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ESCMID Postgraduate Technical Workshop Intracellular bacteria: from biology to clinic Villars-sur-Ollon, 26-30

Référence des rickettsioses, de la fièvre Q et des bartonelloses Our invisible neighbors 1957: Lwoff defines

1975: Woese classifies living organisms in 3 domains: Eukaryotes, Bacteria, Archae XVII century: van Leeuwenhoek

1925: Chatton separates Procaryotes & Eucaryotes (nucleus absent in prokaryotes) New technologies, new questions

microbial world An unsuspected biodiversity 14000 12000 bacterial species 4000 3500 Candidatus Carsonella ruddii 0.

1 ESCMID Postgraduate Technical Workshop Intracellular bacteria: from biology to clinic Villars-sur-Ollon, August 2013 Our invisible neighbors Rickettsiae around the world Pierre-Edouard Fournier Centre National de Référence des rickettsioses, de la fièvre Q et des bartonelloses Our invisible neighbors 1957: Lwoff defines viruses Size < 200 nm, 1 nucleic acid, no autonomous replication, strictly intracellular, no energy metabolism 1975: Woese classifies living organisms in 3 domains: Eukaryotes, Bacteria, Archae XVII century: van Leeuwenhoek invents the microscope XIX century: Cohn & Koch, founders of bacteriology 1878: Sedillot invents the term «microbe» 1925: Chatton separates Procaryotes & Eucaryotes (nucleus absent in prokaryotes) New technologies, new questions 1980: 1,791 validated bacterial species 16S rrna sequencing 2013: >12,000 species (670% increase) Expansion of the microbial world An unsuspected biodiversity bacterial species Candidatus Carsonella ruddii 0.15 Mb bacterial genomes viral species viral genomes Ktedonobacter racemifer Mb 0 0 1

Genomics => redefinition of life boundaries 1970s Koonin. Virology: Gulliver among the Lilliputians. Curr Biol. 2005 Mar 8;15(5):R167-9 Viruses => 1.2 Mb Prokaryotes (0.15 13 Mb) Eukaryotes (0.

species in 1984, 17 in 2013) R What is a rickettsia? Genus Rickettsia What about intracellular bacteria?

2 Genomics => redefinition of life boundaries 1970s Koonin. Virology: Gulliver among the Lilliputians. Curr Biol Mar 8;15(5):R167-9 Viruses => 1.2 Mb Prokaryotes ( Mb) Eukaryotes (0.5 Mb => ) Puzzling bacteria Few culture systems Few phenotypic characteristics Any intracellular bacterium, including Chlamydiae, is a rickettsia Pathogenic species are all known (Rickettsia: U 7 species in 1984, 17 in 2013) R What is a rickettsia? Genus Rickettsia What about intracellular bacteria? 1909: Ricketts 1 st describes a pathogenic intracellular bacterium that replicates autonomously and causes Rocky Mountain spotted fever Dermacentor andersonii Major pathogens majeurs: C. burnetii, C. trachomatis, M. leprae, R. prowazekii Rickettsiales R. rickettsii R. prowazekii R. tsutsugamushi R. quintana C. burnetii E. canis E. phagocytophila E. sennetsu C. ruminantium N. helminthoeca W. pipientis W. persica R. grylli B. bacilliformis G. talpae A. marginale E. ovis H. felis Rickettsia rickettsii R. prowazekii Orientia tsutsugamushi Ehrlichia chaffeensis E. ewingii E. canis E. ruminantium Anaplasma phagocytophilum A. marginale Wolbachia pipientis Neorickettsia sennetsu N.helminthoeca Bartonella quintana B. henselae B. talpae B. bacilliformis Brucella melitensis Coxiella burnetii Rickettsiella grylli Legionella pneumophila «Wolbachia persica» Francisella tularensis Eperythrozoon ovis Hemobartonella felis Mycoplasma pneumoniae Ureaplasma urealyticum No respect for taxonomy Genus Rickettsia Gram + Gram-negative bacteria Stained by the Gimenez method Size x 0.3 µ Strictly intracellular Associated to endothelial cells -Proteobacteria Associated to arthropods: ticks, fleas, lice, mites 26 validated species, incl. 17 pathogens > 100 unclassified Rickettsia sp. Major human pathogens B List of potential bioterrorism agents 2

One rickettsiosis per continent Tick-borne rickettsioses in 2013 R. massiliae R. conorii caspia R. parkerii R. rickettsii R. aeschlimannii R. helvetica R. mongolitimonae R. conorii conorii R.

japonica Phenotypic characteristics Complement fixation Plotz 1944, Pickens 1965 Toxinic neutralization Bell 1960 Proteic profiles (SDS-PAGE) Eisemann 1976, Pedersen 1978 Spotted fever group -

3 One rickettsiosis per continent Tick-borne rickettsioses in 2013 R. massiliae R. conorii caspia R. parkerii R. rickettsii R. aeschlimannii R. helvetica R. mongolitimonae R. conorii conorii R. africae R.slovaca R. conorii israelensis R. sibirica sibirica R. conorii conorii R. australis R. honei Taxonomy R. sibirica mongolitimonae R. conorii indica R. japonica Phenotypic characteristics Complement fixation Plotz 1944, Pickens 1965 Toxinic neutralization Bell 1960 Proteic profiles (SDS-PAGE) Eisemann 1976, Pedersen 1978 Spotted fever group - Associated to ticks -Spotted fevers - Motile - Optimal growth temperature 32 C - Cross-reaction with Proteus vulgaris strain OX-2 Mehrej et al Few phenotypic characteristics Typhus group - Lice-fleas - Typhus - Non motile - 35 C -with OX-19 Phenotypic characteristics Mouse serotyping Philip 1978 Western-blot (rompa & rompb) Gilmore 1991 Monoclonal antibodies Anacker 1987 (Fang R. CDLI 2003) Fang R. CDLI % R. rickettsii R. conorii 7 R. conorii M R. honei R. massiliae R. akari R. felis R. australis R. montanensis R. japonica R. canadensis R. typhi R. prowazekii 3

4 Antibiotic susceptibility Erythromycin Rifampin R S R. sibirica R S R. africae R S R. conorii R S R. slovaca R S R. honei R S R. rickettsii R S R. japonica R R Bar 29 R R R. massiliae R R R. rhipicephali R R R. aeschlimanii R R R. montana R S R. helvetica R S R. australis R S R. akari S S R. typhi S S R. prowazekii R S R. canadensis R S R. bellii Few phenotypic characteristics Few genetic studies Rolain et al. AAC 1998;42: Poorly reproducible discriminatory and comparable tests Bergey s manual 1984: 10 validated species 7 pathogenic Overall Pathogenic vs non pathogenic No Other genus rrs > 98.1% AND glta > 86.5% New bacterium with at least one validated Rickettsia species Yes Member of the Rickettsia genus Genomic characteristics Species GC % DNA-DNA Hybridization R. rickettsii R. prowazekii R. conorii % - R. rickettsii % R. sibirica % - R. akari % - R. canadensis % 45% R. prowazekii % - R. typhi % 72% Taxonomy Official criteria for defining bacterial species are not adequate Bacterial species = DNA-DNA hybridization: > 70% + G+C content: divergence < 5% + 16S rrna: divergence < 1.3% DNA-DNA hybridization: R. montanensis, R. rickettsii, R. sibirica et R. conorii = same species -G+C content: SFG32-33%, TG 29-30% - 16S rrna: divergence < 2% among Rickettsia => The Rickettsia genus would only comprise 3 species (vs 26 in 2013) Taxonomic recommandations for classifying rickettsial isolates Fournier et al. J.Clin.Microbiol.2003;41: Raoult et al. Ann.NY.Acad.Sci.2005;1063: Yes Belongs to a validated species > 1 of these criteria rrs > 99.8% glta > 99.9% with a validated ompa > 98.8% ompb > 99.2% Rickettsia species sca4 > 99.3% No Potential new species Rickettsioses Isolated in pure culture Isolated in pure culture Yes SPD > 3 with respect to the type strain AND Specific epidemio- clinical characteristics No (molecular data only or isolate lost upon subcloning) Strain of Candidatus the species Yes Describe phenotypic characters (geographic distribution, vector, pathogenicity (if any), mouse serotype with respect to genetically closest validated species) Emerging zoonoses Yes Subspecies Deposit of the type strain into two independent official culture collections AND publication in Int. J. Syst. Evol. Microbiol. or publication in another peer-reviewed journal and in a Validation list in Int. J. Syst. Evol. Microbiol. Unvalidated "species" or "subspecies" No Yes Validated species or subspecies 4

Tick hypostoma inserted in the skin of a patient Tick-borne

pathogens Mediterranean Spotted fever (MSF) One of the oldest

description of the eschar in Marseille (Boinet and Pieri) 1930 s: -

5 Tick hypostoma inserted in the skin of a patient Tick-borne rickettsioses Europe and Mediterranean area Europe 2012: emergent pathogens Mediterranean Spotted fever (MSF) One of the oldest recognized vectorborne infectious diseases MEDITERRANEAN SPOTTED FEVER Rickettsia conorii subsp. conorii 1909: first cases in Tunisia (Conor and Brush) 1925: first description of the eschar in Marseille (Boinet and Pieri) 1930 s: - identification of the vector: Rhipicephalus sanguineus (Olmer Durand & Conseil) - identification of the agent : R. conorii (Brumpt) 5

Global distribution of MSF/ Rh. sanguineus R. conorii conorii Rh.

htm Many gaps in the Ecology of MSF: Rh.

endemic in Mediterranean area Rickettsia c. conorii animal reservoir?

wild <1%, with exception in endemic region: small foci?

phenomenon for R. conorii => tick lethality Socolovschi et al.

conorii detected in the tick ovaries by electronic microscopy

rural 1/3 Tick-bite is often unnoticed (larva, nymphs++) Seasonal

6 Global distribution of MSF/ Rh. sanguineus R. conorii conorii Rh. sanguineus Many gaps in the Ecology of MSF: Rh. sanguineus has a worldwide distribution but MSF is known to be only endemic in Mediterranean area Rickettsia c. conorii animal reservoir? Which host for Rh. sanguineus? 2009: 100% vertical transmission of R. conorii in naturally infected Rh. sanguineus over 12 generations BUT Prevalence of infected ticks in the wild <1%, with exception in endemic region: small foci? Effect of climatic conditions on survival of infected tick Reactivation phenomenon for R. conorii => tick lethality Socolovschi et al. 2009; Socolovschi et al R. conorii detected in the tick ovaries by electronic microscopy Mediterranean spotted fever : clinical aspects Urban disease 2/3 - rural 1/3 Tick-bite is often unnoticed (larva, nymphs++) Seasonal zoonosis: warmest period Males Incidence 50/ hbts Incubation 6 7 days Fever 100%, flu-like symptoms Inoculation eschar (single?) 72 % Conjunctivitis Skin rash 97% Peak in August MSF rash involves palms-soles, not face May occur at any age 6

Most often mild 6% => malignant forms Diabetes, immunodeficiency, G6PD deficiency, elderly, alcoholism, delayed antibiotics Mortality 2-3% Mouffok et al.

indica Changing epidemiology of MSF!!! Global warming => effect on tick behavior period of activity of Rh.

sanguineus-transmitted diseases Fatal cases 2 6%, and reach 30% in hospitalized patients Socolovschi et al. 2009. Parola et al.

sanguineus ticks, Human cases: Inoculation eschar 4% Imported fatal cases: UK (from Portugal), Switzerland (from Lybia) R. conorii subsp. caspia Astrakhan fever Kosovo, France: Rh.

7 Most often mild 6% => malignant forms Diabetes, immunodeficiency, G6PD deficiency, elderly, alcoholism, delayed antibiotics Mortality 2-3% Mouffok et al. Int J Infect Dis 2009; 13: Evolution Other Rickettsia conorii subspecies Rickettsia conorii subsp. israelensis Rickettsia conorii subsp. caspiensis Rickettsia conorii subsp. indica Changing epidemiology of MSF!!! Global warming => effect on tick behavior period of activity of Rh. Sanguineus aggressiveness biting of unusual hosts (humans) => Multiple eschars, severe forms => incidence of Rh. sanguineus-transmitted diseases Fatal cases 2 6%, and reach 30% in hospitalized patients Socolovschi et al Parola et al Fatal MSF French in 2003 heat wave: 22 attached ticks on a man Multiple eschars Other Rickettsia conorii subspecies R. conorii subsp. israelensis Israeli spotted fever Sicily, Portugal: Rh. sanguineus ticks, Human cases: Inoculation eschar 4% Imported fatal cases: UK (from Portugal), Switzerland (from Lybia) R. conorii subsp. caspia Astrakhan fever Kosovo, France: Rh. sanguineus ticks (cluster of spotted fever) No human cases in Europe Imported cases: France (from Chad) R. conorii subsp. indica Indian tick typhus Sicily : one human case Imported cases: French traveler (from India) Chai JT JTM 2008; Boillat et al. 2008; Levin et al. 2012; Alexandre N et al.2011; Tarasevich 1991; Parola et al. 2001; Renvoisé et al SENLAT - TIBOLA - DEBONEL 1997: R. slovaca as human pathogen (Lakos. Lancet. 1997) (scalp eschar + enlarged cervical lymph nodes) SENLAT (SCALP ESCHAR AND NECK LYMPHADENOPATHY) Rickettsia slovaca, Rickettsia raoultii, Rickettsia «rioja» 2002: Lakos proposed the name of this syndrome TIBOLA (TIck-BOrne LymphAdenopathy) 2006: R. raoultii = 2 nd agent of this syndrome and Ibarra et al. proposed the name DEBONEL (Dermacentor-Borne Necrosis Erythema Lymphadenopathy) 2010 : Identification of 3 patients with Bartonella henseae infection Raoult proposed the name SENLAT (Scalp Eschar and Neck LymphAdenopaThy) Raoult et al. 1997; Lakos et al. 2002; Ibarra et al. 2006; Angelakis et al. 2010; Parola et al

SENLAT R. slovaca and R. raoulti infection Endemic: Europe Vecteur: Dermacentor marginatus and D.

2009 Unusual agents of SENLAT Bartonella henselae : 3 patients, France Francisella tularensis : 1 patient, France Rickettsia rioja : 1

slovaca-borrelia burgdorferi : 8 patients, Spain Lakos et al. 2002;, Parola et al. 2009; Angelakis et al. 2010; Edouard et al.

2009) (Lymphangitis-associated rickettsiosis) Rickettsia sibirica mongolitimonae R.

8 SENLAT R. slovaca and R. raoulti infection Endemic: Europe Vecteur: Dermacentor marginatus and D. reticulatus The peak incidence: March-May & September- November Parola et al Unusual agents of SENLAT Bartonella henselae : 3 patients, France Francisella tularensis : 1 patient, France Rickettsia rioja : 1 patient, Spain Co-infections: R. slovaca-coxiella burnetii: 1 patient, France R. slovaca-borrelia burgdorferi : 8 patients, Spain Lakos et al. 2002;, Parola et al. 2009; Angelakis et al. 2010; Edouard et al. 2012; Perez-Perez et al R. sibirica mongolitimonae LAR (Parola et al. 2009) (Lymphangitis-associated rickettsiosis) Rickettsia sibirica mongolitimonae R. sibirica mongolitimonae 1996: 1 st human case, France, tick vector? >20 cases to date 2005: first case in Algeria Detection in H. truncatum Niger Migratory birds? 1991: Detection in Hyalomma asiaticum, Mongolia 2004: First case in South Africa Mediterranean area (France, Greece, Portugal, Spain) Potential vector in Europe: Hyalomma anatolicum excavatum (Greece, Cyprus) Rh. pusillus (France, Portugal) France Greece Portugal Spain R. sibirica mongolitimonae in ticks Human infection Imported case: French traveler (from Egypt) Raoult et al. 1996; Pretorius et al Psaroulaki et al. 2005; Chochlakis D. et al. 2012; de Sousa et al; 2006; Edouard et al

LAR (Lymphangitis-associated rickettsiosis) Men 70% Fever 100% Headache and myalgia: 87% Skin rash 90% Inoculation eschar: 87%, including multiple eschars 15% Fournier et al.

sanguineus and later from other Rhipicepalus spp. in Europe, Africa, America Infection rate in ticks: up to 92% in France R.

massiliae 2008: 2 nd patient in France 2010: 3 rd patient in Argentina Vitale et al. 2005, Parola et al. 2008; Garcia-Garcia et al. 2010; Socolovschi et al. 2012 Others spotted fever rickettsioses R.

9 LAR (Lymphangitis-associated rickettsiosis) Men 70% Fever 100% Headache and myalgia: 87% Skin rash 90% Inoculation eschar: 87%, including multiple eschars 15% Fournier et al EID; Socolovschi et al Lymphangitis expanding from eschar 32% Painful enlarged lymph nodes 65% Rickettsia massiliae 1992: first isolated from Rh. sanguineus and later from other Rhipicepalus spp. in Europe, Africa, America Infection rate in ticks: up to 92% in France R. massiliae in ticks Human infection 2006: Identification of a SFG rickettsia isolated from blood in a Sicilian patient in 1984 as R. massiliae 2008: 2 nd patient in France 2010: 3 rd patient in Argentina Vitale et al. 2005, Parola et al. 2008; Garcia-Garcia et al. 2010; Socolovschi et al Others spotted fever rickettsioses R. massiliae, R. monacensis, R. aeschlimannii, R. helvetica Rickettsia monacensis 2002 : first isolated from Ixodes ricinus ticks collected in Germany Infection rate in ticks: 2.4 % in Spain to 52.9% in Bulgaria R. monacensis in ticks Human infection 2007: identification from blood in 2 patients from Spain 2012: 1 patient in Sardinia Madeddu et al Rickettsia aeschlimannii Rickettsia helvetica 1992: first isolated from Hyalomma marginatum marginatum ticks from Morocco 1979: 1 st isolation from I. ricinus ticks from Switzerland 17 Infection rate in ticks: 64.7% in Corsica (H. m. rufipes) Transported through Europe by migratory birds (France, Germany, Spain) R. aeschlimannii in ticks Several infection cases reported in Europe and Asia, but no isolation of the bacterium from clinical samples R. hevetica in ticks 2002: 1 st human case, a French traveller to Morocco with MSF-like illness No autochtonous case in Europe Raoult et al. 2002; Fernandez-Soto et al. 2003; Rumer et al. 2011; Socolovschi et al. 2012; => presumably associated with human illnesses 9

10 R. africae (Raoult et al. N.Engl.J.Med. 2001;344: ) Africa Epidemiologie (Raoult et al. N.Engl.J.Med. 2001;344: ) Amblyomma : attack strategy (CO 2 ) grouped cases (74%) attack rate 4-14% multiple eschars Distribution of R. africae similar to that of Amblyomma Sub-saharan Africa (Parola et al. N.Engl.J.Med.1998;338:1391; Parola et al. Am.J.Trop.Med.Hyg.1999;60:888-93; Kelly et al. 2003;unpublished data) Prevalence = 1,7-72% (Tissot-Dupont et al. Am.J.Trop.Med.Hyg. 1994;50:373-80; Beati et al. J.Med.Entomol. 1995;32: ; Clin.Infect.Dis.1995;21: Parola et al. Emerg.Infect.Dis. 2001;7: ) Seroprevalence = 27 52% (Tissot-Dupont et al. Clin.Infect.Dis.1995;21: ) Most frequent SF rickettsiosis Importation of Amblyomma Clinical manifestations Raoult et al. N.Engl.J.Med. 2001;344: ) Sex-ratio H/F 1.6, mean age 45 years Silent incubation, 6-7 days Sudden onset, fever 88% Rash 46% = MP 51%, vesicular 45%, purpuric 4% Clinical manifestations Raoult et al. N.Engl.J.Med. 2001;344: ) Inoculation eschar 95%, multiple 54%, limbs 73% Distribution of R. Africae in West Indies Parola P, et al. N Engl J Med Satellite enlarged lymph nodes 43% Mild disease (recovery 100%, no sequellae) + Reunion island, New Caledonia 10

11 America R. parkeri 1 st isolation in Amblyoma maculatum in 1931 Prevalent tick in south-eastern USA One isolate in a patient dead from RMSF in Ohio partially similar to R. parkeri (Ralph et al. Ann N Y Acad Sci -1990) Patients in Florida, Uruguay, Argentina Fever, rash, eschar, R. parker isolated from a skin biopsy (C. Paddock, CID, 2004) Other tick-borne rickettsioses Rocky Mountain Spotted Fever Rickettsia rickettsii Vector = Dermacentor andersoni summer USA : 500 cases/year (south-east) central America, Brazil, Argentina, Colombia Fever, rash, no eschar Mortality 5-80 % without treatment R. parkeri Raoult D, Paddock C. N.Engl.J.Med. 2005: Disease Rash Rash specificity Eschar Enlarged lymph nodes Rocky Mountain spotted 90 % 45 % purpuric very rare no fever Mediterranean spotted fever 97 % 10 % purpuric 72 % rare Siberian tick typhus 100% 77 % yes Queensland tick typhus 100 % vesicular 65 % yes Israëli spotted fever 100 % rare no Flinder s Islands spotted 85 % 8 % purpuric 28% yes fever Astrakhan fever 100 % 23 % no African tick-bite fever 30 % vesicular 100 % yes multiple Japanese spotted fever 100 % 90 % no Rickettsia mongolotimonae yes yes no Rickettsia slovaca no yes yes Rickettsia helvetica no - no no R. heilongjangensis yes yes yes R. aeschlimannii yes - Yes no (multiple?) R. parkeri yes - yes? p p > 5 p

conorii complex R. canadensis R. heilongjiangensis R. honei R. hoogstraalii R. helvetica R. japonica R. massiliae R. monacensis R. montanensis R. parkeri R. peackockii R. philipii 364D R. raoultii R.

12 Diagnostic tricks Triad fever eschar rash But Absence of characteristic symptoms No rash (SENLAT) Neither rash nor eschar (R. helvetica) Coexistence of several rickettsioses in an area Acknowledgements : Pr. Didier Raoult Pr. Philippe Parola THANK YOU! R. aeschlimannii R. africae R. asiatica R. australis R. bellii R. conorii complex R. canadensis R. heilongjiangensis R. honei R. hoogstraalii R. helvetica R. japonica R. massiliae R. monacensis R. montanensis R. parkeri R. peackockii R. philipii 364D R. raoultii R. rhipicephali R. rickettsii R. sibirica complex R. slovaca R. tamurae R. felis? R. prowazekii? Ixodidae tick R. hoogstraalii Rickettsia sp. AvBat R. felis? Flea R. felis R. typhi R. africae? Other potential arthropod vectors? R. prowazekii Human body louse Argasidae Mesostigmatid tick Booklice mites R. felis R. akari R. felis Whitefly Rickettsia sp. MEAM Mosquito R. felis Mehrej et al

ESCMID Online Lecture Library. by author

ESCMID Online Lecture Library. by author 23.03.2013 CHYPRE «Emerging Rickettsioses» Didier Raoult Marseille - France didier.raoult@gmail.com www.mediterranee-infection.com Gram negative bacterium Strictly intracellular Transmitted by arthropods: