Bacterial Species Library with Validly Published Names (Green Curve); and Sequenced Viral Genomes (Blue)

17 March 2014 ESCMID-ESCAR PGEC MARSEILLE «Ignorance and blindness for a post-modern microbiology ! »

« 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

p.64 By F.Rohwer 6 Viruses: Essential Agents of Life by Günther Witzany

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

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

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 infection. Using molecular tools, we found evidence for Rickettsia felis- associated illness in © the by initial author days of infection in febrile Senegalese patients without malaria. ESCMID Online Lecture Library

19 Human Infection with Rickettsia felis, Kenya. Richards AL, Jiang J, Omulo S, Dare R, Abdirahman K, Ali A, Sharif SK, Feikin DR, Breiman RF, Njenga MK. Emerg Infect Dis. 2010 Jul;16(7):1081-6. Abstract To determine the cause of acute febrile illnesses other than malaria in the North Eastern Province, Kenya, we investigated rickettsial infection among patients from Garissa Provincial Hospital for 23 months during 2006-2008. Nucleic acid preparations of serum from 6 (3.7%) of 163 patients were positive for rickettsial DNA as determined by a genus- specific quantitative real-time© by author PCR and were subsequently confirmed by molecular sequencing to be positive for RickettsiaESCMID felis. The Online 6 febrile Lecture patients' symptoms Library included headache; nausea; and muscle, back, and joint pain. None of the patients had a skin rash.

20 Common Epidemiology of Rickettsia felis Infection and Malaria, Africa. Mediannikov O, Socolovschi C, Edouard S, Fenollar F, Mouffok N, Bassene H, Diatta G, Tall A, Niangaly H, Doumbo O, Lekana-Douki JB, Znazen A, Sarih M, Ratmanov P, Richet H, Ndiath MO, Sokhna C, Parola P, Raoult D. Emerg Infect Dis. 2013 Nov;19(11):1775-83.

This study aimed to compare the epidemiology of Rickettsia felis infection and malaria in France, North Africa, and sub-Saharan Africa and to identify a common vector. Blood specimens from 3,122 febrile patients and from 500 nonfebrile persons were analyzed for R. felis and Plasmodium spp. We observed a significant linear trend (p<0.0001) of increasing risk for R. felis infection. The risks were lowest in France, Tunisia, and Algeria (1%), and highest in rural Senegal (15%). Co-infections with R. felis and Plasmodium spp. and occurrences of R. felis relapses or reinfections were identified. This study demonstrates a correlation between malaria and R. felis infection regarding geographic distribution, seasonality, asymptomatic infections, and a potential vector. R. felis infection should© be bysuspected author in these geographical areas where malaria is endemic. Doxycycline chemoprophylaxis against malaria in travelers to sub-Saharan Africa also protects against rickettsioses; thus, empirical treatmentESCMID strategies for Onlinefebrile illness forLecture travelers and residents Library in sub-Saharan Africa may require reevaluation.

21 Origin of the human malaria parasite Plasmodium falciparum in gorillas. Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, Keele BF, Ndjango JB, Sanz CM, Morgan DB, Locatelli S, Gonder MK, Kranzusch PJ, Walsh PD, Delaporte E, Mpoudi-Ngole E, Georgiev AV, Muller MN, Shaw GM, Peeters M, Sharp PM, Rayner JC, Hahn BH. Nature. 2010 Sep 23;467(7314):420-5.

© by author Molecular evidence for the presence of Rickettsia felis in the feces of wild-living African apes. ESCMIDAK.Keita,C.Ahuka Online-Mundeke,P.Ratmanov,C.Butel,A.Ayouba,B Lecture Library-I Inogwabini,JJ.MuyembeTamfum,E.MpoudiNgole,E.Delaporte,M.Peeters,F.Fenollar,D.Raoult. PlosOne in press.

22 FASTIDIOUS BACTERIA

• Rickettsioses

• T.whipplei  • Bartonella and TB

23 GROWING THE BACTERIA

 Je Ne Sais Pas protocol (I dont know what I am growing) - Inoculate - 1 ml/blood - crushed biopsy sample - Shells vials seeded with human cell lines - fibroblasts (HEL) - endothelial cells (ECV) - Every week observation of cytopathic effect, and different staining (Gimenez,© by Gram,author Giemsa), for 6 months - Generated in 1992 for blood culture negative, IE ESCMIDRaoult D, et al. Cultivation Online of the bacillus Lecture of Whipple's disease. Library N Engl J Med. 2000;342(9):620-5. Gouriet F, et al. Use of shell-vial cell culture assay for isolation of bacteria from clinical specimens: 13 years of experience. J Clin Microbiol. 2005;43:4993-5002. 24 GROWING THE BACTERIA

 We did not work on W.D.  1999: from Tom Marrie, Canada  42 year old man  IQ of 54, encephalitis during childhood  Rheumatic fever ?  1997: severe aortic insufficiency cardiac insufficiency © by author  May 1998: vegetation on mitral valve at the echography  NoESCMID fever, no diarrhea Online Lecture Library  Surgery of the valve  Valve inoculation PAS staining Raoult D, et al. Cultivation of the bacillus of Whipple's disease. N Engl J Med. 2000;342(9):620-5. 25 © by author ESCMID Online Lecture Library Fenollar F, Puéchal X, Raoult D. Whipple’s disease. New Engl J Med. 2007; 356:5566

26 ANTIBODIES

Monoclonal antibody which recognizes formalin fixed T. whipplei in Dr Whipple’s patient (1907!)

© by author  100 years later ESCMID Online Lecture Library Immunodetection of Tropheryma whipplei in intestinal tissues from Dr. Whipple's 1907 patient. Dumler JS, Baisden BL, Yardley JH, Raoult D. N Engl J Med. 2003 Apr 3;348(14):1411-2

27 Bentley SD, Maiwald M, Murphy LD, Pallen MJ, Yeats CA, Dover LG, Norbertczak HT, Besra GS, Quail MA, Harris DE, von Herbay A, Goble A, Rutter S, Squares R, Squares S, Barrell BG, Parkhill J, Relman DA. Sequencing and analysis of the genome of the Whipple's disease bacterium Tropheryma whipplei. : Lancet. 2003 ;361(9358):637-44.

© by authorRaoult D, Ogata H, Audic S, Robert C, Suhre K, Drancourt M, Claverie JM. Tropheryma whipplei twist: a human pathogenic actinobacteria with a reduced genome. ESCMID Online LectureGenome Res. Library 2003 ;13(8):1800-9.

28 Renesto P, et al. Genome-based design of a cell-free culture medium© by author for Tropheryma whipplei. LancetESCMID 2003;362(9382):447- Online9. Lecture Library

29 -Fenollar F, Laouira S, Lepidi H, Rolain JM, Raoult D. Value of T. whipplei Quantitative PCR assay for the Diagnosis of Whipple Disease. Clin Infect Dis 2008;47:659-7. Analysis of 4,118 samples in our center using repeat-PCR including= 299 stools, 432 saliva, and 380 duodenal biopsies

. Asymptomatic carriage of T. whipplei on stools : General population in France: 2-4% Underground sewer workers in France: approximately 12%

. Asymptomatic carriage of T. whipplei in saliva : General population in France: 0.2% Sewer workers© in by France: author approximately 2.2% Only observed for people with a carriage in stools

ESCMID Online Lecture Library . Asymptomatic carriage of T. whipplei on duodenal biopsies: 0.25%

30 © by author ESCMID Online Lecture Library

31 © by2011 authorMay;66(5):1188-9. Epub 2011 Mar 8.

TROPHERYMA WHIPPLEI PNEUMONIA IN A PATIENT

ESCMID OnlineWITH HIV-2 Lecture INFECTION Library Andreas STEIN, Mahammadou DOUTCHI, Florence FENOLLAR, Didier RAOULT. Am J Respir Crit Care Med. 2013

32 We isolated 41 strains of T.Whipplei

- Whipple’s disease:

1907: Metabolic disease

1991: Infectious disease caused by a rare bacterium

2010: Infection caused ©by aby frequent author bacterium, T. whipplei

- Main hypothesis: ESCMID Online Lecture Library A specific immunodeficiency to a common bacterium

33 FASTIDIOUS BACTERIA

• Rickettsioses

• T.whipplei

• Bartonella and TB  © by author • Fastidious amoebal pathogens ESCMID Online Lecture Library • Future culture strategy

34 LONG INCUBATION OF BLOOD AGAR AND BARTONELLA

We isolated 26 species and 640 strains of Bartonella

Blood agar and Mycobacterium tuberculosis: the end of a dogma. © by authorDrancourt M, Carrieri P, Gévaudan MJ, Raoult D. J Clin Microbiol. 2003 ESCMID Online Lecture Apr;41(4):1710-Library 1.

35 RAPID CULTURE OF MYCOBACTERIUM TUBERCULOSIS

Pr DRANCOURT • Optimized composition of new media for rapid culture of M. tuberculosis (MOD4). • Optimized conditions for growth of M. tuberculosis : – Microaerophilic atmosphere © by author – pH 6.8 –ESCMIDT°C = 37°C Online Lecture Library • Microcolonies detection by Autofluorescence

36 Dramatic reduction of culture time of Mycobacterium tuberculosis. Ghodbane R, Raoult D, Drancourt M. Sci Rep. 2014 Feb 28;4:4236.

Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic- acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture. © by author ESCMID Online Lecture Library

37 942 +/- 51 µm 596 +/- 57 µm 2 mm

2 mm

272.9 +/- 19 µm

Naked eyes colony Binocular observation

Micro- colony © by author 6.4 +/- 3.5 105 mycobacteria

ESCMID Online6.6 +/- 1 104 mycobacteria Lecture Library

48 h for culture and antibiotic susceptibility

7.88 +/- 5.28 103 mycobacteria 38 FASTIDIOUS BACTERIA

• Rickettsioses

• T.whipplei

• Bartonella and TB

39 LLAP – We tested by serology all these LLAP with TJ. Marrie on a collection of sera from 255 Canadian patients with pneumonia L. lytica L. drancourtii and found that sera reacted with many of these LLAP © by author – L. drancourtii seroprevalenceESCMID (4.3%) Online in Lecture Library pneumonia was higher than in 511 controls  Marrie TJ, Raoult D, La Scola B, Birtles RJ, de Carolis E; Canadian Community-Acquired Pneumonia Study Group. Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia. Emerg Infect Dis. 2001;7(6):1026-9. 40 Parachlamydia

– By 16S rRNA sequencing – Surprisingly one of the LLAP isolate was a Chlamydia like agent – Named since Parachlamydia acanthamoebae that cause rare cases of pneumonia © by author

 Birtles RJ,ESCMID Rowbotham TJ, Storey C, Marrie TJ,Online Raoult D. Chlamydia-like Lecture obligate parasite of free-living amoebae.Library Lancet. 1997;349:925 -6.  Greub G, Berger P, Papazian L, Raoult D.Parachlamydiaceae as rare agents of pneumonia.Emerg Infect Dis. 2003; 9(6):755-6.

41 FASTIDIOUS BACTERIA

• Rickettsioses

• T.whipplei

• Bartonella and TB

 42 Cell extract-containing medium for culture of intracellular fastidious bacteria. Singh S, Kowalczewska M, Edouard S, Eldin C, Perreal C, Weber P, Azza S, Raoult D. J Clin Microbiol. 2013 Aug;51(8):2599-607. doi: 10.1128/JCM.00719-13. Epub 2013 Jun 5.

The culture of fastidious microorganisms is a critical step in infectious disease studies. We evaluate this empirical medium containing eukaryotic cell extract for the culture of C. burnetii as a proof-of-concept experiment. We highlight exponential growth of several strains, including the C. burnetii Nine Mile phase I and phase II and C. burnetii isolates from humans and animals. Low oxygen tension, the presence of small hydrophilic molecules and short peptides were critical to facilitate growth. Furthermore, antigenicity was conserved, revealing the potential for this culture medium to be used in diagnostic tests and in the elaboration of vaccines for C.© burnetii. by Weauthor were also able to grow most tested intracellular and fastidious bacteria, including Tropheryma whipplei, Mycobacterium bovis, Leptospira sp, Borrelia sp and most putative bioterrorism agents.ESCMID In contrast, Rickettsia Online africae and Lecture Legionella sp . Librarywere unable to grow in this medium.The versatility of this medium should encourage its use as replacement for the cell-based culture systems currently used for a number of facultative and putative intracellular bacteria. 43 Next target:

ANAEROBES

44 © by author ESCMID Online Lecture Library

45 NEW BACTERIA

1- 16SrDNA

2- Intracellular and fastidious

3- MALDI-TOF  © by author 4- Culturomics ESCMID Online Lecture Library

46 Ongoing revolution in bacteriology: routine identification of bacteria by matrix- assisted laser desorption ionization time-of-flight mass spectrometry Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, Raoult D. Clin Infect Dis. 2009 Aug 15;49(4):543-51

Abstract Since > 300 publications BACKGROUND: Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. METHODS: We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. RESULTS: Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of© 1 isolate by was 6author minutes for an estimated 22%-32% cost of current methods of identification. CONCLUSIONS: MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising > or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace GramESCMID staining and biochemical Online identification in the near Lecture future. Library

Direct identification of bacteria in positive blood culture bottles by matrix- assisted laser desorption ionisation time-of-flight mass spectrometry. La Scola B, Raoult D.PLoS One. 2009 Nov 25;4(11):e8041 47 IDENTIFICATION

Timetables of all the bacterial identification techniques used throughout the 10-year study In La Timone Hospital

ESCMID Online Lecture Library

16S rDNA/ Rpob sequencing 48 Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, Fournier PE, Drancourt M, La Scola B, Raoult D. J Clin Microbiol. 2013 Jul;51(7):2182-94. During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold© and theby cost authorby 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases.ESCMID The ability to rapidly identifyOnline bacterial species Lecture rarely described Libraryas pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

49 Evolution of the number of clinical isolates identified in our clinical laboratory from 2002 to 2012

600 species

© by author ESCMID Online Lecture Library Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, Fournier PE, Drancourt M, La Scola B, Raoult D. J Clin Microbiol. 2013 Jul;51(7):2182-94. 50 NEW BACTERIA

1- 16SrDNA

2- Intracellular and fastidious

3- MALDI-TOF © by author 4- Culturomics ESCMID Online Lecture Library

51 Background : Gut microbiotal analysis tools

Culture : first method to characterize bacterial ecosystems

• 300 species in 33 persons, Finegold, Am J Clin Nutr, 1974 • 113 species in 20 persons, Moore, Appl Microb, 1974

1% of bacteria can be grown easily Pyrosequencing et metagenomic

• 130 à 200 phylotypes, Eckburg, Science, 2005 • >157 phylotypes, Qin, Nature, 2010 © by author

ESCMID Online Lecture Library

80% of bacteria uncultured

52 « Depth bias »

METAGENOMICS 53 Microbial culturomics: paradigm shift in the human gut microbiome study. Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, Bittar F, Fournous G, Gimenez G, Maraninchi M, Trape JF, Koonin EV, La Scola B, Raoult D. Clin Microbiol Infect. 2012 Sep 4. doi: 10.1111/1469-0691.12023

Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular© studies.by author Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 ofESCMID which overlapped Online with the microbiome Lecture identified Library by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

54 Identification of bacteria in the human gut by culturomics and metagenomics

ESCMID Online Lecture80% cultured species Library are undetected by molecular technique

55 THE GENOMES OF THE RECORDS

The largest human The largest The largest archea human virus Human bacteria

Methano Microvirga Senegalvirus massiliicoccus 9.3 Mb 0.37 Mb ESCMID2.6 Mb Online Lecture Library

56 Total Culturomics: Samples

Number of Number of colonies % of isolates Sample Origin Clinical context colony identified unidentified Number of New New Culture with MALDI-TOF species Condition Number tested by 16S rRNA sequencing method isolated species genus s 1 Senegal healthy 3000 32 3,20 99 2 0 56 2 Senegal healthy 13000 246 5,68 219 18 3 124 3 France morbid obesity 16500 214 3,89 191 6 2 150 4 France anorexia nervosa 12700 206 4,87 133 9 2 88 5 Daguestan XDR tuberculosis 4000 4 0,30 39 1 0 73 6 France Coxiella burnetii vascular infection 5400 7 in progress 50 0 1 69 7 France ICU 3 months, antibiotics cocktail 11000 22 in progress 104 3 0 54 8 France ICU 10days, imipenem 11200 18 in progress 107 3 0 73 9 France HIV type 1-200 LT-CD4/mm3 10290 49 1,43 122 3 0 70 10 France HIV type 1-2 LT-CD4/mm3 16090 5 0,09 54 0 0 70 11 France HIV type 1-345 LT-CD4/mm3 10630 1 0,03 63 0 0 53 French 12 Polynesia excess weight 21000 20 0,29 139 2 2 74 French 13 Polynesia healthy 10674 0 0,00 37 0 0 42 14 Amazonia healthy 17338 2 0,03 66 0 0 58 15 Amazonia healthy 34700 5 0,04 131 2 0 114 16 India healthy 12500 18 0,43 98 0 0 53 17 Saudia Arabia obesity 1840 in progress in progress 20 0 0 11 18 Saudia Arabia obesity ©23476 by authorin progress in progress 66 5 0 66 19 Saudia Arabia lean 14280 in progress in progress 62 0 0 42 20 Saudia Arabia lean 4356 in progress in progress 40 0 0 10 21 Niger Kwashiorkor 576 in progress in progress 15 0 0 3 22 Niger Marasme 384 in progress in progress 10 0 0 1 23 TouaregESCMID Touareg Online2742 Lecturein progress in Libraryprogress 32 0 0 8 24 Amazonia lucien 1020 in progress in progress 17 0 0 8 25 Saudia Arabia obesity 1152 in progress in progress 19 0 0 8 26-372 Senegal 162/347 patients: diarrheae 2753 32 3,49 189 2 0 9 Necrotizing enterocolitis in preterm 373-387 France neonates 3000 in progress in progress 50 1 0 27

57 TOTAL CULTUROMICS: 1200 29 stool samples

1065

1000

800 727

627 Culturomics : 340 species newly 600 isolated in the human gut (31.9%)

400

200 350

300 287 (45,8%) 0 Species isolated in Before culturomics Total Culturomics human gut study © by author250 200

143 (22,8%) 150 131 (20,9%)

ESCMID Online Lecture100 Library 66 (10,5%)

0 58 Human Gut Human (other sites) First isolation in New species and human genera 1200 1065

1000

800 727 627 438 species not isolated using culturomics concept 600

400

200

0 Species isolated in Before culturomics Total Culturomics human gut study

Proteobacteria Firmicutes Actinobacteria © by author Verrucomicrobia Bacteroidetes Proteobacteria +++ Spirochaetes Firmicutes Fusobacteria ActinobacteriaESCMID Online Lecture LibraryChlamydiae Euryarchaeota Tenericutes Lentisphaerae

59 Total Culturomics: 66 new species and genera (in purple) Firmicutes (40) Actinobacteria (16) Bacteroidetes (7) Proteobacteria (3) Anaerococcus obesiensis Actinomyces grossensis Alistipes marseilloanorexicus Enterobacter massiliensis Anaerococcus senegalensis Actinomyces polynesiense Alistipes obesihominis Herbaspirillum massiliense Anaerosalibacter massiliensis Aeromicrobium massiliense Alistipes senegalensis Microvirga massiliensis Bacillus casamancensis Blastococcus massiliensis Alistipes timonensis Bacillus jeddamassiliensis Brevibacterium senegalense Bacteroides neonatalis Bacillus massilioanorexicus Cellulomonas massiliensis Bacteroides timonensis Bacillus massiliosenegalensis Collinsella massiliensis Butyricimonas massiliensis Bacillus saudimassiliensis Corynebacterium reamasilliensis Bacillus timonensis Corynebacterium saudimassiliensis Brevibacillus massiliensis Enorma massiliensis Clostridium anorexicamassiliensis Enorma timonensis Clostridium anorexicus Nesterenkonia massiliensis Clostridium dakarense Nocardioides massiliensis Clostridium jeddamassiliensis Senegalemassilia anaerobia Clostridium massilioamazoniensis Soleaferrea massiliensis Clostridium polynesiense Streptomyces massiliensis Clostridium saudimassiliensis Clostridium senegalense Dielma fastidiosa Dorea massiliensis Holdemania massiliensis Kallipyga massiliensis Kurthia massiliensis Kurthia senegalensis Kurthia timonensis Megasphaera massiliensis © by author Nosocomiicoccus massiliensis Oceanobacillus massiliensis Paenibacillus antibioticophyla Paenibacillus reamassiliensis Paenibacillus senegalensis ESCMIDPeptoniphilus grossensis Online Lecture Library Peptoniphilus obesi Peptoniphilus senegalensis Peptoniphilus timonensis Polynesia massiliensis Pytheasella massiliensis Ruminococcus massiliensis Stoquefichus massiliensis 60 Timonella senegalensis 1st human 1st case isolation Ref Fastidious bacteria 4 22 Anaerobe Anaerobic bacteria NA 6 2011 16S rRNA 16 11 JCM 2004 16S rRNA NA 18 JCM 2000 Others described at URMITE 2 26 MALDI-TOF 30 0 JCM 2013 Culturomics 131 66 Total 183 149 332 © by author Our Laboratory isolated 16.2% of species isolated ESCMIDfrom humansOnline for Lecture the first time.Library Including 7.5 % species Humans

61 Viruses: Essential Agents of Life by Günther Witzany

p.64 By F.Rohwer 62 Diversity of cultivable methanogenic archaea in the human gut

• Molecular screening (PCR identification)

≈ 850pb (tm=60°C) Sulfo_for-CTGAGACAAGGGCCCAGG (18pb) Sulfo_rev-CACGCGGGCTACAATGGG (18pb) Desulfococcales

≈ 600 pb (tm=60°C) Methanobacteriales_for-AGCAGGCGCGAAACCTCC (18pb) Methanomicrobiales_revCCTGGGAAGTACGGTCGC(18pb)

Methanobacteria ≈ 800pb (tm=60°C)

Methanococci Methanococcales_for-GGGCGCAGCAGGCGCGAA (18pb) © by authorMethanococcales_ rev-GTTAAGTCAGGYAACGAGCGAG (21pb)

≈ 800 pb (tm=58°C) Halo_for-CAGCCGCCGCGGTAATAC(18pb) ESCMID Online LectureHalo_rev-CATGAAGCTGGATTCGGTAG(20pb) Library Halobacteria ≈ 800 pb (tm=60°C) Methanomicrobiales_for-TACGGGTTGTGGGAGCAA (18pb) Methanomicrobia Methanomicrobiales_rev-CTGAGAGGAGGTGCATGG (18pb) 63 A versatile medium for cultivating methanogenic archaea. Saber Khelaifia, Didier Raoult, and Michel Drancourt. PONE-D-13-03759-revised 100 Halorubrum sp. B6 [EF077636] 4 out of 6 found in the Halobacterium noricense [DSM 15987]

gut 3 out of 6 from 100 Methanogenic archaeon clone E18_16S2_Mx-03[FJ752571] humans 1 new species Methanogenic archaeon clone E20_16S1_Mx-04 [FJ752572]

Methanogenic archaeon clone E20_16S2_Mx-02 [FJ752573] 100 98 Methanomassiliicocus luminyensis B-10 [CSUR P135T ] 100 Uncultured archaeon clone MX-01 [EU662200]

90 Methanosaeta concilii [AF414037] Methanosphaera stadtmanae [84488831]

Methanobrevibacter smithii JMR02 [CSUR P1713] 90 Methanobrevibacter smithii [148642060] 100 Methanobrevibacter oralis JMR01 [CSUR P1714] 0.1 ©90 by author Methanobrevibacter oralis [DQ251043]

Methanobrevibacter arboriphilicus [AB065294] ESCMID Online100 Methanobrevibacter Lecture arboriphilicus Library ANOR1 [CSUR P1715 ] Methanobrevibacter millerae ZA-10T [16643]

100 Methanobrevibacter millerae CMR-6 [CSUR P1712]

Euryarchaeote clone LL2-3-6 [FJ458322] From M.DRANCOURT 64 Culture aérobie à 150g/L de NaCl

First halophilic archaea in humans! 65 Viruses: Essential Agents of Life by Günther Witzany

p.64 By F.Rohwer 66 THE FUNGI: 1, 2, 3 … 5.1 MILLION SPECIES?

Meredith Blackwell American Journal of Botany 98(3): 426–438. 2011

ESCMID 330Online biomedical Lecture species Library Numbers of known fungi from the Dictionary of the Fungi (editions 1 – 10, 1950 – 2008). Authors state that the large increase in species numbers in the 10th edition may be infl ated because asexual and sexual forms were counted separately and molecular techniques that distinguish close taxa have been used. 67 Viruses: Essential Agents of Life by Günther Witzany

p.64 By F.Rohwer 68 Science. 2011 Mar 25;331(6024):1513

Expanding universe. The tally of known viruses is exploding, and this graph doesn’t

even include the incredible number that prey on bacteria. 69 PNEUMONIA

– The causative agent of pneumonia unknown in  40% – Free living amoebae in water may select pathogenic agents – We decided to test bacteria associated with amoebae as agent of pneumonia With Prof. Bernard La Scola © by author ESCMID Online Lecture Library

70 Amoeba for© pathogenby author isolation ESCMIDLegionella Online and Lecture Mimivirus Library Raoult D, et al. The 1.2-megabase genome sequence of Mimivirus. Science. U R 2004;306:1344-50. La Scola B, et al. A giant virus in amoebae. Science;299:2033. 71 Mimivirus

 In 1992 Rowbotham working on relationships between amoebae and Legionella isolated a new Gram positive coccus in a cooling tower after a pneumonia outbreak in Bradford, England, using amoebae as a culture system.

 This was considered a LLAP (Legionella Like Amoebal Pathogen) with© others by author

 TheESCMID LLAP collection Online was bring in Lecture Library Marseilles-France by Richard Birtles an English post-doc.

72 The 1.2-megabase genome sequence of Mimivirus Didier Raoult et al. Science. 2004 Nov 19;306(5700):1344-50.

Map of the Mimivirus chromosome. The predicted protein coding sequences are shown on both strands and colored according to the function category of their matching COG. Genes with no COG match are shown in gray. Abbreviations for the COG functional categories are as follows: E, amino acid transport and metabolism; F, nucleotide transport and metabolism; J, translation; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane biogenesis; N, cell motility; O, posttranslational © by author modification, protein turnover, and chaperones; Q, secondary metabolites biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknownESCMID. Small red arrows Online Lecture Library indicate the location and orientation of tRNAs. The A+C excess profile is shown on the innermost circle, exhibiting a peak around position 380,000 (2) (fig. S1). U R73 Giant viruses have been forgotten when assessing viromes through metagenomics

Viruses of a size < that of •Other viruses of a size < the filter’s pores that of the filter’s pores

Since Ivanovsky’s experiment, viruses were not considered as belonging to the world of visible particles by light microscopy (Raoult et al., CID 2007). This paradigm has led to a considerable bias in recent studies of viral metagenomics.ESCMID Indeed, these studies Online have fully considered Lecture the dogma regarding Library the small size of viruses through filtering the samples prior to their analysis (Edwards and Rohwer, Nat Rev Microbiol 2005 3 504; Angly et al. Plos Comput Biol 2009 e1000593 ; Thurber et al. Nature Protocols 2009). Such procedures are inevitably preventing the detection of viruses larger than the pores of the filters used, i.e. 0.2 to 0.45 µm (Zhang et al. Plos Biology 2006; Dinsdale et al. Nature 2008; 452 Willner and al, PLoS One 2009 4 (10); Thurber et al. Nature Protocols 2009).

74 THE VIROPHAGE AS A UNIQUE PARASITE OF THE GIANT MIMIVIRUS

BL Scola et al. Nature 000, 1-5 (2008) doi:10.1038/nature07218

Different morphological aspects of mamavirus and Sputnik. 75 First isolation of Mimivirus in a patient with pneumonia. Saadi H, Pagnier I, Colson P, Cherif JK, Beji M, Boughalmi M, Azza S, Armstrong N, Robert C, Fournous G, La Scola B, Raoult D. Clin Infect Dis. 2013 Aug;57(4):e127-34.

A B

C Figure 1A-B. Visualization of lysis plaque, on agar plate coated with A. polyphaga, due to Mimivirus (A) and negative control (B). Figure 1C. LBA111 virus staining with ruthenium red. 76 © by author ESCMID Online Lecture Library Emerg Infect Dis,2005;11:449452

77 Acanthamoeba polyphaga mimivirus Virophage Seroconversion in Travelers Returning from Laos Parola P, Renvoisé A, Botelho-Nevers E, La Scola B, Desnues C, Raoult D. Emerg Infect Dis. 2012 Sep;18(9):1500-2.

Conclusion: We look for human pathogens and find human pathogens 78 AMEBA-ASSOCIATED KERATITIS, FRANCE. Cohen G, Hoffart L, La Scola B, Raoult D, Drancourt M. Emerg Infect Dis. 2011 Jul;17(7):1306-8.

79 Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Boyer M, Yutin N, Pagnier I, Barrassi L, Fournous G, Espinosa L, Robert C, Azza S, Sun S, Rossmann MG, Suzan- Monti M, La Scola B, Koonin EV, Raoult D. Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21848-53.

Abstract Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of "Marseille" virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes.© by Theauthor genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are "melting pots" of microbial evolution where diverse forms emerge, includingESCMID giant viruses with Online complex gene Lecturerepertoires of various Library origins.

80 Immunofluorescence using Marseillevirus antibodies on viral particles purified from serum 27725

Cytoblock Gelose inclusion of purified viral particles

Paraffin inclusion Thin slice section Giant virus in the blood Green : MarV polyclonal Ab Red : DAPI 100X Marseillevirus-like virus recovered© by from bloodauthor donated by asymptomatic humans. Popgeorgiev N, Boyer M, Fancello L, Monteil S, Robert C, Rivet R, Nappez C, Azza S, Chiaroni J, Raoult D, Desnues C. J Infect Dis. 2013 Oct 1;208(7):1042-50. ESCMIDMarseillevirus prevalence Online in multitransfused Lecture patients suggests bloodLibrary transmission. Popgeorgiev N, Colson P, Thuret I, Chiarioni J, Gallian P, Raoult D, Desnues C. J Clin Virol. 2013 Dec;58(4):722-5

Marseillevirus adenitis in an 11-month-old child. Popgeorgiev N, Michel G, Lepidi H, Raoult D, Desnues C. J Clin Microbiol. 2013 Dec;51(12):4102-5. 81 Marseillevirus adenitis in an 11-month-old child. Popgeorgiev N, Michel G, Lepidi H, Raoult D, Desnues C. J Clin Microbiol. 2013 Dec;51(12):4102-5.

A Marseillevirus (giant virus of amoeba) has been found in the blood and stool samples of individuals who otherwise appear to be healthy. During an attempt to define a serological cutoff for Marseillevirus by enzyme-linked immunosorbent assay (ELISA) in children, we serendipitously detected high antibody responses to Marseillevirus in an 11-month-old boy suffering from adenitis. Marseillevirus DNA was then found in his blood using PCR and with a unique sequence. We identified Marseillevirus in a lymph node using fluorescence in situ hybridization (FISH) and immunohistochemistry, and the lymph node was removed surgically. The child was declared to be cured 1 year later. We conclude that adenitis during early childhood may be caused by Marseillevirus. © by author ESCMID Online Lecture Library

82 Marseillevirus prevalence in multitransfused patients suggests blood transmission. Popgeorgiev N, Colson P, Thuret I, Chiarioni J, Gallian P, Raoult D, Desnues C. J Clin Virol. 2013 Dec;58(4):722-5.

BACKGROUND: Emerging viral infections in humans are appearing at an increasing rate. Recently, we identified a new Marseillevirus, named Giant Blood Marseillevirus (GBM), by performing viral metagenomics on asymptomatic blood donors. OBJECTIVES: To study and compare the prevalence of Marseillevirus between asymptomatic blood donors and thalassemia patients. DESIGN: Here, we present a combined molecular and serological study on 174 asymptomatic blood donors and 22 patients with thalassemia who receive repeated blood transfusions to estimate the prevalence of Marseillevirus in these two populations. RESULTS: We identified Marseillevirus genomic DNA in 4% of donors, whereas 9.1% of the thalassemia patients were positive© for thisby virus. author Moreover, IgG seropositivity was detected in 22.7% of patients in the thalassemia group, whereas this seropositivity was observed in 12.6% of the blood donor population. CONCLUSION: TheseESCMID results suggest that MarseillevirusOnline infection Lecture is not rare in healthyLibrary persons and may be transmitted by transfusion, thus raising speculation regarding the long-term consequences of this viral infection, particularly in patients requiring repeated blood transfusions.

83 Application to Hartmanella vermiformis

 Inoculation of sewage samples on Hartmanella vermiformis using High Throughput Cytometry on 90 samples/ 6 detected lysis

 Detection in 4 of a « new giant » virus family: provisionnal Faustovirus

84 Faustovirus DNApol

85 Phylogenetic and Phyletic Studies of Informational Genes in Genomes Highlight Existence of a 4th Domain of Life Including Giant Viruses Mickaël Boyer., Mohammed-Amine Madoui., Gregory Gimenez, Bernard La Scola, Didier Raoult

PLoS One. 2010 Dec 2;5(12):e15530 Abstract: The discovery of Mimivirus, with its very large genome content, made4? it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation,© weby found author in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supportsESCMID existence of a cladeOnline containing LectureNCLDVs clearly Librarydistinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.

Phylogenetic tree of the RNA polymerase II beta subunit 87 Gene content analysis of informational genes

Hierarchical clustering of Eukarya (blue), Bacteria (purple), Archaea (green) and NCLDVs (red) by phyletic pattern 88 TRUC or the need for a new microbial classification. Raoult D.Intervirology. 2013;56(6):349-53.

Exceptions to the eukaryote/prokaryote and virus/microbe definitions.

Hierarchical clustering of Eukarya (blue), Bacteria (purple), Archaea (green), and NCLDVs (red) by phyletic pattern. 89 4 CONCLUSION

Finally defining TRUC (« thing » in French) with 4 parts, bacteria, eukarya, archae and megavirales. As an acronym of© by author Things Resisting Uncomplete Classification (includingESCMID Megavirales Online Lecture). Library

90 Viruses: Essential Agents of Life by Günther Witzany

p.64 By F.Rohwer 91 • TABASCO, Hot Ketchup and human stool • Positive detection by RT-PCR

9/ TEM ~ 10 ml

• Is it multiplying in humans? © by author • Is it causing something?ESCMID Online Lecture Library P.Colson.Pepper Mild Mottle Virus, a Plant Virus Associated with Specific Immune Responses, Fever, Abdominal Pains, and Pruritus in Humans.Plos one.2010

92 UR Pepper mild mottle virus, a plant virus associated with specific immune responses, Fever, abdominal pains, and pruritus in humans. Colson P, Richet H, Desnues C, Balique F, Moal V, Grob JJ, Berbis P, Lecoq H, Harlé JR, Berland Y, Raoult D. PLoS One. 2010 Apr 6;5(4):e10041.

93 4- Virulence factor

94 • Shigella as a paradigm • Less metabolic activity, genetically considered the same species than E. coli • Described "plasmid of virulence" • Motility quoted as a virulence factor

Goldberg MB, Theriot JA, Sansonetti PJ. Regulation of surface presentation of IcsA, a Shigella protein essential to intracellular movement and spread, is growth phase dependent.© by Infect author Immun. 1994;62(12):5664-8.

ESCMIDHale TL, Sansonetti PJ,Online Schad PA, AustinLecture S, Formal SB.Library Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli. Infect Immun. 1983 ;40(1):340-50.

95 Shigella

• ↓ metabolic activity • ↓ genome size • ↓ LDC © by author Maurelli AT, Fernández RE, Bloch CA, Rode CK, Fasano A. "Black holes" and bacterial pathogenicity: a large genomic deletion that ESCMIDenhances the virulence Online of Shigella Lecture spp. and enteroinvasive Library Escherichia coli. Proc Natl Acad Sci U S A. 1998;95(7):3943-8. U R 96 Facteur de virulence Rickettsia prowazekii

• The big killer versus R. conorii less pathogenic – Motility – genome comparison – Rick A – Motility in Shigella and Listeria are considered virulence factors ! © byRickettsia author conorii : Actin-based motility

ESCMID Online Lecture Library

Science 2001; 293: 2093-2098.

98 R. prowazekii is a subset of R. conorii 767 11 44 552 R. conorii

R. prowazekii 767 23 14 30

R. conorii 1374 ORFs R. prowazekii 834 ORFs

804 FunctionsESCMID for 104 Online Lecture FunctionsLibrary for 3 ORFs ORFs

More non coding DNA for R. prowazekii 99 Free-living bacterial community

264 lost COGs

Facultative host-associated Bacteria GS 2.92 ± 1.6 GC 43.0 ± 11.7 Op 3.36 ± 2.6 TR 65.7 ± 56.0

615 lost COGs

Merhej V, et al. ESCMID Online Lecture LibraryBiol Direct . 2009 Apr 10;4:13.

101 Number of protein toxin in 24 studied species

All of the « bad bugs » have more toxins than « controls » 102 Significant difference p value = 0.0470 but no « virulence factors » 5- Phylogeny et Taxonomy

103 DOGMA - Phylogeny reflects taxonomy.

- A cut off of 98.7% of simility in 16Sr DNA reflects a new species.

FACTS - But there are recombinations of 16S (Bartonella henselae), bacteria whose 2 copies of 16S have a divergence higher than 1,3%.

- This is not adapted to the© definitionby author of the species: there are 9000 bacterial species validated against 1,5 million eucaryotes.

- TheESCMID contents genomic Online are not Lectureenvisaged by Library phylogeny.

104 R. conorii R. prowazekii M. tuberculosis M. leprae 10.000 years Orientia spp. Chimpanzee Bonobo Human 10.000 Gorilla 400.000 years 400.000

0.02% 1 million years 1 million Apes/ monkeys 5 million

mammals

50 million years 1-2% Rickettsia spp. 50 million

vertebrates

500 million 500 million

multicellular eukaryotes

2 billion years protoeukaryotes 2 billion ESCMID40% Onlinealpha - proteoabacteria Lecture Library 50% Mycobacteria 62% Actinobacteria

4 billion years 80% 4 billion

TIME SCALE 16S rRNA TIME SCALE divergence Figure 4 105 ORFans and new genes current creation of genes and species

• Arbre Darwin

It is a biblical concept 106 Genealogy of R. felis genes

107 Rhizome (or mycelium) of life

D.Raoult.The post-Darwinist rhizome of life Lancet. 2010 Jan 9;375(9709):104-5.

109 DELICATE INTELLECT HARDENED INTELLECT Rationalist (which is based on principles) Empiricist (which is based on facts) Intellectualist Sensationalist Idealist Materialist Optimistic Pessimism Religious Irreligious Supporter of free will Fatalist Monist Pluralist Dogmatic Skeptical Culture for discovery!

 P.90  Thank E.Bapteste 110