Download Mycoplasma and Fastidious Gram Negative Bacteria

Mycoplasmas and Fastidious
Gram-negative Bacteria
Haemophilus * Bordetella
Legionella *
Moraxella * Mycoplasma
Ureaplasma* Brucella * Francisella
Ken B. Waites, M.D. F(AAM)
Objectives
• To review and discuss
• microbiological characteristics
• epidemiology
• virulence factors
• associated diseases
• laboratory detection
Haemophilus
Bordetella
Mycoplasma
Ureaplasma
Legionella
Brucella
Franciscella
Haemophilus
• “Blood-loving”
• Gram-negative
coccobacillus
• Facultative anaerobe
• Non-hemolytic
• Invasive strains
• Non-encapsulated H.
influenzae & other spp.
common URT flora
Haemophilus influenzae
Haemophilus: Pathogenesis
• Respiratory aerosol dissemination
• Endogenous infection
• Antiphagocytic capsule (type b)
– major virulence factor
– 6 capsular serotypes (a-f) H. influenzae
• Endotoxin
– damages respiratory epithelium leading to
bacteremic spread
• No exotoxins
• IgA protease
• Beta lactamase in 30% of strains
Detection:
H. influenzae
• Growth on chocolate
but not blood or
MacConkey agar
• 5-10% CO2 required
• X (hemin) & V( NAD)
– varies among
species
• Satellitism with S.
aureus
Diseases: H. influenzae
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Otitis media
Sinusitis
Bacteremia
Epiglottitis
• Laryngotracheobronchitis
• Meningitis
• Exacerbation of chronic
bronchitis in COPD
• Pneumonia
• Cellulitis
• Otitis media
• Conjunctivitis
Prevention: H. influenzae
• Invasive disease rare in child > 5 yrs
• Hib conjugate vaccine
– polysaccharide capsule type b
– protein carrier
– given in infancy (3-4 doses) since 1987
– reduced invasive disease > 90%
– No impact on non-typeable H. influenzae
infections which are still common
Other Haemophilus sp
• H. ducreyi
–chancroid - genital ulcer
• H. aegyptius
– conjunctivitis
Bordetella pertussis
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Encapsulated
Gram-negative coccobacillus
Slow-growing
Fastidious
Strict aerobe
Non-fermentative
Oxidizes amino acids
B. pertussis: Whooping Cough
• 5-21 day incubation - very contagious
– Catarrhal stage - cough & sneeze (1-2 wk)
– Paroxysmal stage (1-6 wks)
– Convalescent stage (months)
• Lymphocytosis
• Recovery confers immunity
http://www.whoopingcough.net/sound%20of%20whoopi
ng%20cough%20with%20some%20whoop.htm
Pertussis Epidemiology
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Outbreaks described in 16th Century
Highly contagious respiratory droplets
> 285,000 deaths worldwide in 2001
> 10,000 US cases in 2007
No environmental or animal reservoir
Adolescents and adults > 50% of cases
Older persons often spread to children
Pertussis in Alabama
Reported cases
82 cases in 2005
49 cases in 2004
19 cases in 2003
37 cases in 2002
37 cases in 2001
Reasons Pertussis is Increasing
 Under-vaccination in infants
 Under- or misdiagnosis
 Waning Immunity from childhood
vaccination
 Increased recognition among adolescents
and adults, which contributes to the
disease reservoir
Pertussis Pathogenesis
• Attachment to ciliated
respiratory epithelium by
various adhesins
– Filamentous
hemagglutinin (FHA)
– Pertussis toxin (PTx)
• Evasion of host defense
– impaired chemotaxis
• Local tissue damage &
systemic disease due to
exotoxins
Pertussis Toxins
• Pertussis toxin
• Facilitates adherence
• Adenyl cyclase/hemolysin
• Increases cAMP in cells
• Inhibits phagocytic killing & monocyte
migration
• Lethal toxin
• Inflammation & local necrosis
• Tracheal cytotoxin
• Kills respiratory epithelial cells
• Stimulates release of IL-1 (fever)
• Endotoxin (LPS)
• Activates alternate complement pathway
• Stimulates cytokine release
B. pertussis: Detection
• NP swab collected at bedside
• Bordet-Gengou or Regan-Lowe enriched
horse blood-charcoal medium
– Incubate 3-7 days in moist environment
– Identify by immunofluorescence or slide
agglutination
• Measurement of serum antibody titers
• PCR – in addition to culture
• DFA on NP secretions – low sensitivity
Pertussis Prevention
• Acellular vaccine during infancy “DaPT” (1996)
– FHA, PTx, pertactin, type 2 fimbriae
– Antibody prevents attachment
• 5 doses: 2 mo; 4 mo; 6 mo; 15-18 mo; 4-6 yr
• Adolescent/ adult formulations “Tdap” (2005)
– Ages 11 through 64 yrs – single dose
– Td, protects against tetanus & diphtheria, but
not pertussis - recommended every 10 yrs
Legionella
pneumophila
• Fastidious
• Catalase-negative
• Facultatively intracellular
Gram-negative bacillus
• Nonfermentative
• Stains poorly with safranin
• > 30 species
• Multiple serogroups
• 1st described in 1976
Pathogenesis of Legionellosis
• Organism inhaled from environment
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C3 deposits on bacteria
bacteria bind macrophage C3 receptor
bacteria uptaken by macrophages
prevent phagolysosome fusion
intracellular multiplication
bacteria produce enzymes
cell dies & bacteria are released
• No person to person transmission
• Acute purulent pneumonia
& abscesses
Legionella Culture
• Buffered charcoal yeast
extract agar + cysteine
• Good for all species
• 3-7 d or more required
• ETA, TTA > sputum
• ID species by
agglutination or
immunofluorescence
Legionella on BCYE agar
Legionella Detection
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Silver staining of histopathology specimens
Direct fluorescent antibody (poor sensitivity)
Urine polysacharide antigen
Serology (IFA) - paired sera required
PCR
Legionnaires Disease
• 5-10% of CAPs: 10-20,000 cases/yr in US
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Point source outbreaks
Cooling towers
Hospital water supplies
Hot tubs
• Purulent alveolar exudate
• GI & renal manifestations
• Risk factors
– Older men with COPD
– Immunosuppressed (transplant recipients)
• Summer months (AC)
Prevention of Legionellosis
• No vaccine
• CMI more important than AB
• Identify sources and eliminate them
Moraxella catarrhalis
• Gram-negative coccus
• May be carried in URT of
healthy children
• Causes bronchitis, CAP,
sinusitis, otitis
• Occasional cause of
non-respiratory
infections
• Most strains produce
beta lactamase
MYCOPLASMA
PNEUMONIAE
Eukaryotic Cell
Herpes
virus
HIV
Mycoplasma
Chlamydia
elementary body
Treponema
Streptococci
1 m
Mycoplasma
• Smallest free-living
organisms
• > 150 species
• Genome of 816,394 bp; 687
genes
• Lack cell wall - pleomorphic
• Specialized cell membrane
• Evolved from Grampositives
• Extracellular on mucosal
surfaces
Pathogenesis
• Cytadherence
– P1 & other proteins
– Immunogenic
• Cytotoxicity (H2O2) hemolysin  ciliostasis
• Induction of inflammation
• Cytokine cascade
• Antigenic variation
• Autoimmunity
• Superoxide anions inhibit host catalase
increasing oxidative damage
• MP CARDS toxin - ADP ribosyl transferase
induces vacuolation & ciliostasis of epithelium
Mycoplasma Detection
• Enriched agar
medium - SP4 +
serum (sterols)
• Slow growth - 5-20
days
• Glucose hydrolysis
• ID colonies by PCR
• Serology – IgM +
(children) or
seroconversion
• PCR
Microscopic spherical colonies
< 100 m
M. pneumoniae Disease
• Tracheobronchitis
• Atypical interstitial “Walking” pneumonia
• All ages affected but more common in younger
persons
• 20-50% of all CAP
• Clinically similar to other pneumonias
• Extrapulmonary disease
• Spread through households
• Outbreaks in closed populations
• Role in asthma?
• Reinfection common – no protective immunity
Other Mycoplasmas
• Mycoplasma hominis
• Mycoplasma genitalium
• Ureaplasma species
Ureaplasma
M. hominis
Diseases due to Genital
Mycoplasmas
• Commensals in lower
urogenital tract in normal
sexually active adults
Diseases due to Genital Mycoplasmas
Condition
Ureaplasma
M. hominis
M. genitalium
Male urethritis
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Prostatitis
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Urinary calculi
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Pyelonephritis
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Bacterial vaginosis
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Cervicitis
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Pelvic inflam. disese
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Infertility
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Chorioamnionitis
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Spont. Abortion
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Low birth weight
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Postpartum fever
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Neonatal
pneumonia/meningitis
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Extragenital disease
adults (arthritis)
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Detection of Genital Mycoplasmas
and Ureaplasmas
• Culture – good for rapid growing M.
hominis and Ureaplasma
• Serology – not useful
• PCR – needed for M. genitalium
Brucella
• Small gram-neg. coccobacilli
– B. abortus (cattle)
– B. melitensis (goats, sheep)
• most common cause of human infection
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– B. suis (pigs, deer)
– B. canis (dogs, foxes, coyotes)
Intracellular pathogen - grows in macrophages,
inhibits lysosome fusion
Nonmotile
Encapsulated
Fastidious; slow-growing
Strict aerobe
Brucellosis
 Worldwide distribution
 Spread:
 direct contact with organism
 consumption of contaminated food product
 inhalation
 Clinical
 Acute vs. chronic infection
 Intermittent “undulant” fevers
 Multisystem involvement
 Manifestation & severity varies with species
Brucellosis
• Lab Detection
– Blood & bone marrow culture
– Prolonged incubation
• Grows on enriched blood agar after > 3 days
• Positive oxidase & urease
• ID by antibody agglutination
– Serology
• Control of human infection:
– animal vaccination for B. abortus & B. melitensis
– elimination of infected herds
– avoiding unpasteurized dairy products
Bioterrorism agent
Francisella tularensis
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Small gram-neg. coccobacilli
Fastidious
Strict aerobe
Grows in > 3 days
Intracellular pathogen – grows in
macrophages, inhibits lysosome fusion
• Encapsulated
• Common in many animals, birds,
arthropods
• AR, OK, MO are most common US
locations ~ 100 cases/yr
Tularemia
• Clinical Manifestations
– Ulceroglandular
– Oculoglandular
– Typhoidal
– Pneumonic
– Oropharyngeal
– Gastrointestinal
• Lab Detection
– Growth on chocolate or BCYE media
– Serology – four-fold rise in titer
• Vaccine available but rarely used
• Bioterrorism agent