1. Michel DRANCOURT, Professor, HDR
2. Gérard ABOUDHARAM, Associate Professor; HDR
3. Louis HOFFART, Associate Professor
4. Stéphan COHEN-BACRIE, Assistant Professor
5. Cécilia BEBEACUA, Postdoc.
Our team developed specific skills in the microbiology of the fastidious microorganisms, which will be declined in 4 directions during the next four-year contract. Concerning mycobacteria (Direction 1), we are going to develop new systems of culture based on enriched media (patented) [Drancourt M, Raoult D. Cost-effectiveness of blood agar for isolation of mycobacteria. PLoS Negl Trop Dis. 2007;1:e83] after decontamination by new decontaminants (chlorexhidine; derivatives of squalamine – in collaboration with team of Pr. Jean-Marc ROLAIN team) with the objective to shorten by 50 % the average delay for isolation and culture of Mycobacterium tuberculosis. This project will be done by a PhD student Ramzi Ghobdane. Combining the genomic data in GenBank with high throughput phenotyping (Biolog) of the Mycobacterium tuberculosis complex members, will contribute to refine the composition of culture media on the model previously established in our laboratory for Tropheryma whipplei [Renesto P. and al. 2003 Lancet; 362 447-9]. This project will be done by a PhD student. Simultaneously, we will develop protocols for the identification of mycobacteria by MALDI-TOF mass spectrometry in the perspective of its direct use on the Ziehl-positive specimens [PhD students Ramzi Ghobdane and Stéphan Cohen-Bacrie]. Also, our leader's position in the field of Mycobacterium avium and Mycobacterium abscessus complexes will be consolidated by the sequencing of the genomes of all these species, with the objective of studying mycobacteriophages of the complex M. abscessus and the VII secretion systems by genomic and structural approaches and the role of these systems in the resistance of M. abscessus complex organisms, in particular in cystic fibrosis patients [Roux AL, Catherinot E, Ripoll F, Soismier N, Macheras E, Ravilly S, Bellis G, Vibet MA, Le Roux E, Lemonnier L, Gutierrez C, Vincent V, Fauroux B, Rottman M, Guillemot D, Gaillard JL; Jean-Louis Herrmann for the OMA Group. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in france. J Clin Microbiol. 2009;47:4124-8](collaboration IFR48 with Christian CAMBILLAU, AFMB laboratory; collaboration with Pr. JM ROLAIN team; PhD student Mohamed Sassi; post-doc student Cécilia Bebeacua). The third project concerns the role of the cellulases of mycobacteria following the observation of their paradoxical preservation among Mycobacterium tuberculosis complex members (collaboration with B. Henrissat who was the first to highlight this preservation, laboratory AFMB, Marseille) [Medie FM, Vincentelli R, Drancourt M, Henrissat B. Mycobacterium tuberculosis Rv1090 and Rv1987 encode functional β-glucan-targeting proteins. Protein Expr Purif. 2010 Sep 6; Mba Medie F, Ben Salah I, Drancourt M, Henrissat B. Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms. Microbiology. 2010;156:1468-75] and we are going to study the role of these cellulases as for mycobacteria–amoeba relationships since the amoebal cyst wall is composed of cellulose [Salah IB, Ghigo E, Drancourt M. Free-living amoebae, a training field for macrophage resistance of mycobacteria. Clin Microbiol Infect. 2009;15:894-905] and as a potential factor for plant- mycobacteria interactions (collaboration Cindy Morris, INRA UR 407, Avignon; PhD student: Thiam).
The second direction concerns the sequencing of the genome of Borrelia crocidurae (ANR project; collaboration with F. Renaud, Genetics and Evolution of Infectious diseases, Mixed Unit of Research - Institute of Research for Development / Centre of the Scientific Research 2724, Montpelier) [Lescot M and al. The genome of Borrelia recurrentis, the agent of deadly relapsing fever louse-border, is a degraded subset tick-borne Borrelia duttonii. PLoS Genet. 2008; 4:e1000185; Thein M and al. Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes. J Bacteriol. 2008; 190:7035-42] in the perspective to clarify the mechanisms underlying the characteristic antigenic variability of this group of organisms and to work-out the specific tools of detection and identification of recurrent fever Borrelia applied for the point-of-care diagnosis in western Africa (collaboration JF Trappe, UMR 6236, Dakar; PhD students Haitham Elbir and Stéphan Cohen-Bactrie). As a preliminary result, we observed a 250-bp region which is specific for B. crocidurae among recurrent fever borreliae; re-annotation of B. duttonii and B. recurrentis genomes possible found also a region specific for this organism. If confirmed, these specific regions will be checked for their specificity among bacteria, for their sensitivity including in the presence of human blood and they could be incorporated into a rapid, multiplex real-time PCR for the rapid detection of recurrent fever borreliae in patients (and in ticks) in Africa. Such a clinical test would be incorporated into the point-of-care (POC) that URMITE is implementing in rural Senegal (collaboration JF Trappe and Oleg Mediannikov, UMR 6236, Dakar; and Stéphan Cohen-Bacrie).
The third direction concerns Archaea, for which we demonstrated the presence of methanogenes Archae in all of the individuals [Dridi B, Henry M, El Khechine A, Raoult D, Drancourt M. High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS One. 2009; 4:e7063] (PhD student Bédis Dridi). These tools are going to be used to investigate the variety of intestinal Archae and the Archaea in the respiratory tract, in particular non-methanogen Archaea. The project consists into the molecular detection of new Archaea [Dridi B, Didier Raoult, Michel Drancourt. Improved protocol for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of host-associated Archaea. PLoS ONE, submitted for publication] and culture and isolation of new Archaea as well as the general-purpose culture using numerous media and anaerobic atmospheres containing a variable proportion of d' H2, N2 and CO2 (PhD student Saber Khelaifia). Our preliminary, non-published data indicate isolation and culture of a new methanogen Archaea Methanomassiliibacterium luminyensis from the intestinal microbiote. The genome of this fourth ever Archaea cultured in humans will be determined and specific molecular tools for detection will be developped for its detection in individuals as well as in potential sources. Further cultures will be done by using strict anaerobe protists such as Trimyema. Also, clinical specimens other than digestive tract specimens will be tested, including cerebral abscesses for which our preliminary data indicate the presence of M. smithii (PHRC and collaboration Pr. PE FOURNIER).
The fourth direction concerns Planctomycetes for which we have established the in-vitro antibiotic susceptibility profile in order to incorporate suitable antibiotic in culture media [Cayrou C, Raoult D, Drancourt M. Broad-spectrum antibiotic resistance of Planctomycetes organisms determined by Etest. J Antimicrob Chemother. 2010;65:2119-22] as well as the MALDI-TOF identification protocol [Caroline Cayrou, Didier Raoult, Michel Drancourt. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of environmental organisms: the Planctomycetes paradigm. Environ Microbiol. Reports 2010; on line]. Preliminary, non-published data indicate the detection of at lesat 3 different phylotypes of Planctomycestes in stools (PhD student Caroline Cayrou).
The detection and the culture of Archaea and Plancomycetes will be applied to the digestive, respiratory, ocular and bone specimens, in accordance with the corresponding "diagnostic kits" routinely used in the laboratory. New Archaea and Planctomycetes organisms will be deposited in collections and described by using a polyphasic taxonomic approach. They will be sequenced by high throughput sequencing for a rapid characterization and their phenotype will be determined by phenotypic tests by MALDI-TOF and BioLog technologies.
The current data of the medical literature show the importance of the complex flora that modern technologies allow to handle in new perspectives. These data go in line with the data that we acquired in the field of microbiology of cerebral abscesses [Al Masalma M et al. The expansion of the microbiological spectrum of brain abscesses with use of multiple 16S ribosomal DNA sequencing. Clin. Infect Dis. 2009; 48:1169-78]. We will develop the analysis of complex flora in three clinical situations: cerebral abscesses, chronic sinusitis and keratitis. A first global approach relies on on-going DNA chip "any procaryotes" PANPRO allowing to detect > 90 % of the known variety of bacteria and archae, which we intend to apply to complex flora specimens (PhD student, Emilie Donatin). Preliminary data indicate that such a chip has to contain approximately 156.000 different phylotypes. In order to accomplish latter work the group of ophthalmologists headed by Doctor Louis Hoffart (PHU, Université de la Méditerranée, Marseilles) joined our team. This project can be divided into 3 research directions. The 1st direction concerns isolation, culture and identification of protozoons responsible for cerebral abscesses, chronic sinusitis and keratitis for which the preliminary data show 3 different genera and 5 species. Medical literature indicates that there are few molecular tools for the identification of these protists. Therefore the first research direction concerns the development of their isolation and culture and the development of molecular and high throughput phenotyping tools (BioLog) (PhD student N. Gouba). The second area of research concerns the isolation, identification and characterization of pathogenic intra-protozoons bacteria. The third area of research concerns the detection of "all pathogens" by high throughput pyrosequencing (metagenomic 454-Roche) using our sequencing platform IBiSA.
We are going to develop new approaches for the massive detection of pathogens in the ancient samples. From the dental pulp, we are going to set up the massification of the detection of highly contagious pathogens (smallpox, plague, anthrax, typhoid fever agents) by real-time PCR in order to resolve the epidemics of unknown origin for which we have acquired the dental pulp samples through our network of collaborations with different anthropological research teams in France. The second project concerns the detection of the genome of Yersinia pestis by very high throughput pyropsequensing (454-Roche) from the samples of plague mass graves (PhD student Tung Nguyen-Hieu); as well as their differential analysis by mass spectrometry (PhD student Thi-Nguyen-Ny Tran). The third project concerns the development of non-directed techniques of detection of microorganisms containing on the one hand, the RDA methods and, on the other hand, the methods of massive sequencing by the microbiogenomic platform IBiSA (454-Roche). In parallel, we will develop the tools of phenotypic detection of microorganisms and we obtained preliminary data of the detection (by ELISA method and by immuno-PCR method) of Yersinia pestis in samples of ancient dental pulp extracted from presumed medieval plague mass graves in Europe. In between of these two research topics, we will use the chip PANPRO as a first-line exploration tool of the microbial flora in human coprolithes dating from the Neolithic period of 15th century (in collaboration with University of Franche-Comté, UFR Sciences and Techniques, UMR CNRS 6249 Chrono-Environnement; Matthieu Lebailly and University of Reims, Prof. Françoise Boucher) and at the same time in order to analyze by high throughput screening (sequencing) the evolution of human intestinal microbiote during the centuries.