Legionella and Bordetella
6 other recognized species
... (dont need to know too much)
Legionnaires' disease
Febrile disease with pneumonia Sporadic cases Epidemic outbreaks Nosocomial clusters 70% cases: L. pneumophila Differential diagnosis from other pneumonia causing agents
Pontiac fever
Febrile disease without pulmonary involvement Non-pneumonic form Incubation period: 2 days Previously healthy; flu-like symptoms: fever, headache, myalgia (2 to 5 days) that subsides without medical intervention L. pneumophila
B. pertussis and B. parapertussis Virulence factors
Filamentous hemagglutinin (FHA) and pertactin (outer membrane protein) facilitate attachment to ciliated epithelial cells **Pertussis toxin (PT): protein exotoxin; modification of host proteins Adenylate cyclase toxin: inhibits host epithelial and immune effector cells Tracheal cytotoxin: ciliostasis, inhibits DNA synthesis, promotes cell death
Protocol
Gram stain suspicious colonies growing on BCYE Subculture to BCYE with L-cysteine and SBA or BCYE without L-cysteine
BCYE
Grayish white or blue-green, convex, glistening Dissecting microscope: central portion of young colony exhibits **"ground glass appearance"** periphery has pink or light blue or bottle green bands with a furrowed appearance Examine plates daily UV light for suspicious colonies (366 nm)-autofluorescence
Legionella diagnosis
Isolation using special media Urine antigen detection Direct fluorescent antibody (DFA) Serology - immune response so takes time
Extra-pulmonary infections with or without pneumonia
Kidneys, liver, heart, CNS, lymph nodes, spleen, bone marrow, cutaneous abscesses
Nosocomia infections
L. pneumophila, L. micdadei, L. longbeachae, L. dumoffii Atypical pneumnia L pnemophilia most significant
Rapid methods
Urine antigen test Radioimmunoassays, microplate enzyme immunoassays, rapid immunochromatographic assays Detects soluble ag of L. pneumophila serogroup 1 Ag detected by day 3 of infection and can persist for 1 year
Legionella background
1976: American Legion Convention in Philadelphia 52 species; more than 70 serogroups Primary human pathogen: L. pneumophila (16 serogroups)
Incubation period
2 to 10 days-nonproductive cough, fever, headache, myalgia, pulmonary infiltrates L. pneumophila serogroup 1 (4, 6); L. longbeachae, L. micdadei
Isolation Methods
Acid treatment of specimen before culture (sputum) - gets rid of other things 35°C to 37°C for at least 7 days Fastidious, aerobic DO NOT grow on SBA
B. epidemiology
Acquired through respiratory tract via respiratory droplets or direct contact with infectious secretions Orgs uniquely adapted to adhere to and replicate on ciliated respiratory epithelial cells One of most highly communicable diseases of childhood Infants less than 6 months old are at highest risk Adolescents and adults serve as reservoirs
B. hinzii
Avian commensal
Bordetella background
B. pertussis and B. parapertussis whooping cough or pertussis
BCYE with L-cysteine
BEST for isolation!! Nonselective Semi-selective: polymyxin B, anisomycin, vancomycin Charcoal: neutrolize metabolic toxins from orgs
Virulence Factors
Enter, survive, and multiply within host cell (bronchoalveolar macrophages) Production of proteolytic enzymes Can survive in extracellular environments
Antimicrobial susceptibility
Erythromycin Azithromycin
Regan lowe
Charcoal, horse blood, cephalexin
Conventional
Demonstrate requirements for L-cysteine, Gram stain, DFA
Epidemiology
Found worldwide in aquatic sources: lakes, rivers, hot springs, mud L. longbeachae: exposure to gardening materials (compost and potting soil); New Zealand and Australia
CAREFUL!!
Haemophilus spp. can sometimes breakthrough on this media
B. holmesii
Immunocompromised bacteremia
B. trematum
Immunocompromised bacteremia + B. petrii
Isolation method
Incubate at 35°C in ambient air for minimum of 7 days 3-5 days Ensure adequate moisture
Infections
Legionnaires disease pontiac fever Asymptomatic infection
Legionella Clinical significance
Mild upper respiratory tract infections to pneumonia 2%-15% of community acquired pneumonia
Colonization of these sources
Multiplies over temp range of 20°C to 43°C and survive for varying periods at 40°C to 60°C Adheres to pipes, rubber, plastics, sediment; persists in piped water systems even when flushed Survives and multiplies within free-living protozoa and in the presence of commensal bacteria and algae
Antimicrobial Susceptibility
Not standardized or routinely performed Macrolide (azithromycin or fluoroquinolone)
Microscopic Examination
Only by DFA Use only with culture Prepare directly from swab specimens Polyclonal fluorescent labeled conjugates for B. pertussis and B. parapertussis
Serologic testing
Outbreaks and document seroconversion Lack of association between ab titers and immunity
Differentiating charac
Oxidase, urease Pertusisis oxidase posiive Urease neg Parapertusus Opposite!
Nucleic acid detection
PCR-primary rapid diagnostic strategy Use at least 2 DNA targets In house verification and validation studies
B. clincial maniefstations
Pertussis or whooping cough 1-3 week incubation period (7-10 days) Catarrhal phase: sneezing, mild cough, runny nose, conjunctivitis INFANTS can develop apnea or respiratory distress Paroxysmal phase: severe repetitive coughing followed by "whoop"; can be followed by vomiting Convalescent phase: within 4 weeks of onset
Legionella characteristics
Ubiquitous Gram-negative bacilli Acquired through inhalation Wide variety of conditions Asymptomatic to life threatening disease
Transport media
Plate directly to culture media or transfer to appropriate transport system at bedside Transport at room temp and transfer to culture media immediately
Microscopic Examination
Pleomorphic weakly staining Gram-negative bacilli Extend safranin counterstaining to 10 mins L. micdadei-wealy acid fast DFA
B. avium
Resp tract pathogen of wild and domestic birds
B. bronchiseptica
Resp tract pathogen of wild and domestic birds
DFA
Samples from lower respiratory tract FITC conjugate binds to ags on cell surface Sensitivity and specificity issues (cross reactivity)
Common phenotypic characteristics
Slow growth (3-5 days) Ground glass morphology Lightly staining GNR Asaccharolytic Catalase or oxidase: weakly positive
Specimen collection and handling
Sputum, BAL, bronchial washings, tissue (saline or buffer should not be used-sodium has inhibitory effects) Refrigerate if testing delayed more than 2 hours Blood Urine (L. pneumophila serogroup 1) Environmental sources
ID methods
Tiny Gram-negative coccobacilli that can become elongated if recovered from media containing cephalexin Increase safranin counterstaining to 2 minutes Fluorescent staining (colony)
Nasopharyngeal aspirates or swabs
calcium alginate or Dacron with flexible wire shaft) is specimen of choice for culture, DFA, and PCR
RESIST WATER TREATMENT
can tolerate high chlorine concentrations Human-made water supplies and distribution systems, hot water systems, cooling towers, evaporative condensers Cold water systems, ornamental fountains, whirlpool spas, humidifiers, respiratory therapy equipment
Amie
charcoal
**Bordet-Gengou
potato infusion agar with glycerol and horse or sheep blood B. pertussis and B. parapertussis are hemolytic
Charcoal agar
supplemented with 10% horse blood and cephalexin (similar to Regan-Lowe transport media) Shelf life of 8 weeks Smooth, glistening, silver (resembles mercury droplets) Colonies turn whitish-grey as they age
Predisposition
suppressed immune system, chronic lung disease, alcoholism, heavy smoking