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