1. LegionellaceaeLegionellaceae Fastidious Gram-Negative Bacilli

2. 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

3. 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

4. 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% CO 2 Colonies pinpoint, ground-glass appearance observe under dissecting microscope

5. 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

6. 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

7. 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%)

8. 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)

9. 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

10. 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

11. C. burnetti: Virulence Factors Resistant to lysosomal enzymes – survive and replicate in phagolysosome of cell Intracellular growth – protected from host immune defense

12. 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

13. C. burnetti: Treatment and Prevention Doxycycline for acute infection Vaccination of animal herd

14. 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,

15. 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/mm 3 (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.

16. 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?

17. Class Assignment Textbook Reading: Chapter 18 Haemophilus and Other Fastidious Gram-Negative Rods –B. Legionella Key Terms Learning Assessment Questions

18. Final Lecture Exam Tue., 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.