Diapositive 1

29.04.2013 ESCMID-BERLIN «Culturomics»

We received

-220,000 samples for culture (bactéria, fungi, viruses)

- 200,000 PCR were performed

- 115,000 serological testing were tested © by author Real-time laboratory surveillance of sexually-transmissible infections in Marseille

University hospitals reveals rise of gonorrhea, syphilis and HIV seroconversions in 2012. PhilippeESCMID Colson1,2 , Frédérique Online Gouriet1,2 Lecture , Sékéné Badiaga 2,3Library, Catherine Tamalet 1,2, Andreas Stein2,4, Didier Raoult1,2 *. Eurosurveillance 2013

2 Culture has been negleted in clinical microbiology, very few new media have been recently very few introduced but it is still central for:

 Causality

 Suceptibility testing

 Genome sequencing© by author

ESCMID Pathophysiology Online Lecture Library

3 NEW IDENTIFICATIONS

Helicobacter pylori • Peptic ulcer disease • Cancer of the stomach, grown in 1983 © by author

ESCMIDSeen sinceOnline the Lecture 19th century Library

4 © by author ESCMID Online Lecture Library

5 PROGRESSES MADE IN MICROBIOLOGY FROM 1979 TO 2012 THANKS TO THE DEVELOPMENT OF NEW TECHNOLOGIES

a) the ESCMIDleft ordinate axis refers toOnline the cumulative numbers Lecture of bacterial species Library with validly published names (green curve); the right ordinate axis refers to the cumulative numbers of sequenced bacterial genomes (purple) and sequenced viral genomes (blue);

6 © by author b) the left ordinate axis refers to the numbers of articles containing “metagenome” as keyword (red) and of articles containing “microbiota” as keyword (grey); the right ordinate axisESCMID refers to the numbers Online of articles containing Lecture “MALDI-TOF” andLibrary “clinical microbiology” as keywords (orange).

7 Number of species contained by each database 1000 900 800 700 600 500 400 300 200 100 0

Progression 2500 © by author 2000 1500 1000 500 ESCMID Online Lecture Library 0

8 Viruses: Essential Agents of Life by Günther Witzany

p.64 By F.Rohwer 9 Research strategy for identification and detection of new pathogens

Culture-dependent Culture-independent

Genome ESCMID Online Lecture Library sequencing

Identification Detection 10 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

11 Anaerobic bacterial culture: litterature

12 Anaerobic bacterial culture: litterature

KH2Po4 NaCl MgSo4 CaCl2 (Nh 4)2So4 MnCl2 CoCl2 © by author ESCMID Online Lecture Library

13 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP Bacteria (BCNEI) 210 Anaerobes techniques Culture AMIBES AMIBES AMIBES axenic BK (Afipia) (virus) intracellular Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia

Kaeberlein Simulting environment 14 Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture. Marrero M, Raoult D. Am J Trop Med Hyg. 1989 Feb;40(2):197-9.

The shell vial technique for isolation of cytomegalovirus was adapted to detect Rickettsia conorii in blood culture using human fibroblast monolayers. The inoculation was performed with low speed centrifugation and the rickettsiae demonstrated by immunofluorescence 24-120 hr after inoculation. R.conorii was identified in 11 of 13 patients with Mediterranean spotted fever in 48-72 hr.

15 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. 16  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 PAS staining  Valve inoculation Raoult D, et al. Cultivation of the bacillus of 17 Whipple's disease. N Engl J Med. 2000;342(9):620-5. 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 authorC)

 And we used XTC2 cells obtained from Xenopus growing at 28ESCMIDC 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 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Axenic extract Shellvial Shellvial XTC medium BK a special medium TW BK and blood media

JNSP Bacteria (BCNEI) 210 Anaerobes techniques Culture AMOEBA AMOEBA AMOEBA axenic BK (Afipia) (virus) intracellular Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia © by author T.v er a ENVIRONMENT Rappe Culture Epstein ESCMIDP.ubique Online in sea Lectureshort Library water peptide

Kaeberlein Simulting environment 19

Pneumonia

– The causative agent of pneumonia unknown in > 40% – Free living amoebae in water may select pathogenic agents © by author – We decided to test bacteriaESCMID associated Online with Lecture Library amoebae as agent of pneumonia 20 Free-living amoebae Phagocytic protists and wild predating Amoebae feed bacteria

21 These resisting bacteria are prepared to resist U macrophages and do the opposite of filtration R U Amoeba phagocyte everything! R

22 Key dates for the role of amoeba

Date First authors 1978 - Amoebae and Mycobacteria: Krishnan-Prasad 1979 - Amoebae and Cryptococcus neoformans Bunting 1980 - Amoebae and Legionella Rowbotham 1981 - Amoebae and Enterovirus Danes 1986 - Role of amoebae as virulence selecting agent Rowbotham 1988 - Role of amoeba as a chlorination protective agent King 1995 - Role of amoeba© in modulatingby author bacterial antibiotic susceptibility Barker 1997ESCMID - Parachlamydia Online as an emerging Lecture pathogen Library Birtles 2003 - Mimivirus the giant intra amoebal virus La Scola

 Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev. 2004 ;17(2):413-33. 23 Microorganisms INTRODUCTION...... 413 resistant to free-living FREE-LIVING AMOEBAE AS A TOOL FOR ISOLATION OF AMOEBA-RESISTANT amoebae. INTRACELLULAR MICROORGANISMS...... 415 Culture of Amoebae for Detecting ARB ...... 415 Greub G, Raoult D. Practical Use of Amoebae for ARB Culture...... 417 Clin Microbiol Rev. 2004 AMOEBA-RESISTANT MICROORGANISMS...... 419 Holosporaceae ...... 419 Apr;17(2):413-33. Bradyrhizobiaceae...... 420 Review. Legionellaceae ...... 420 Legionella pneumophila...... 420 Legionella anisa and other Legionella spp...... 421 Legionella-like amoebal pathogens...... 421 Pseudomonaceae ...... 422 Parachlamydiaceae ...... 422 Mycobacteriaceae ...... 423 Mimivirus...... 423 Enterovirus ...... 424 Other Endosymbionts of Free-Living Amoebae...... 424 Rickettsia-like endosymbionts ...... 424 Members of the Cytophaga-Flavobacterium-Bacteroides phylum...... 424 proteobacteria naturally infecting free-living amoebae...... 424 Other Microorganisms Shown In Vitro To Resist Destruction by Free-Living Amoebae...... 424 Burkholderiaceae ...... 425 Coxiella burnetii...... 425 Francisella tularensis...... 425 Enterobacteriaceae ...... 425 Vibrionaceae...... 426© by author Listeria monocytogenes ...... 426 Helicobacter pylori ...... 426 Mobiluncus curtisii ...... 426 Cryptococcus neoformans ...... 426 ESCMIDMicroorganisms Online Recovered Using Lecture Amoebal Coculture ...... Library 426 FREE-LIVING AMOEBAE AS A RESERVOIR OF AMOEBA-RESISTANT MICROORGANISMS...... 426 TRANSMISSION OF AMOEBA-RESISTANT MICROORGANISMS...... 427 FREE-LIVING AMOEBAE AS AN EVOLUTIONARY CRIB ...... 427 Induction of Virulence Traits...... 427 Adaptation to Macrophages ...... 428 CONCLUSION...... 429 REFERENCES ...... 429 24 Amoeba as Reservoir of Known Pathogens - Legionella sp. 1980 Key dates - Listeria monocytogenes 1990 - Vibrio cholerae 1992 - Ehrlichia-like 1995 - Rickettsia-like 1997 - Ralstonia picketti 1997 - Chlamydia pneumoniae 1997 - Mycobacterium sp. © by author1998 - Burkholderia sp. 2000 - CoxiellaESCMID burnetii Online Lecture2001 Library - Coxiella burnetii in Pseudomonas aeruginosa 2002 amoebae. La Scola, - Francisella tularensis 2003 CMI, 2001 25

Amoeba as a tool for isolation of bacteria for known bacteria • First description by T. Rowbotham – Shell vial system – Well adapted to single samples • Allow to isolate bacteria in contaminated samples • Isolation of L. pneumophila • From sputum and stool samples • When bacteria grow only in amoeba • Named Legionella-like amoebal pathogen (LLAP) Modified in our laboratory © by author

 La Scola B, Mezi L, Weiller PJ, Raoult D. Isolation of Legionella anisa using an ESCMID Onlineamo ebicLecture coculture procedure. J Clin Microbiol.Library 2001 ;39(1):365-6.

26 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-li ke 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.

27 Amoeba as a bacteriological tool for new pathogens

New alpha proteobacteria • facultative – Af ipia birgiae – Afipia massiliae – Bosea massiliensis – Bosea eneae – Bosea vestrisii – Rhodobacter massiliensis – Rhizobium massiliae – Rosomonas massiliae© by author

ESCMIDOther Online gram negative Lecture bacteria Library

– Amoebinatus massiliae – Chryseobacterium massiliae 28

AMOEBAL COCULTURE OF "MYCOBACTERIUM MASSILIENSE" SP. NOV. FROM THE SPUTUM OF A PATIENT WITH HEMOPTOIC PNEUMONIA. Adékambi T, Reynaud-Gaubert M, Greub G, Gevaudan MJ, La Scola B, Raoult D, Drancourt M. J Clin Microbiol. 2004 Dec;42(12):5493-501. A nonphotochromogenic, rapidly growing Mycobacterium strain was isolated in pure culture from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia by using axenic media and an amoebal coculture system. Both isolates grew in less than 7 days at 24 to 37 degrees C with an optimal growth temperature of 30 degrees C. The isolates exhibited biochemical and antimicrobial susceptibility profiles overlapping those of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium immunogenum, indicating that they belonged to M. chelonae-M. abscessus group. They differed from M. abscessus in beta-galactosidase, beta-N-acetyl-beta-glucosaminidase, and beta- glucuronidase activities and by the lack of nitrate reductase and indole production activities, as well as in their in vitro susceptibilities to minocycline and doxycycline. These isolates and M. abscessus differed from M. chelonae and M. immunogenum by exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that of M. abscessus and >99.6% similarity with those of M. chelonae and M. immunogenum. Further molecular investigations showed that partial hsp65 and sodA gene sequences differed from that of M. abscessus by five and three positions over 441 bp, respectively. Partial rpoB and recA gene sequence analyses showed 96 and 98% similarities with M. abscessus, respectively. Similarly, 16S-©23S rRNAby internal author transcribed spacer sequence of the isolates differed from that of M. abscessus by a A-->G substitution at position 60 and a C insertion at position 102. Phenotypic and genotypic features of these two isolates indicated that they were representative of a new mycobacterial species within the M. chelonae-M. abscessus group. Phylogenetic analysis suggestedESCMID that these isolates were Online perhaps recently Lecture derived from M. abscessus Library. We propose the name of "Mycobacterium massiliense" for this new species. The type strain has been deposited in the Collection Institut Pasteur as CIP 108297(T) and in Culture Collection of the University of Goteborg, Goteborg, Sweden, as CCUG 48898(T). 29 Amoeba-resisting bacteria and ventilator-associated pneumonia. La Scola B, Boyadjiev I, Greub G, Khamis A, Martin C, Raoult D. Emerg Infect Dis. 2003 Jul;9(7):815-21.

To evaluate the role of amoeba-associated bacteria as agents of ventilator- associated pneumonia (VAP), we tested the water from an intensive care unit (ICU) every week for 6 months for such bacteria isolates; serum samples and bronchoalveolar lavage samples (BAL) were also obtained from 30 ICU patients. BAL samples were examined for amoeba-associated bacteria DNA by suicide- polymerase chain reaction, and serum samples were tested against ICU amoeba- associated bacteria. A total of 310 amoeba-associated bacteria from 10 species were isolated. Twelve of 30 serum samples seroconverted to one amoeba- associated bacterium isolated in the ICU, mainly Legionella anisa and Bosea massiliensis, the most common isolates from water (p=0.021). Amoeba-associated bacteria DNA was detected in ©BAL bysamples author from two patients whose samples later seroconverted. Seroconversion was significantly associated with VAP and systemic inflammatory response syndrome, especially in patients for whom no etiologic agent was found by usual microbiologic investigations. Amoeba-associated bacteria might be aESCMID cause of VAP in OnlineICUs, especially Lecture when microbiologic Library investigations are negative.

30 Amoeba for© pathogenby author isolation ESCMIDLegionella Online and Lecture Mimivirus Library Raoult D, et al. The 1.2-megabase genome sequence of Mimivirus. Science. 2004;306:1344-50. U La Scola B, et al. A giant virus in amoebae. Science;299:2033. R 31 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 Onlinewas bring in Lecture Library Marseilles-France by Richard Birtles an

English post-doc. U R 32 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP Bacteria (BCNEI) 210 Anaerobes techniques Culture AMIBES AMIBES AMIBES axenic BK (Afipia) (virus) intracellular Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia

Kaeberlein Simulting environment 33 Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Rappé MS, Connon SA, Vergin KL, Giovannoni SJ. Nature. 2002 Aug 8;418(6898):630-3.

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation- independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities© by into author very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in cultureESCMID. Online Lecture Library

34 Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Kaeberlein T, Lewis K, Epstein SS. Science. 2002 May 10;296(5570):1127-9.

The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory. Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives. We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment. Colonies of representative marine organisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. This observation may help explain the nature of microbial uncultivability©. by author ESCMID Online Lecture Library

35 Short peptide induces an "uncultivable" microorganism to grow in vitro. Nichols D, Lewis K, Orjala J, Mo S, Ortenberg R, O'Connor P, Zhao C, Vouros P, Kaeberlein T, Epstein SS. Appl Environ Microbiol. 2008 Aug;74(15):4889-97.

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time©. Growthby authorinduction of MSC 33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and appliedESCMID research. Access toOnline cultures of some Lecture of these species Libraryshould be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.

36 Use of ichip for high-throughput in situ cultivation of "uncultivable" microbial species. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, Kanigan T, Lewis K, Epstein SS. Appl Environ Microbiol. 2010 Apr;76(8):2445-50.

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion© chambers,by author each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significantESCMID phylogenetic Online novelty. The newLecture method allows Library access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.

37 © by author Isolation chip, or ichip, for high-throughput microbial cultivation in situ. (A) Dipping a plate with multiple through-holes into a suspension of mixed environmental cells leads to capturing (on average) a single cell (B). (C) Ichip assembly: membranes cover arrays of through-holes from each sideESCMID; upper and bottom Online plates with matching Lecture holes press Librarythe membranes against the central (loaded) plate. Screws provide sufficient pressure to seal the content of individual through-holes, each becoming a miniature diffusion chamber containing (on average) a single cell. (Artwork by Stacie Bumgarner, Whitehead Institute for Biomedical Sciences, Cambridge, MA.) 38 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP 210 Bacteria Anaerobes (BCNEI) techniques

Culture axenic AMIBES AMIBES AMIBES intracellular BK (Afipia) (virus) Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia T.v er a © by author ENVIRONMENT Rappe Culture Epstein P.ubique in sea short ESCMIDwater Online Lecturepeptide Library

Kaeberlein Simulting environment 39 Axenic culture of fastidious and intracellular bacteria Sudhir Singh*, Carole Eldin*, Malgorzata Kowalczewska, and Didier Raoult Trends in Microbiology. In press

Figure 1. Strategy for the design of Tropheryma ESCMID Online Lecture Librarywhipplei axenic medium. Circular representation of the T. whipplei twist genome, reproduced, with permission, 40 from [29]. Abbreviation: Ab, antibody. Renesto P, et al. Genome-based © by author design of a cell-free culture medium for Tropheryma whipplei. LancetESCMID 2003;362(9382):447 Online-9. Lecture Library

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

Figure 3. Mycobacterium tuberculosis colonies on blood agar tubes. 42 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP 210 Bacteria Anaerobes (BCNEI) techniques

Culture axenic AMIBES AMIBES AMIBES intracellular BK (Afipia) (virus) Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia

Kaeberlein Simulting environment 43 Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era. La Scola B, Fournier PE, Raoult D. Reintroducing Anaerobe. 2011 Jun;17(3):106-12. Anaerobes culture The isolation of anaerobes from patients has declined in recent years, whereas their detection by molecular techniques has increased. In the present work, we analyzed the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing to routine identification of anaerobes in clinical microbiology laboratory. We identified 544 isolates of 79 species by routine culture from deep samples in our hospital. MALDI-TOF MS allowed identification of 332 isolates (61%). The remaining 212 (39%) were identified by 16S rRNA gene sequencing, allowing identification of 202 at the species level. The most common anaerobes were Propionibacterium spp. (12%), Finegoldia magna (4%), Fusobacterium spp. (6%) and Bacteroides spp. (6%). However, among the 79 identified species, seven were new species or genera, including two Prevotella© conceptionensis by author, a species previously detected by our team by amplification and sequencing, five Anaerococcus sp. and one Prevotella sp. Beyond the identification of these new species, we also identified several uncommon or previously not described associations between species and specificESCMID pathologic conditions Online. MALDI -LectureTOF MS-based identification,Library which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology.

44 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP 210 Bacteria Anaerobes (BCNEI) techniques

Culture axenic AMIBES AMIBES AMIBES intracellular BK (Afipia) (virus) Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia © by author T.v er a ENVIRONMENT Rappe Culture Epstein P.ubique in sea short water peptide ESCMID Online Lecture Library Kaeberlein Simulting environment

45 C.burnetii

Figure 2. Strategy for the design of the Coxiella burnetii axenic medium © by author ACCM2. The figure shows successive steps in development of the medium development and major factors found to optimize the medium. Abbreviation: ACCM, ESCMID Online Lecture acidifiedLibrary citrate cysteine medium; ACCM2, acidified citrate cysteine medium 2; CCM, complex Coxiella medium; FBS, fetal bovine serum. 46 Host cell-free growth of the Q fever bacterium Coxiella burnetii. Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA. Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4430-4.

The inability to propagate obligate intracellular pathogens under axenic (host cell- free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log(10)) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious© for by Vero authorcells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q ESCMIDfever preventatives Online such as an effective Lecture subunit vaccine.Library Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.

47 Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, Gordon JI. Proc Natl Acad Sci U S A. 2011 Apr 12;108(15):6252-7 The proportion of the human gut bacterial community that is recalcitrant to culture remains poorly defined. In this report, we combine high-throughput anaerobic culturing techniques with gnotobiotic animal husbandry and metagenomics to show that the human fecal microbiota consists largely of taxa and predicted functions that are represented in its readily cultured members. When transplanted into gnotobiotic mice, complete and cultured communities exhibit similar colonization dynamics, biogeographical distribution, and responses to dietary perturbations. Moreover, gnotobiotic mice can be used to shape these personalized culture collections to enrich for taxa suited to specific diets. We also demonstrate that thousands of isolates from a single donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections. Retrieving components of a microbiota ©that byhave authorcoexisted in single donors who have physiologic or disease phenotypes of interest and reuniting them in various combinations in gnotobiotic mice should facilitate preclinical studies designed to determine the degree to which tractable bacterial taxa are able to transmit donor traitsESCMID or influence host biology Online. Lecture Library

Rumen 48 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP 210 Bacteria Anaerobes (BCNEI) techniques

Culture axenic AMIBES AMIBES AMIBES intracellular BK (Afipia) (virus) Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia © by author T.v er a

ENVIRONMENT Rappe Culture Epstein short ESCMIDP.ubique Online in sea Lecturepeptide Library water

Kaeberlein Simulting environment 49

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 th e 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 syste m). It may replace ESCMIDGram 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 50 IDENTIFICATION

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

ESCMID Online Lecture Library

51 51 16S rDNA/ Rpob sequencing Evolution of the number of clinical isolates identified in our clinical laboratory from 2002 to 2012

600 species

52 Clinical sample distribution of total 284992 isolates

90000

78493 80000

70000

60000 54478 52036

50000

40000

30000

20722

20000 16135

9757 10000 7892 7346 6071 5573 4512 3692 2544 2096 1687 1169 1141 1039 929 902 876 863 799 658 389 372 367 338 335 263 191 165 153 142 139 136 126 111 88 63 59 51 31 22 17 11 8 5 0 Ear Nail Nose Sinus Stools Biopsy Others Cornea Genital Sputum Abscess Pharynx Placenta

Joint bone © by author Skin wound Conjonctive Lung biopsy Lung Colic biopsy Liver biopsy Gastric fluid Human milk Bloodstream Rectal swabs Cardiac valve Lymph nodes Myringotomy Amniotic fluid Pleural biopsy Human semen Peritoneal fluid Biliary drainage Pericardial fluid Aortic aneurysm Aortic Cerebral abscess Cerebral Surgical drainage Pericardial biopsy Intrauterin device Cerebrospinal fluid Cardiac pacemaker Endobuccal samples Bone marrow culture Intravascular catheter Intravascular Urinary tract infection Bronchoalveolar lavage Umbilical cord bloodstream Surgical composite material Tracheobronchial aspiration ESCMID Online Lecture Library Intracellular cacteria culture isolats (n=)

53 Article:

First results:

Top 50 of the most frequent isolated well identified bacteria for 10 years (more than 300 time per year).

Top 50 of the identified bacteria for the 10-year study

Stenotrophomonas maltophilia Staphylococcus 242 haemolyticus 261 231 Acinetobacter baumannii 234 245 Mycobacterium 266 223 Serratia marcescens tuberculosis Klebsiella oxytoca 268 16462 Streptococcus pneumoniae Citrobacter koseri Morganella 277 Haemophilus influenzae morganii 1360 Others 283 Enterobacter aerogenes Enterococcus faecium Streptococcus agalactiae 287 (B group) 953 Proteus mirabilis 293 887 Enterobacter cloacae 316 856 Enterococcus © by author faecalis 317 840 352 Klebsiella Staphylococcus pneumoniae aureus 598 Staphylococcus ESCMID Online Lecture448 Library warneri Staphylococcus 539 Escherichia coli 578 epidermidis 536 Pseudomonas aeruginosa 54 Method: Selection of the 10-year period, utilization of the nb.si function, suppression of duplicates based on the name of the bacteria and selection54 of the top 50 Before MALDI With MALDI

7,6 years 3 years

159 rare species identified (≤ 50 PubMed) 39 55 66 species species species

Before MALDI With MALDI © by author 159 rare species identified (≤ 10 PubMed) 14 15 29 ESCMID Online speciesLecture species Library species

(De Piseth) 55 Comparison by phylum

Phylum Before MALDI Both With MALDI Actinobacteria 11 26 14 Bacteroidetes 7 8 8 Firmicutes 8 16 28 Fusobacteria 2 2 1 Proteobacteria 15 16 22 © by author ESCMID Online Lecture Library

56 First results:

Evolution of the number of new identified bacteria species over the period of study (2002-2011)

600

500

400 Number of new bacteria species found for the selected year 300 Cumulated number of bacteria species found the past year(s) 200

100

Method: SelectionESCMID of each year one by one using Online text files, suppression Lecture of the duplicates based Library on the NOM_BACT and creation of a new column called NOM_BACT_BIS to put the deduplicated list in. From 2004, creation of cumulative sheets containing all the deduplicated bacteria species list NOM_BACT_BIS of the years before the selected year. Finally, after the deduplication of this list (after filling it with the same list of the past years), the RECHERCHEV function was started based on the column NOM_BACT_BIS of the cumulative year (except for 2002) and the NOM_BACT_BIS of the selected year, a tag column showing if the bacteria species of the selected year were new (NA) or already found (the tag chosen). 57 57

« Depth bias »

samples Axenic culture Temperature Culture media Atmosphere 70 best culture conditions © by author

No identification by MALDI-TOF ESCMID Online Lecture Library

Pyrosequencing 59 PCR 16SrRNA 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© studiesby .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.

60 Culture conditions

Conventional technique

138 bacteria whose 1 new species Anaerobes bacteria

Innovating techniques : to recreate an artificial environment Bacteria whose 3 news genera, 4 news species Anaerobies bacteria

Hemoculture bottles: 35 species whose 54% Anaerobies, 1 new kind, 2 news© species by author Rumen’s fluid: 14 species, (8 anaerobies) whose 2 news kind and 2 news species Stools filtrate medium: 2 species ESCMIDAnaerobies Online enclosure: 3 Lecturespecies Library

Fastidious culture of bacteria is possible!

61 Innovative culture conditions: Protocol of rumen's fluid

16 paunch’s sheep 1st filtration in a funnel cover with a Solid content thrown out digestive content laboratory sock

rumen's fluid © by author ESCMID Online Lecture Library

Centrifugation Suspension in Suspension filtered 3 successive filtration : 10.000 rpm during bootles (12Liter) through a funnel cover 0,8μm + pré filter, 90min with a laboratory hat 0,45μm, 0,2μm 62 Rationalization of culture conditions

Blood culture bottle

29 supplementary species including 3 new species and 3 new genera © by author E. Coli lytic phages ESCMID Online Lecture Library 1 new species

63 Analysis of the 212 culture conditions used

Nb espèces

400 350 300 250 200 Number 150 Nb of species 100 espèces 50 0 © by author

ESCMID10 conditions20 conditions30 conditionsOnline40 conditions50 conditions Lecture60 conditions70 conditions Library

70 culture conditions = 100% of species

64 20 « best » conditions

Columbia microaérophilie 37°C Inocultaion blood culture bottle with thioglycolate, 4 days, Columbia Anaerobe 37°C Columbia anaérobie 37°C Columbia Ae 28°C Inoculation blood culture bottle ana 5 days, Columbia ANA 37°C Schaedler Ana 37°C Columbia aérobie 37°C PVX Ae 37°C 5% CO2 Orange Ae 37°C Mueller Hinton Ae 37°C Thermic shock 65°C Columbia Ae 37°C Inoculation blood culture bottle 26 days with rumen and sheep blood, COLUMBIA anaerobe 37°C Inoculation blood culture bottle 3 days with 5 ml sheep blood, Columbia anaerobe 37°C Marin Ae 28°C Inoculation in blood culture bottle anaerobe 14 days with 8ml rumen fluid, COLUMBIA anaerobe 37°C Inoculation in blood culture bottle anaerobe© 3 daysby with 8mlauthor rumen fluid, COLUMBIA anaerobe 37°C Inoculation blood culture bottle ana 10 days, Brain Heart Infusion ANA 37°C BHI 57°C Ae Inoculation blood culture bottle 3 days with rumen and sheep blood, Columbia, anaerobe 37°C BrainESCMID Heart Infusion + NaCl 1g/l Online37°C Ae Lecture Library 20 culture conditions : 73% of species

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

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

66 In a single lab with 3 persons during 2 years • 212 different culture conditions, 32,500 colonies tested by MALDI-TOF

• 342 bacterial species – 7 phylums, 117 genera, – 178 never described in human gut, – 32 new species including 6 new genera, – 5 fungi, – 2 species from rare phylums and – the first human giant virus – 32 new species = 42% of the new species validated by IJSEM during the 10 last years from human gut

• Genome sequencing – 15,000 unknown genes which will help the future molecular studies and allowed to identify the largest bacterial© genome by (Microvirga author massiliensis : 9Mb) from a human and to increase of – Increase to 3,3% the number of sequenced bacteria from human gut.

• PyrosequencingESCMID : 698 phylotypes Online including 282Lecture phylotypes of knownLibrary bacteria and 416 phylotypes of uncultured bacteria but with a dramatically divergence with culturomics 67 – only 51 species were common with the 342 species cultured. – microbial culturomics may increase of 30% the repertoire knowledge of human gut only studied by pyrosequencing.

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

68 Culturomics 502 bacteria and 20 fungal species Including 55 new bacterial species And 250 species first described from human gut

69 Repartition of species isolated with culturomics 300

248 250

200 NHG

150

HG: Human Gut NHG: Non Human Gut 70 The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics. Dubourg G, Lagier JC, Armougom F, Robert C, Hamad I, Brouqui P, Raoult D. Eur J Clin Microbiol Infect Dis. 2013 Jan 6

Gut microbiota consists of 10(10) bacteria per gram of stool. Many antibiotic regimens induce a reduction in both the diversity and the abundance of the gut flora. We analyzed one stool sample collected from a patient treated for drug-resistant Mycobacterium tuberculosis and who ultimately died from pneumonia due to a Streptococcus pneumoniae 10 months later. We performed microscopic observation, used 70 culture conditions (microbial culturomics) with identification by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF) and 16S rRNA amplification and sequencing, pyrosequencing, and 18S rRNA amplification and clone sequencing. Electron and optical microscopic observations revealed the presence of yeast, but no bacterial species were observed. By culture, only 39 bacterial species were identified, including one new species, as well as three species that have not been previously observed in the human gut. The pyrosequencing showed only 18 phylotypes, detecting a lower number of bacterial species than the culture techniques. Only two phylotypes overlapped with culturomics. In contrast, an amount of chloroplasts was found. Additionally, specific molecular eukaryote detection found three fungal species. We recovered, for the first time, more cultivable than non-cultivable bacterial species in a patient with a low bacterial load in the gut, demonstrating© theby depth author bias of pyrosequencing . We propose that the desertification of gut microbiota in this patient is a reflection of the total body microbiota and may have contributed to the invasive infection of S. pneumoniae. This finding suggests that caution should be applied when treating patients with broad-spectrum antibiotics, and preventive measures should be taken in order to avoid ESCMIDinvasive infection. Online Lecture Library

71 Butyricimonas massiliensis ABCE Actinobaculum massiliae AC Marseille new Casaltella massiliensis ABCE Actinobaculum timonae AC species found in Cellumonas massiliensis ABCE Actinomyces grossensis ABCE Chryseobacterium massiliae AC Actinomyces massiliensis ABCE humans Chryseobacterium timonae AC Actinomyces polynesiense ABCE = 115 Clostridium anorexicamassiliense ABCE Actinomyces timonensis ABCE Clostridium anorexicus ABCE Aeromicrobium massiliense ABCE Clostridium dakarense ABCE Alistipes marseilloanorexicus ABCE Clostridium polynesiense ABCE Alistipes obesi ABCE Clostridium reamassiliensis ABCE Alistipes senegalensis ABCE Clostridium senegalense ABCE Alistipes timonensis ABCE Collinsella massiliensis ABCE Amoebinatus massiliae AC Corynebacterium massiliense ABCE Anaeroccoccus senegalensis ABCE Corynebacterium reamasilliensis ABCE Anaerococcus obesiensis ABCE Corynebacterium timonense ABCE Anaerococcus pacaensis ABCE Dielma fastidiosa ABCE Anaerococcus provenciensis ABCE Diplorickettsia massiliensis ABCE Bacillus marseilloanorexicus ABCE Dorea massiliensis ABCE Bacillus massiliensis ABCE Enorma massiliensis ABCE Bacillus massiliosenegalensis ABCE Enorma timonensis ABCE Bacillus timonensis ABCE Enterobacter massiliensis ABCE Bacteroides massiliensis ABCE © by authorFenollara massiliensis ABCE Bacteroides timonensis ABCE Halomonas phocaeensis AC Bosea massiliensis ABCE Helcobacillus massiliensis ABCE Brevibacillus massiliensis ABCE Herbaspirillum massiliense ABCE Brevibacterium massiliense ABCE Holdemania massiliensis ABCE BrevibacteriumESCMID senegalense ABCE Online LectureKallipyga Library massiliensis ABCE Kurthia massiliensis ABCE Kurthia senegalensis ABCE Kurthia timonensis ABCE

72 Massilia timonae ABCE Peptostreptococcus massiliae AC Megasphaera massiliensis ABCE Phocaeicola abscessus ABCE Methanomassilicoccus luminyensis ABCE Polynesia massiliensis ABCE Microvirga massiliensis ABCE Prevotella conceptionensis ABCE Mycobacterium aubagnense ABCE Prevotella massiliensis ABCE Mycobacterium barassiae ABCE Prevotella timonensis ABE Mycobacterium bolletii ABCE Pytheasella massiliensis ABCE Mycobacterium bouchedurhonense ABCE Rhizobium massiliae AC Mycobacterium conceptionense ABCE Rhodobacter massiliensis AC Mycobacterium marseillense ABCE Rhodobacter massiliensis ABC Mycobacterium massiliense ABCE Rickettsia aeschlimannii ABCE Mycobacterium phocaicum ABCE Rickettsia africae ABCDE Mycobacterium timonense ABCE Rickettsia conorii- caspia ABCDE Nesterenkonia massiliensis ABCE Rickettsia felis BDE Nocardioides massiliensis ABCE Rickettsia heilongjianghensis BCDE Nordella oligomobilis ABCE Rickettsia massiliae ABCE Nosocomiicoccus massiliensis ABCE Rickettsia raoultii ABCDE Oceanobacillus massiliensis ABCE Roseomonas massiliae AC Paenibacillus antibioticophyla ABCE Senegalemassilia anaerobia ABCE Paenibacillus massiliensis ABCE Soleaferrea massiliensis ABCE Paenibacillus provencensis ABCE © by authorStaphylococcus massiliensis ABCE Paenibacillus reamassiliensis ABCE Stenotrophomonas africana ABE Paenibacillus sanguinis ABCE Stoquefichus massiliensis ABCE Paenibacillus senegalensis ABCE Streptococcus massiliensis ABCE Paenibacillus timonensis ABCE Streptomyces massiliensis ABCE PaenibacillusESCMID urinalis OnlineABCE LectureTeichococcus Library massiliensis AC Parachlamydia acanthamoebae ACE Timonella senegalensis ABCE Peptoniphilus grossensis ABCE Tropheryma whipplei ABE Peptoniphilus obesi ABCE Peptoniphilus senegalensis ABCE Peptoniphilus timonensis ABCE 73  1971 bacterial species isolated in human  756 species isolated in the human gut

Number of human species described since 1980 2500

2000

1500

0 ESCMID Online Lecture Library

74 Archae - Archae is inappropriate name as it induces the opinion that this organism an ancestral and are extramophiles. - It produces most of the methane on the earth - In fact there are more archae cells in human body then Eukaryotic cells! - 10 % of the prokaryotes of the gut

Contrôle de la culture par microscopie optique: Les ESCMID Onlineméthanogènes Lecture sont visualisés Library par la fluorescence de l'un de leurs co-enzymes uniques, le F420. (Culture de Methanobrevibacter marseillense à partir d’une selle). M.Drancourt 75 A versatile medium for cultivating methanogenic archaea Saber Khelaifia, Didier Raoult, and Michel Drancourt PONE-D-13-03759-revised

76 100 Halorubrum sp. B6 [EF077636] 4 out of 6 found in the gut 3 out of 6 from Halobacterium noricense [DSM 15987] humans 1 new species 100 Methanogenic archaeon clone E18_16S2_Mx-03[FJ752571] 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 © byMethanobrevibacter author oralis [DQ251043] Methanobrevibacter arboriphilicus [AB065294]

100 Methanobrevibacter arboriphilicus ANOR1 [CSUR P1715] ESCMID OnlineMethanobrevibacter Lecture millerae ZA-10T [16643]Library 100 Methanobrevibacter millerae CMR-6 [CSUR P1712]

Euryarchaeote clone LL2-3-6 [FJ458322] From M.DRANCOURT 77

Largest Archae from human (Methanomassilicoccus luminyensis) Size genome: 2,6 Mb 4th species isolated Complete genome sequence of Methanomassiliicoccus luminyensis, the largest genome of a human-associated Archaea species. Gorlas A, Robert C, Gimenez G, Drancourt M, Raoult D. J Bacteriol. 2012 Sep;194(17):4745.

The present study describes the complete and annotated genome sequence of Methanomassiliicoccus luminyensis strain B10 (DSM 24529(T), CSUR P135), which was isolated from© human by fecesauthor. The 2.6- Mb genome represents the largest genome of a methanogenic euryarchaeon isolated from humans. The genome data of M. luminyensis reveal unique features and horizontal gene transfer events, which might have occurred during its adaptation and/or evolutionESCMID in the human Onlineecosystem. Lecture Library

78 1984 1992 1999 2000 2002 2003 2008 2009 2011 2012 2013 OUR LAB New Vero Shellvial Shellvial XTC Axenic extract medium TW BK and blood medium

JNSP 210 Bacteria Anaerobes (BCNEI) techniques

Culture axenic AMIBES AMIBES AMIBES intracellular BK (Afipia) (virus) Medium archae OTHER HUMANS Heinzen Gordon Hackstadt C.burnetii Rumen Chlamydia © by author T.v er a

ENVIRONMENT Rappe Culture Epstein short ESCMIDP.ubique Online in sea Lecturepeptide Library water

Kaeberlein Simulting environment 79

Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium. Omsland A, Sager J, Nair V, Sturdevant DE, Hackstadt T. Proc Natl Acad Sci U S A. 2012 Nov 27;109(48):19781-5.

Chlamydia trachomatis is among the most clinically significant human pathogens, yet their obligate intracellular nature places severe restrictions upon research. Chlamydiae undergo a biphasic developmental cycle characterized by an infectious cell type known as an elementary body (EB) and an intracellular replicative form called a reticulate body (RB). EBs have historically been described as metabolically dormant. A cell-free (axenic) culture system was developed, which showed high levels of metabolic and biosynthetic activity from both EBs and RBs, although the requirements differed for each. EBs preferentially used glucose-6-phosphate as an energy source, whereas RBs required ATP. Both developmental forms showed increased activity when incubated© by under author microaerobic conditions. Incorporation of isotopically labeled amino acids into proteins from both developmental forms indicated unique expression profiles, which were confirmed by genome-wide transcriptional analysis. The described axenic culture system will greatly enhance biochemicalESCMID and physiological Online analyses ofLecture chlamydiae. Library

80 Arch Med Vet 44, 273-277 (2012) COMUNICACION Un nuevo medio de cultivo líquido para el patógeno Piscirickettsia salmonis

A new liquid medium for the pathogen Piscirickettsia salmonis

T Vera*, A Isla, A Cuevas, J Figueroa Laboratorio de Biologia Molecular de Peces, Instituto de Bioquimica y Microbiologia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.

SUMMARY Piscirickettsia salmonis is the ethiologic agent of the Salmonid Rickettsial Syndrome (SRS), which was first described in cultured Coho salmon in the south coast of Chile in 1989 and since then has been reported in different places around the world. Initially, this bacterium was described as an obligate intracellular pathogen, able to grow only in cytoplasmic vacuoles in host cell where it replicates by binary fission, so their in vitro culture is performed mainly in cell lines derived from fish. Recently, it has been demonstrated that this bacterium can grow in cell-free media containing sheep blood or fish and high levels of cysteine; h owev e r, due to the presence of cellular elements these culture media present drawbacks regarding handling, storage© and by high authorcosts. We propose an artificial culture medium supplemented with an iron salt to replace the blood that is traditionally used; it reported successful growth of the bacterial reference strain and of a strain isolated from the environment, which was evaluated by optical density (OD 600), while purity and identity of the culture were checked by using Gram stain,ESCMID immunofluorescence Online and PCR. Developing Lecture a blood-free cultureLibrary medium facilitates the cultivation of P. salmonis and allows the growth of this bacterium in a liquid medium, which provides various microbiological and biotechnological applications.

81 Vero Extract A – Axenic medium for intracellular bacteria CB avec CM tous les 2 jours en gazpack Nbre de Cb / mL CB avec CM tous les 2 jours en gazpack gim 1/10 inoculum 1,00E+06 14 1.00E+06 J0 1,00E+05 20 J2 5,00E+05 17 CB avec CM tous les 2 J4 1,00E+06 19,5 1.00E+05 jours en gazpack J7 2,00E+05 19 CB avec CM tous les 2 jours en gazpack J9 1,00E+05 22 1.00E+04 J12 1,00E+05 23 CB avec CM tous les 2 jours en gazpack 1.00E+03 gim 1/10 inoculum 1,00E+05 17 J0 1,00E+04 21 1.00E+02 J2 5,00E+04 21 J4 1,00E+05 23 1.00E+01 J7 1,00E+05 21 J9 1,00E+04 25 J12 1,00E+04 25 1.00E+00 J0 J2 J4 J7 J9 J12 CB avec CM tous les 2 jours en O2 contrôlée gim 1/10 Nbre de Cb / mL CB avec CM tous les 2 jours en O2 contrôlée inoculum 1,00E+06 14 J0 1,00E+05 19 1.00E+06 J2 1,00E+05 22 CB avec CM tous les 2 J4 1,00E+06 23 jours en O2 contrôlée J7 5,00E+05 19 © by1.00E+05 author J9 1,00E+05 16 Datenreihen2 J12 1,00E+05 23 1.00E+04 CB avec CM tous les 2 jours en O2 contrôlée ESCMIDgim 1/10 Online1.00E+03 Lecture Library inoculum 1,00E+05 17 J0 1,00E+04 21 1.00E+02 J2 1,00E+04 26 J4 1,00E+05 26 1.00E+01 J7 5,00E+04 22 82 J9 1,00E+04 26 1.00E+00 J12 1,00E+04 26 J0 J2 J4 J7 J9 J12 New Vero Extract Tropheryma whipplei Twist strain Yes

Bartonella henselae Houston-1 strain (ATCC Yes

49882T)

Bartonella quintana Oklahoma strain Yes

Francisella tularensis F. tularensis holartica LVS Yes

Rickettsia africae CSUR R15 No growth

Mycobacterium bovis BCG CIP 105050 Yes

Leptospira interrogans M 20 Yes

Borrelia crocidurae CSUR P235 Yes

Borrelia burgdorferi B31 Yes

Brucella melitensis B115 Yes

Yersinia pestis EV76 Yes

Bacillus anthracis CIP 77.2 Yes ESCMIDShigella dysenteriae OnlineCIP 57.28 Lecture YesLibrary Bordetella pertussis CSUR P237 Yes

Campylobacter fetus CSUR P236 Yes

Campylobacter jejuni CSUR P239 Yes 83 Legionella pneumophila CIP 103854T No growth Figure 4. Evolution of bacterial culture techniques

84 PATENTS

Culture - Cell culture for Tropheryma whipplei (2000)

- Culture for Mycobacterium tuberculosis (2008)

- Culture for Archae (2012)

85 CONCLUSION

There is a lot to fish as new microbes by culture.