LegionellaceaeLegionellaceaeLegionellaceaeLegionellaceae

FastidiousFastidious

Gram-Negative BacilliGram-Negative Bacilli

Legionellaceae: Genera• Legionella pneumophilia

– Pontiac Fever – febile illness• 1968, people working in Public Health

Department, Pontiac, Michigan• Self-limited disease, no pulmonary involvement

– Legionnaires Disease – acute pneumonia• 1976, outbreak American Legion convention,

Philadelphia• 221 cases, 34 deaths (15%)

• Coxiella burnetii – Q fever– Intracelluar growth, originally classified

with Rickettsia– Now shown related to Legionella

Legionella pneumophila: Gram Stain and Characteristics• G(-) small pleomorphic rods• Stain poorly by Gram stain• Ubiquitous aquatic saprophyte - lakes,

streams; air conditioning, water systems, shower heads, hot tubs, humidifiers

• Facultative intracellular parasite– Free-living amoebae in nature– Human alveolar phagocyte, epithelial cell

• Sporadic, epidemic, nosocomial infection

L. pneumophilia: Lab Culture• Fastidious - no growth ordinary lab media,

requires cysteine for growth• Buffered charcoal yeast extract (BCYE) -

primary isolation, selective by antibiotics• Growth enhanced in 3-5% CO2

• Colonies pinpoint, ground-glass appearance observe under dissecting microscope

L. pneumophilia: Lab ID• Previously not recognize – stains poorly, no

growth on common lab media• BCYE culture - “gold standard” detection MO from

clinical specimen• Rapid Urine Antigen Test – enzyme immunoassay

(EIA), lower specificity, sensitivity; test of choice • Direct fluorescent antibody (IFA) test - specific

antibody tagged with dye to ID MO under UV microscope; lower sensitivity

• Serology Test – specific Legionella antibody detected by FA, EIA; require 4x rise antibody titer in paired patient serum

• DNA amplification test – specific, sensitive; enzyme inhibitors in RT secretions give false (-); currently not available

L. pneumophilia: Virulence Factors

• Endotoxin – LPS of Gram(-) cell wall• Infect & replicate in macrophage

– Enter cell by endocytosis– Inhibit phagolysosome fusion, not killed– Multiply in cell vacuole– Produce proteolytic enzymes, kill & lyse cell

• Proteolytic enzymes - hemolysin, protease, nuclease, phosphatase, lipase

• Collagenase – spreading factor

L. pneumophilia: Pontiac Fever

• Airborne transmission - inhale aerosols from environment; no person-to-person transmission; attack rate low (<5%)

• Acute, self-limited febrile illness• Incubation 24-36 hours• Fever, chills, malaise, myalgia,

headache; no pulmonary disease• Persist 2-5 days, resolve spontaneously

without treatment• Minimal morbidity, low mortality (<1%)

L. pneumophilia: Legionnaires Disease

• Airborne transmission - inhale aerosols from environment; no person-to-person transmission; attack rate high (>90%)

• Incubation 2-10 days• Abrupt fever, chills, malaise, myalgia,

headache, dry cough, vomiting, diarrhea, abdominal & chest pain

• Primary manifestation – pneumonia, multilobar consolidation

• Hospitalization usually required in 3-5 days• Without antibiotics, fatality rate 15-20%,

higher in transplant patients• Occurs more in males >60 years age,

immune compromised individual• At risk are elderly, patients with

compromised pulmonary function (smokers), decreased CMI (transplant patients)

L. pneumophilia: Treatment and Prevention• Use antibiotics able to penetrate

macrophages – macrolides (azithromycin), fluoroquinolones (ciprofloxacin)

• Identify environmental source, then reduce number of MO

• Water treatment – hyperchlorination, heating, copper-silver ionization

• Elimination of all MO in water not possible, reduce to low level not able to cause infection

Coxiella burnetti: Gram Stain and

Characteristics• Gram(-) small rod• Stain weakly• Infection of mammal, bird, tick• Facultative intracellular parasite • Extremely stable, survive in

environment long time

C. burnetti: Virulence Factors

• Resistant to lysosomal enzymes – survive and replicate in phagolysosome of cell

• Intracellular growth – protected from host immune defense

C. burnetii: Q Fever• 1935 Australia - “query” unexplained

fever in cattle, slaughterhouse workers• Disease worldwide, uncommon USA• Human infection - contact infected

cattle, sheep, goats, dogs, cats• Via inhalation, ingest contaminated

milk• Most infections AS, <5% acute disease• Incubation 14-26 days, sudden onset

fever, chills, headache, no rash• Atypical pneumonia 5-14 days, low

mortality• Potential Bioterrorist weapon

– Extraordinarily infectious (~1 MO)– Prolonged disease– Difficult to diagnose

C. burnetti: Treatment and Prevention

• Doxycycline for acute infection• Vaccination of animal herd

Case Study 10 - Legionella

• A 73-year-old man was admitted to the hospital because of breathing difficulties, chest pain, chills, and fever of several days’ duration. He had been well until 1 week before admission, when he noted the onset of a persistent headache and a productive cough. The patient smoked two packs of cigarettes a day for more than 50 years and drank a six-pack of beer daily; he also had a history of bronchitis. Physical examination results revealed an elderly man in severe respiratory distress with a temperature of 39ºC,

Case Study 10 - Legionella

• pulse of 120 beats/minute, respiratory rate of 36 breaths/minute, and blood pressure of 145/95 mm Hg. Chest radiograph revealed an infiltrate in the middle and lower lobes of the right lung. The white blood cell count was 14,000 cells/mm3 (80% polymorphonuclear neutrophils). Gram stain of the sputum showed neutrophils but no bacteria, and routine bacterial cultures of sputum and blood were negative for organisms. Infection with Legionella pneumophilia was suspected.

Case Study - Questions• 1. What laboratory tests can be used to

confirm this diagnosis? Why were the routine culture and Gram-stained specimen negative for Legionella organisms?

• 2. How are Legionella species able to survive phagocytosis by the alveolar macrophages?

• 3. What environmental factors are implicated in the spread of Legionella infections? How can this risk be eliminated or minimized?

Class Assignment• Textbook Reading: Chapter 18

Haemophilus and Other Fastidious Gram-Negative Rods– B. Legionella

• Key Terms• Learning Assessment Questions

Final Lecture ExamTue., March 20, 2012

8:30 – 10:30 am• Mycobacterium thru Ureaplasma • Lecture, Reading, Key Terms, Learning

Assessment Questions• Case Study 7, 8, 9, 10 (Mycobacterium,

Clostridium, Chlamydia, Legionella)• Exam Format:

– Multiple Choice– Terms– True/False Statements– Short Essay

• Review, Review, Review!• Repetition is the key to retention.