Legionella: Then and NoLegionella • The first known outbreak • Discovery of the causative agent...

Legionella: Then and Now

Development of Knowledge About Legionella

• The first known outbreak • Discovery of the causative agent • Development of a medium to grow Legionella • Knowledge of the reservoir and transmission

of the bacterium • Realization that outbreaks still occur • Preventive measures?

Historical Significance

Legionella: Molecular Biology (2008) http://www.q-net.net.au/~legion/Legionnaires Disease_Worlds_First-Outbreak.htm

Fraser, D.W., T.R. Tsai, W. Orenstein, W.E. Parkin, P.H.J. Beecham, R.G.Sharrar, J. Harris, G.E. Mallison, S.M.Martin, J.E. McDade, C.G. Shepard, P.S. Brachman, and The Field Investigation Team. 1977. Legionnaires’ Disease: Description of an Epidemic of Pneumonia. N. Eng. J. Med. 297:1189-1197.

McDade, J.C. Shepard, D. Fraser, et. Al. 1977. Legionnaires’ Disease: Isolation of a bacterium and demonstration of its role in other Respiratory diseases. N. Eng. J. Med. 297:1197-1203.

References on Legionella Media and Recovery

• Feeley, J.C., R.J. Gibson, G.W. Gorman, N.C. Langford, J.K. Rasheed, D.C. Mackel, and W.B. Baine. 1979. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10:437-441

• Barbaree, J.M., G.W. Gorman, W.T. Martin, B.S. Fields, and W.E. Morrill. 1987. Protocol for sampling environmental sites for Legionellae. Appl. Environ. Microbiol. 53:1454-1458.

• Fields, B.S., J.M. Barbaree, W.T. Martin, W.E. Morrill, and G.N. Sanden. 1992. Procedures for the Reovery of Legionella from the Environment. CDC publication.

• Wadowsky, R.M. and R.B. Yee. 1981. Glycine-containing Selective medium for isolation of Legionellaceae from

environmental specimens. Appl. Environ. Microbiol. 42:768-772. • Others

BCYE agar: Charcoal 2.0 g YE 10.0 g ACES buffer 10.0 g Ferric Pyrophosphate 0.25 g L-Cysteine 0.40 g Agar 17.0 g Potassium alpha- ketoglutarate 1.0 g Dist. Water 1L Yeast Extract GPAV: Glycine 0.3% Poylymyxin B 100U/ml Vancomycin 5ug/ml Anisomycin 80 ug/ml

1 = BCYE alpha; 2=PAV; 3=GPAV; 4=PAV

The Genus Legionella • >48 species,70 serogroups • Small, G- bacilli • Aquatic • require L-cysteine and soluble iron • Facultative intracellular pathogens • Motile • Energy source = amino acids • Aerobic, capnophilic • high [phospholipid] in outer membrane

Filtering and Air Sampling for Legionella

IFA and DFA Tests IFA: 4-fold rise in serum titer

DFA

Wilkenson, H.W., B.J. Fikes, and D.D. Cruce. 1979. Indirect immunofluorescent test for serodiagnosis of Legionnaires’ Disease. Evidence for serogroup diversity of Legionnaires’ Disease bacterial antigens and multiple specificity of human antibodies. J. Clin. Microbiol. 9:379-383

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Pontiac Fever

• Influenza like illness • Incubation period: 1-2 days • Symptoms: fever, chills, muscle pain, lethargy,

and headache • Symptoms will develop over a 12 hour time period

and persist for 2-5 days • Will resolve without antibiotics

Legionnaires’ Disease

• Usually causes pneumonia • Severe multisystem disease

• GI, CNS, liver and kidneys

• Incubation period: 2-10 days • Symptoms: fever, chills, dry and non-productive

cough and headache. • Multi-organ disease is common

• Requires antibiotics • Prevalent in late summer or autumn

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Acceptable Lab Tests for LD

• Culture • *DFA on biopsy or secretion • 4-fold rise in antibody titer • **Urine Antigen

Where is Legionella?

• Lakes, rivers, streams, etc. • Cooling towers • Hot tubs • Decorative fountains • Plumbing systems • Evaporative Coolers • Humidifiers • Other

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Life-cycle of Legionella pneumophila in protozoa and human macrophages

Jamie Bartram, Yves Chartier, J. V. L., and K. P. and S. Surman-Lee. 2007. Legionella and the prevention of legionellosis. WHO Libr. Cat. Data.

Pathogenesis

Ira Schwartz and Gary P. Wormser . Bacterial Pathogenesis: A Molecular Approach, Second Edition Clin Infect Dis. (2002) 35 (5): 638-639 doi:10.1086/342198

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Protozoa Interactions • Rowbotham, T.J. 1980. Preliminary report on the pathogenicity of

Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33:1179-1183.

• Anand, C.M., A.R. Skinner, A. Malik, and J.B. Kurtz. 1983. Interaction of Legionella pneumophila and a free-living amoeba (Acanthamoeba palestinensis). J. Hyg. (Cambridge) 91:167-178.

• Barbaree, J.M., B.S. Fields, J.C. Feeley, G.W. Gorman, and W.T. Martin. 1986. Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl. Environ. Microbiol. 51:422-424.

• Fields, B.S., G.N. Sanden, J.M. Barbaree, W.E. Morrill, R.M. Wadowsky, E.H. White, and J.C. Feeley. 1989. Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr. Microbiol. 18:131-137.

Monoclonal Ab Subtyping of Legionella pneumophila serogroup 1

A

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• Direct immunofluorescence

assays

https://courses.cit.cornell.edu/biomi290/ microscopycases/methods/fabs.htm

Diagnosing legionellosis using nucleic acid detection

• Polymerase chain reaction (PCR) assays – ribosomal RNA (rRNA) genes or their

intergenic spacer regions – a gene coding for heat-shock protein

(dnaJ)

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Legionella PCR

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Diagnosing legionellosis using nucleic acid detection

• Benefits – Legionella PCR could potentially detect all serogroups of L. pneumophila

• useful in the early diagnosis of infections, particularly in nosocomial cases – PCR methods could have important economic benefits.

• Their use in outbreaks of legionellosis could help to rapidly rule out implicated sites • Limitations of PCR assays

– Current data are insufficient for reliably estimating PCR sensitivity and specificity values, or for comparing PCR to other methods

– Requires evaluation and standardization of sample preparation and PCR protocols

• Define primer and probe specifications and assay sensitivities, and to reduce the effect of PCR inhibitors

Jamie Bartram, Yves Chartier, J. V. L., and K. P. and S. Surman-Lee. 2007. Legionella and the prevention of legionellosis. WHO Libr. Cat. Data.

Subtyping Procedures

• Serotyping • Monoclonal antibody panel • Ribotyping • Multilocus alloenzyme • PFGE • Arbitrary Primer PCR • Nested Primer analysis • Sequence base typing • Direct sequence analysis

Preventive Measures

• Cleaning and chlorination • Don’t make aerosols • Maintenance of cooling towers & water systems • Maintain hot tubs (spas) • Consideration of the engineering aspects • Follow guidelines • Connect surveillance and subtyping

Legionella Today

• Outbreaks still occurring • Number of cases reported to the CDC about

15K/yr. • Worldwide awareness • Guidelines available • More assistance at the county/state level • Subtyping more advanced • 2013 7th International meeting on Legionella in

Melborne, Australia