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Mycoplasmas and FastidiousGram-negative Bacteria
Haemophilus * BordetellaLegionella *
Moraxella * MycoplasmaUreaplasma
Ken B. Waites, M.D. F(AAM)2008
Objectives• To review and discuss
• microbiological characteristics• epidemiology• virulence factors• associated diseases• laboratory detectionOf:
HaemophilusBordetellaMycoplasmaUreaplasmaLegionella
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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
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Detection:H. influenzae
• Growth on chocolatebut not blood orMacConkey agar
• 5-10% CO2 required• X (hemin) & V( NAD)
– varies amongspecies
• Satellitism with S.aureus
Diseases: H. influenzae• Otitis media• Sinusitis• Bacteremia• Epiglottitis
• Laryngotracheobronchitis• Meningitis• Exacerbation of chronic
bronchitis in COPD• Pneumonia• Cellulitis• Otitis media• Conjunctivitis
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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
Other Haemophilus sp
• H. ducreyi–chancroid - genital ulcer
• H. aegyptius– conjunctivitis
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Bordetella pertussis
• 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
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Pertussis Epidemiology
• Outbreaks described in 16th Century• Highly contagious respiratory droplets• > 285,000 deaths worldwide in 2001• > 25,000 US cases in 2005 - increasing• No environmental or animal reservoir• Adolescents and adults > 50% of cases• Older persons often spread to children
Pertussis in Alabama
Reported cases82 cases in 200549 cases in 200419 cases in 200337 cases in 200237 cases in 2001
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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 thedisease reservoir
Pertussis Pathogenesis• Attachment to ciliated
respiratory epithelium byvarious adhesins– Filamentous
hemagglutinin (FHA)– Pertussis toxin (PTx)
• Evasion of host defense– impaired chemotaxis
• Local tissue damage &systemic disease due toexotoxins
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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
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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
Legionellapneumophila
• Fastidious• Catalase-negative• Facultatively intracellular
Gram-negative bacillus• Nonfermentative• Stains poorly with safranin• > 30 species• Multiple serogroups• 1st described in 1976
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Pathogenesis of Legionellosis• Organism inhaled from environment
– 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 orimmunofluorescence
LegionellaonBCYEagar
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Legionella Detection• 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
– 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)
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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-respiratoryinfections
• Most strains producebeta lactamase
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MYCOPLASMAPNEUMONIAE
Eukaryotic Cell
Chlamydia elementary body Treponema
Streptococci
Herpesvirus
Mycoplasma
HIV
1 µm
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Mycoplasma• Smallest free-living organisms• > 150 species• Genome of 816,394 bp; 687 genes• Lack cell wall - pleomorphic• Specialized cell membrane• Evolved from Gram-positives• Extracellular on mucosal surfaces
M. pneumoniae:Pathogenesis
• Cytadherence– P1 & other proteins– Immunogenic
• Cytotoxicity (H2O2)hemolysin → ciliostasis
• Altered macromolecularsynthesis
• Induction of inflammation• Cytokine cascade• Antigenic variation• Autoimmunity• Intracellular growth?
MP
P1
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Mycoplasma Detection• Enriched agar
medium - SP4 +serum (sterols)
• Slow growth - 5-20days
• Glucose hydrolysis• ID colonies by PCR• Serology – IgM +
(children) orseroconversion
• PCR
Microscopic spherical colonies < 100 µm
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
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Other Mycoplasmas• Mycoplasma hominis• Mycoplasma genitalium• Ureaplasma species
UreaplasmaM. hominis
Diseases due to GenitalMycoplasmas
• Commensals in lowerurogenital tract in normalsexually active adults
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Diseases due to Genital Mycoplasmas
+++Extragenital diseaseadults (arthritis)
-++Neonatalpneumonia/meningitis
-++Postpartum fever
--+Low birth weight-++Spont. Abortion-++Chorioamnionitis--+Infertility++-Pelvic inflam. disese+--Cervicitis-++Bacterial vaginosis-++Pyelonephritis--+Urinary calculi+-+Prostatitis+-+Male urethritis
M. genitaliumM. hominisUreaplasmaCondition
Detection of Genital Mycoplasmasand Ureaplasmas
• Culture – good for rapid growing M.hominis and Ureaplasma
• Serology – not useful• PCR – needed for M. genitalium