture
– Best if specimen plated at bedside to
appropriate agar (Regan
(Regan-Lowe:
Lowe: looks black
due to charcoal)
– Submit inoculated plate to lab immediately
– Organism must remain viable
– Culture held for 7 days with growth visible at 34 days
Bordetella pertussis
Susceptibility Testing/Vaccine
• No standardized methods available due to
fastidious nature of organism
• Drug of choice = macrolides (erythromycin,
clarithromycin,
l ith
i azithromycin)
ith
i ) and
d SXT
• Prevention: DTaP vaccine (pertussis, diphtheria,
and tetanus)
See CDC:
http://www.cdc.gov/vaccines/vpdvac/pertussis/default.htm
CLS 511 Medical Microbiology
Unit 13 Fastidious Gram-negative rods
Bordetella pertussis
Laboratory Diagnosis
• Direct specimen detection (results within 24
hours)
– Nucleic acid detection (molecular methods)
– Appears to be the most sensitive and specific
test for B. pertussis
– No universal FDA approved test available
– Organism does not need to remain viable for
accurate test results
Bordetella pertussis
Specimen of Choice for All Tests
• Nasopharyngeal aspirate or swab
– Ciliated epithelial cells need to be collected
– Press the swab through the nares until
resistance is met due to contact with the
nasopharynx.
– Rotate the swab gently and allow the swab to
maintain contact with the nasopharynx for 2030 seconds or until coughing is induced.
• A direct specimen technique is usually used
in combination with culture
Fastidious gram-negative rods
Legionella sp.
• Etiologic agent of
– Pontiac Fever: self-limited, influenzaelike illness
– Pneumonia: severe (death in 15%
immunocompetent and up to 75% in
immunocompromised)
• Acquire: inhalation of organism from
water source
• Person-to-person spread not
documented
3
Legionella sp.
Legionnaires Disease
• At risk:
– >40 years-of-age or older
– Compromised pulmonary function
– Decreased cellular immunity (transplant
patients)
– Nosocomial pathogen (hospital water
system)
Legionella sp.
Legionnaires Disease - Prevention
• Identification of environmental source
• Hyperchlorinate water
• Maintenance of elevated water
temperatures
• Immunosuppressed patients
– Run water to flush out pipes prior to using
(showers)
Legionella sp.
Laboratory Diagnosis - Culture
• Specific Legionella culture must be
ordered
• BAL fluid inoculated to specific
medium: buffered charcoal yeast
extract agar
Legionella sp.
Legionnaires Disease
• After 2-10 day incubation
– Fever, chills w/pneumonia (multilobar)
– Nonproductive, dry cough (sputum not
produced)
– Multiorgan disease (liver, kidney)
Legionella sp.
Laboratory Diagnosis
• Specimen of choice: bronchial
alveolar lavage (BAL) fluid
– Sputum
p
is often not p
produced
• Direct specimen detection (none are very
sensitive, should be paired with culture)
– Direct fluorescent antibody
– Nucleic acid detection (molecular testing)
– Urine antigen detection: antigens of only L.
pneumophila serogroup 1 shed in urine for up to
1 year
Legionella sp.
Susceptibility Testing
• No standardized methods available
due to fastidious nature of organism
• Drugs of choice
– macrolides
lid (clarithromycin,
( l ith
i azithromycin),
ith
i )
– fluoroquinolones (ciprofloxacin,
levofloxacin)
– Contains L-cysteine and iron salts required for
organism growth
• Organism growth detected at 3-5 days,
cultured held for 10 days
CLS 511 Medical Microbiology
Unit 13 Fastidious Gram-negative rods
4
Fastidious gram-negative rods
Overview
• Most common gram-negative coccobacilli
isolated = Haemophilus influenzae
– Respiratory infections, conjunctivitis, otitis media
– Meningitis (Hib vaccine decreasing incidence)
• Bordetella pertussis: pertussis
• Legionella sp.
– Pontiac fever: self-limited, flu-like
– Legionnaires pneumonia: multi-organ
involvement, nosocomial pathogen for
immunosuppressed
CLS 511 Medical Microbiology
Unit 13 Fastidious Gram-negative rods
5