17 March 2014 ESCMID-ESCAR PGEC MARSEILLE «Ignorance and blindness for a post-modern microbiology ! » © by author ESCMID Online Lecture Library Didier Raoult Marseille - France [email protected] www.mediterranee-infection.com © by author « ESCMIDPostmodern scienceOnline makes Lecture the theory Library of its own evolution as discontinuous, catastrophic, nonrectifiable, paradoxical.» P.97 2 INFECTIONS IN THE XXIST CENTURY Infections in the world : 17 million deaths - 30% - The big 3s : HIV - TB – malaria : no vaccine - Respiratory infections : Etiology? - Digestive infections : Etiology? - Vaccine-prevented infections© by : authorAdhesion - Nosocomial infections : Circuits? - EmergingESCMID infections Online : LectureSource Library - Cancer : Communicable 3 IGNORANCE that decreases but reveals ou ARROGANCE BLINDNES that persists and leads to FALSE DEDUCTIONS © by author and UNVERIFIED PREDICTIONSESCMID Online Lecture Library 4 EMERGING DISEASES IGNORANCE: Major and unique acceleration of knowledge in history Blindness False deductions© by author ESCMID Unverified Online predictions Lecture Library So what ? 5 Viruses: Essential Agents of Life by Günther Witzany © by author ESCMID Online Lecture Library p.64 By F.Rohwer 6 Viruses: Essential Agents of Life by Günther Witzany © by author ESCMID Online Lecture Library p.64 By F.Rohwer 7 PROGRESSES MADE IN MICROBIOLOGY FROM 1979 TO 2012 THANKS TO THE DEVELOPMENT OF NEW TECHNOLOGIES Cultured Sequenced © by author a) the ESCMIDleft ordinate axis refers toOnline the cumulative numbers Lecture of bacterial species Library with validly published names (green curve); and sequenced viral genomes (blue). 8 The human bacteriome Identification of all human-associated bacteria among all bacterial species validated since 1980 (http://www.bacterio.net/). Results obtained from 5 different sources 1 Mediterrannée- 2 Infectious diseases books Infection databases Principles and Routine Culturomics Dictionnaire de Pratice of Infectious Manual of laboratory Strains Maladies Infectious Diseases Cohen Clinical strains 579 Infectieuses D. Diseases and Powderly, Microbiology, th 609 Raoult Mandell et al., 3rd ed 2010 10 ed 2011 Only 275 in common 7th ed 2009 4 Bacterial strain collections 3 Human Microbiome Deutsche Project Collection de Collection de American (NIH, US Dpt of Health & Human Services) Souches de Sammlung von l’Institut Type Culture List of bacterial genomes isolated in l’Unité des Mikroorganismen Pasteur Collection humans (blood, abdomen, urogenital Rickettsies und (CIP) (ATCC) tract, heart, eye, liver, nose, skin, oral (CSUR) Zellkulturen © by author (DSMZ) and wound) 5 Literature search PubmedESCMID search Journal search: OnlineMedline search LectureDeletion of Library for « Rickettsia », JCM, Anaerobes, using queries Candidatus, « Borrelia », EID using established on 2,031 species « Bartonella », « Human» as MESH articles duplicates, « Leptospira » keyword indexation unvalidated names 9 Molecular studies neglect apparently gram- negative populations in the human gut microbiota. Hugon P, Lagier JC, Robert C, Lepolard C, Papazian L, Musso D, Vialettes B, Raoult D. J Clin Microbiol. 2013 Oct;51(10):3286-93 © by author ESCMID Online Lecture Library 10 NEW BACTERIA 1- 16SrDNA 2- Intracellular and fastidious 3- MALDI-TOF © by author 4- Culturomics ESCMID Online Lecture Library 11 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D..J Clin Microbiol. 2000 Oct;38(10):3623-30. Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria. Systematic 16S rRNA gene sequencing of atypical clinical isolates identified 27 new bacterial species associated with humans. Drancourt M, Berger© P, Raoult by D.J Clin Microbiol.author 2004 May;42(5):2197- 202 Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a > or =1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (< or ESCMID=10 cases reported in the literature) Online human pathogens. Lecture Eleven new species, Library"Actinobaculum massiliae ," "Candidatus Actinobaculum timonae," Paenibacillus sanguinis, "Candidatus Bacteroides massiliae," Chryseobacterium massiliae, "Candidatus Chryseobacterium timonae," Paenibacillus massiliensis, "Candidatus Peptostreptococcus massiliae," "Candidatus Prevotella massiliensis," Rhodobacter massiliensis, and "Candidatus Veillonella atypica" were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases. 12 29 new species - 16 first isolation in humans NEW BACTERIA 1- 16SrDNA 2- Intracellular and fastidious 3- MALDI-TOF © by author 4- Culturomics ESCMID Online Lecture Library 13 FASTIDIOUS BACTERIA • Rickettsioses • T.whipplei • Bartonella and TB © by author • Fastidious amoebal pathogens ESCMID Online Lecture Library • Future culture strategy 14 WHAT IS A RICKETTSIA ? Gram negative bacterium Strictly intracellular Shall vid Transmitted by arthropods: ticks, fleas, lice,© mites by author ESCMID Online Lecture Library We isolated and deposited 18 Rickettsia species in our collection (156 strains) and C. burnetii 180 strains 15 TICK-BORNE RICKETTSIOSES R. PA4 R. monacensis R. massiliae R. conorii caspia R. sibirica R. sibirica R. parkerii R. helvetica sibirica mongolotimonae R. mongolotimonae R. conorii indica R.slovaca R. conorii conorii R. conorii israeli R. japonica R. rickettsii © by author R. conorii conorii R. australis ESCMIDR. aeschlimannii Online Lecture Library R. africae R. honei 16 In 1970, changes in the distribution of murine typhus a disease caused by R.typhi transmitted by the rat fleas (Xenopsylla cheopis) Cases are more prevalent in a wealthy county Orange (CA) than in poor counties Opossums are identified as major actors © by author They are infested by cat fleas (CenocephalidesESCMID felis)Online Lecture Library A flea from Ethiopia positive! 17 Growing «R. felis» We failed to grow it at 37°C in HEL cells We suspected that temperature growth was critical as this is reported for many arthropod borne microorganism : Y. pestis (plague) arboviroses Wolbachia pipientis Bartonella bacilliformis Tick borne Rickettsia© by (32 author°C) And we used XTC2 cells obtained from Xenopus growing at 28ESCMID°C used for Arbovirus Online Lecture Library A flea-associated Rickettsia pathogenic for humans. Raoult D, La Scola B, Enea M, Fournier PE, Roux V, Fenollar F, Galvao MA, de Lamballerie X. Emerg Infect Dis. 2001 Jan-Feb;7(1):73-81. 18 Rickettsia felis-associated uneruptive fever, Senegal Socolovschi C, Mediannikov O, Sokhna C, Tall A, Diatta G, Bassene H, Trape JF, Raoult D. Emerg Infect Dis. 2010 Jul;16(7):1140-2. Abstract During November 2008-July 2009, we investigated the origin of unknown fever in Senegalese patients with a negative malaria test result, focusing on potential rickettsial