ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library Latex Agglutination Test

$ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library Latex Agglutination Test$

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 Hystory of Legionnaires’ Disease

• 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. γ-Proteobacteria

© by author ESCMID Online Lecture Library 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

• Aminoacids as C-source • L-Cystein and other Aa • Iron salt

‰Medium αBCYE © by author ESCMID Online Lecture Library Life-Cycle

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

• 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

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

•ESCMID A colony of Legionella Online spp. is put directly Lecture on a steel plate withLibrary a plastic needle Sample preparation

• ESCMIDThe cells are overlaid Online with 0.5 Lecture µl matrix 2,5-dihydroxybenzoic Library acid (DHB) Sample preparation

Species-specific Genus-specific Identification of Legionella spp.

• SuperSpectraTM were created by the National Reference Centre for Legionella with strains previously identified by mip- sequencing • SuperSpectraTM were included in the SARAMIS Database (Vitek MS) and used for species identification

Gaia V, Casati S, Tonolla M. Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting. Syst Appl Microbiol. 2011 Feb;34(1):40-4. Application: rapid screening of colonies

© by author 15-20 min ESCMID Online Lecture Library L. tucsonensis F. bozemanae ATCC 33217 mip L. bozemanii L. anisa ATCC 35292 mip L. parisiensis 97 L. parisiensis ATCC 35299 mip L. anisa L. tucsonensis ATCC 49180 mip L. gormanii F. gormanii ATCC 33297 mip L. cherrii L. steigerwaltii ATCC 35302 mip L. cherrii ATCC 35252 mip L. wadsworthii 89 L. steigerwaltii F. dumoffii ATCC 33279 mip

L. dumoffii 98 L. wadsworthii ATCC 33877 mip

L. adelaidensis L. sainthelensi ATCC 35248 mip L. pneumophila 100 L. longbeachae 1 ATCC33462 mip L. oakridgensis L. cincinnatiensis ATCC 43753 mip L. cincinnatiensis 98 L. pneumophila ATCC 33152 mip L. birminghamensis L. moravica ATCC 43877 mip L. quinlivanii L. fallonii ATCC mip L. micdadei L. maceachernii L. israelensis ATCC 43119 mip L. taurinensis ATCC 700508 mip L. taurinensis 100 L. rubrilucens 100 L. rubrilucens ATCC 35304 mip L. erythra L. erythra ATCC 35303 mip L. gresilensis L. lansingensis ATCC 49751 mip L. londiniensis L. jamestowniensis ATCC 35298 mip L. longbeachae L. hackeliae ATCC 35250 mip L. sainthelensi L. brunensis ATCC 43878 mip

L. jordanis 98 T. micdadei ATCC 33218 mip L. moravica 97 T. maceachernii ATCC 35300 mip L. brunensis © by author L. drozanskii ATCC mip L. beliardensis L. jordanis ATCC 33623 mip

L. israelensis L. feeleii ATCC 35072 mip L. feelei L. beliardensis L. yabuuchiae 99 L. gresilensis L. impletisoli L. quinlivanii ATCC 43830 mip L. geestiana 100 ESCMIDL. lansingensis Online Lecture Library L. birminghamensis ATCC 43702 mip L. jamestowniensis L. adelaidensis ATCC 49625 mip L. oakridgensis ATCC 33761 mip L. hackeliae 85 L. drozanskii L. londiniensis ATCC 49505 mip L. fallonii L. impletisoli Error (%):0.08 Absolute Intensity >0 0.05 557bp Genebank (NCBI) Relative Intensity >0 Neighbour-Joining Massrange from 3000 to 20000 WCMS mip MEGA 4 Is MALDI-TOD MS useful for typing?

• Calibration and standardisation necessary • time- and cost-saving • allows identification and typing of a large number of colonies within minutes • No association with serotyping with raw data • A preliminary study showed © by authorthat MALDI-TOF MS can be useful for subtyping of Legionella pneumophila ESCMID Online Lecture Librarystrains

Gaia V. Evaluation of Whole Cell MALDI-TOF Mass Spectrometry (WCMS) fingerprinting for rapid subtyping of Legionella pneumophila strains. EWGLI Meeting 2011 Vienna. Typing using MALDI-TOF MS

Ward/Euclidean Ward/Euclidean Bootstrap (500 replicates) Bootstrap (500 replicates) L. pneumophila EUL strains

Gaia V. Evaluation of Whole Cell MALDI-TOF Mass Spectrometry (WCMS) fingerprinting for rapid subtyping of Legionella pneumophila strains. EWGLI Meeting 2011 Vienna. Occurrence of Legionella spp. in the environment

Legionellae prefer warm and moist environments •water – water systems in buildings – recycled water – air conditioning – cooling towers •soil © by author – soil – potting soil ESCMID– compost Online Lecture Library Most abundant Legionella species

• Patients – Legionella pneumophila

• Water – Legionella pneumophila – Legionella anisa

• Soil / water /patients© by author – Legionella longbeachae – Legionella pneumophila ESCMID– Legionella bozemanii Online Lecture Library Water

Data from Swiss NRC for Legionella 2012-2013 Gaia V., Casati S. 2013. Bulletin OFSP 16: 253-257. Cooling Towers vs. Patients culture Cooling towers Patients CH 2000-2011 (n=29) (n=153)

Gaia V., EWGLI 2006. Lisbon Gaia V., Casati S. 2013. Bulletin OFSP 16: 253-257. Soil

Casati S, Gioria-Martinoni A, Gaia V. Casati S, Conza L, Bruin J, Gaia V. Commercial potting soils as an alternative infection source of Legionella pneumophila Compost facilities as a reservoir of Legionella pneumophila and other and other Legionella species in Switzerland. Clin Microbiol Infect. 2009 Jun;15(6):571-5. Legionella species. Clin Microbiol Infect. 2010 Jul;16(7):945-7. Composts vs. aerosols co-culture Compost samples Air samples from compost (n=88) facilities (n=88)

Conza L, Pagani SC, Gaia V. Presence of Legionella and free-living Amoebae in composts and bioaerosols from composting facilities. PLoS One. 2013 Jul 2;8(7):e68244. Transmission

• Legionella spp. from the environmental source to man: environmental habitats and microbial ecology © by author ESCMID Online Lecture Library