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ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library Latex Agglutination Test The etiological agent: Legionella pneumophila and other Legionella spp. Valeria Gaia © by author National Reference Centre for Legionella ESCMIDc/o Online Microbiology Lecture laboratory Library Ente Ospedaliero Cantonale Bellinzona - Switzerland © by author ESCMID Online Lecture Library Hystory of Legionnaires’ Disease July 21st 1976 - Philadelphia • 58th Convention of the American Legion at the Bellevue-Stratford Hotel • > 4000 World War II Veterans with families & friends • 600 persons staying at the hotel © by author • ESCMIDJuly 23nd: convention Online closed Lecture Library • Several veterans showed symptoms of pneumonia Searching for the causative agent David Fraser: CDC – Atlanta •Influenza virus? •Nickel intoxication? •Toxin? o 2603 toxicology tests o 5120 microscopy exams o 990 serological tests© by author ESCMIDEverybody seems Online to agree: Lecture it’s NOT a bacterialLibrary disease! July 22nd – August 2nd •High fever •Coughing •Breathing difficulties •Chest pains •Exposed Population =© people by authorstaying in the lobby or outside the Bellevue Stratford Hotel «Broad Street Pneumonia» •221ESCMID persons were Online infected (182+39 Lecture «Broad StreetLibrary Pneumonia» ) 34 patients died (29+5) September 1976-January 1977 Joseph McDade: aims to rule out Q-fever (Rickettsiae) •Injection of “infected” pulmonary tissue in Guinea Pigs microscopy: Cocci and small Bacilli not significant at the time •Inoculation in embryonated eggs + antibiotics to inhibit the growth of contaminating bacteria No growth Microscopy on the same slides: This time he sees Bacilli within a white cell! © by author •Inoculation in embryonated eggs without antibiotics ESCMID Injection in Guinea Online Pigs symptoms Lecture of Legionnaires’ Library Disease •Discovery of Legionella pneumophila Looking back … • Legionella was similar to some « thermophilic bacteria» living as biofilm and in association with algae described by Carl Fliersmans in thermal regions of the Yellowstone National Park, • Previously unresolved outbreaks: – 1968 in Pontiac, Michigan: 95% of the persons working in the same building – 1965 in Washington:© by outbreak author with 34 cases in a hospital – 1957 in Austin, Minnesota: 78 cases ESCMID– 1942 Fort Bragg, Online North Carolina:Lecture 40 soldiers Library The Family Legionellaceae © by author ESCMID Online Lecture Library • Domain: Bacteria • Kingdom: Eubacteria • Phylum: Proteobacteria • Class: γ-Proteobacteria • Order: Legionellales • Family: Legionellaceae© by author • Genus: Fluoribacter / Legionella / Tatlockia • ESCMIDSpecies: 59 officiallyOnline described Lecture in Library 2013 © by author ESCMID Online Lecture Library Ecker DJ et al. The Microbial Rosetta Stone Database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents. BMC Microbiol. 2005 Apr 25;5:19. © by author ESCMID Online Lecture Library γ-Proteobacteria Legionella pneumophila subsp pneumophila Legionella pneumophila subsp fraseri Legionella pneumophila sg 1 to 15 Legionella pneumophila subsp pascullei © by author Tatlockia micadadei Legionella micadadei Tatlockia maceachernii Legionella maceachernii ESCMIDFluoribacter Online bozemanae LectureLegionella Library bozemanii Fluoribacter gormanii Legionella gormanii Fluoribacter dumoffii Legionella dumoffii In 2013: 59 species Legionella pneumophila Legionella spiritensis Legionella londiniensis Legionella bozemanii Legionella jamestowniensis Legionella taurinensis Legionella dumoffii Legionella santicrucis Legionella lytica Legionella micdadei Legionella cherrii Legionella drozanskii Legionella longbeachae Legionella steigerwaltii Legionella rowbothamii Legionella jordanis Legionella rubrilucens Legionella fallonii Legionella wadsworthii Legionella israelensis Legionella gresilensis Legionella hackeliae Legionella quinlivanii Legionella beliardensis Legionella feelei Legionella brunensis Legionella busaniensis Legionella maceachernii Legionella moravica Legionella drancourtii Legionella birminghamensis Legionella gratiana Legionella jeonii Legionella cincinnatiensis Legionella adelaidensis Legionella yabuuchiae Legionella gormanii Legionella fairfielddensis Legionella impletisoli Legionella sainthelensi ©Legionella by shakespearei author Legionella dresdeniensis Legionella tucsonensis Legionella waltersii Legionella nagasakiensis Legionella anisa Legionella genomospecies Legionella steelei Legionella lansingensis Legionella quateirensis Legionella tunisiensis Legionella erythra Legionella worsleiensis Legionella massiliensis LegionellaESCMID parisiensis OnlineLegionella geestiana Lecture LegionellaLibrary cardiaca Legionella oakridgensis Legionella nautarum Phenotypical characteristics • Legionella pneumophila • Catalase + • gram negative rods (faint) • Oxydase – or (+) • 0.3-0.9µm x 2µm long • Mobile (polar or lateral • No fermentation of flagella) carbohydrates • non-sporulating • No Nitrate reduction • noncapsulated • No urease • facultative intracellular • (Degradation of gelatin) parasite © by author • (Autofluorescence) • LPS O antigens – Blue ESCMIDserogroups Online Lecture– Red Library – Jellow-green LPS Legionella pneumophila sg1 © by author ESCMID Online Lecture Library Nutritional requirements • Aminoacids as C-source • L-Cystein and other Aa • Iron salt Medium αBCYE © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Life-Cycle Life-Cycle of Legionella © by author ESCMID Online Lecture Library Life-Cycle of Legionella © by author ESCMID Online Lecture Library Intracellular Phase Planctonic Phase Replicative Phase Infectious Phase Legionella forms found in the environment Form infectious Survival Culturable MIF +++ ++ ++ mature intracellular form FIL + +++ (+) filamentous form Pellets +++ +++ (+) © by author VBNC ? ++ - Viable but not culturable ESCMIDSPF Online Lecture+ + Library+ Stationary phase form Garduño R. et al. Are all the Legionellae found in water equal? – ecology of the various Legionella forms present in water and potential role in transmission of disease. 8th International Conference on Legionella 2013, Melbourne, Australia Identification and typing of Legionella spp. © by author ESCMID Online Lecture Library Confirmation of the identity of L. pneumophila and L. species • Morphology of the colonies ? • Autofluorescence ? • Grows on Blood agar (or without cystein) ? • Latex agglutination test – L. pneumophila ©(Lp1/Lp by 2-15)author – L. anisa • ESCMIDFurther tests Onlineare required Lecture for other Library Legionella species © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Latex agglutination test L. anisa Lpn 1 Lpn 1 Lpn 2-15 © by author Legionella longbeachae 1 & 2 Legionella bozemanii 1 & 2 Lpn 2-15 Legionella dumoffii ESCMID Online LectureLegionella Library gormanii Legionella jordanis Legionella micdadei Legionella anisa Serotyping • Serotyping is based on O antigens of LPS • Immunogenic determinant © by author ESCMID Online Lecture Library Serotyping • Immunofluorescence – Monoclonal antibodies specific for Lp serogroups 1 to 15 – Monoclonal antibodies for subtyping of Lp1 (Dresden scheme) • Philadelphia • France/AllentownMab 3/1 positive • Benidorm = Pontiac group • Knoxville © by author • OLDA • Oxford ESCMID• Heysham Online Mab 3/1 negative Lecture Library • Camperdown • Bellingham Identification of Legionella spp. • Identification of Legionella spp. Used to rely on serological tests (serotyping). Specific antisera are available only in few reference laboratories. • Today, identification of Legionella spp. is based on sequencing of the gene mip (macrophage infectivity potentiator).© by Ratcliffauthor at al. in 1998 • mip sequencing is easy to use thanks to a free ESCMIDdatabase hosted Online by Public Lecture Health England Library (access still available via www.ewgli.org) mip sequencing © by author ESCMID Online Lecture Library www.ewgli.org Ratcliff, R.M., et al., Sequence-based classification scheme for the genus Legionella targeting the mip gene. J Clin Microbiol, 1998. 36(6): p. 1560-7. © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Identification of Legionella spp. • Identification of Legionella spp. can also be performed by MALDI-TOF MS • Specific databases for bacterial identification are available. Legionella spp. will soon be included or implemented © by author ESCMID Online Lecture Library Molecular Typing: Sequence-based typing (SBT) • 7 genes – flaA (flagellum subunit gene) – pilE (pilus biogenesis gene) – Asd (aspartate-semialdehyde dehydrogenase ) – mip (macrophage infectivity potentiator) – mompS (major outer membrane protein) – proA (Zn-metalloprotease) – neuA (N-acylneuraminate cytidylyl transferase) • Free database for automatic© by sequence author analysis – 8405 strains submitted (64% clinical/34% environmental) – 1708 STs • ESCMIDAnalysis tool is hosted Online by Public Lecture Health England Library(access still available via www.ewgli.org) CLINICAL ST1 ENVIRONMENTAL BOTH © by author ESCMID Online Lecture Library CLINICAL ST47 ENVIRONMENTAL BOTH © by author ESCMID Online Lecture Library MALDI-TOF MS Time-Of-Flight Vacuum Time-of-flight ionisation detector + Laser beam [M+H] UV laser 337 nm 3 ns Intact, protonized biomolecule α-Cyano- © by author 4-hydroxy- cinnamic Collision acidESCMID Online Lecturereactions Library Matrix (CHCA) molecules Matrix-Assisted Laser Desorption/Ionization Positive linear mode Positive linear ++ © by author ESCMID
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