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Diapositive 1 29.04.2013 ESCMID-BERLIN «Culturomics» © by author ESCMID Online Lecture Library Didier Raoult Marseille - France [email protected] www.mediterranee-infection.com As samples in 2012 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 Online Lecture Library Pathophysiology 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 © by author 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 © by author ESCMID Online Lecture Library p.64 By F.Rohwer 9 Research strategy for identification and detection of new pathogens Culture-dependent Culture-independent © by author Cloning 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 © by author ESCMID Online Lecture Library 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 © by author T.v er a ENVIRONMENT Rappe Culture Epstein P.ubique in sea short ESCMID waterOnline Lecturepeptide Library 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. © by author ESCMID Online Lecture Library 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 © by author ESCMID Online Lecture Library 21 These resisting bacteria are prepared to resist U macrophages and do the opposite of filtration R U Amoeba phagocyte everything! R © by author ESCMID Online Lecture Library 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...........................................................................................................................
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