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Microbiology & Humanities Sciences

Team 1: leader Didier Raoult, PU-PH

Team composition

 

  1.  Didier RAOULT, PU-PH, HDR
  2.  Gilbert HABIB, PU-PH, HDR
  3.  FlorenceFENOLLAR, MCU-PH, HDR 2010
  4.  Bernard DAVOUST, vétérinaire militaire
  5.  Jean-Lou MARIE, vétérinaire militaire
  6.  Jean-Paul CASALTA, PH
  7.  Pierre-Yves LEVY, PH
  8.  Franck THUNY, PHU
  9.  Fadi BITTAR, Maître de Conférence, Chaire d’excellence IRD

The unit of Rickettsies is the first research unit which was built in the current URMITE. It is very deeply associated with the National Reference Centre for Rickettsia, Coxiella, Ehrlichia and Bartonella and extended the spectrum of its activity towards the emerging pathogens.

Five projects gathering partners working together for a long time will be developed. The first project will be centered by the National Centre of Rickettsioses and the emergent zoonosis, including a therapeutic part which will be the subject of a second specific project. The Whipple’s disease for which we are the world leaders will constitute the third project. The continuation of our activities on the research of causes of infections and the strategies of management, in particular cardiologic infectious diseases, will be the subject of a fourth project. The fifth project is developed for the first time in the URMITE in collaboration with the IRD in Senegal, centered on the study of the digestive tract microbiota in Africa.

 

Project 1. National Reference Center for Rickettsioses and emerging zoonoses - (DR, BD, JLM)


            Our situation of center of reference for rickettsioses, as well on the national plan as on the international plan (as a WHO collaborative center on rickettsia and arthropod-borne bacteria) lead us to be frequently solicited to help in the identification of various pathogens in misunderstood situations, challenging our permanent efforts for the development of new diagnostic tools.

The first step of the diagnosis is based mainly on the serologic techniques, which are also often at the origin of the question of the potential pathogenicity of the micro-organisms. This first step requires confirmation by direct diagnostic elements: molecular diagnosis or culture. This approach misses sometimes sensitivity and specificity linked to frequent cross reactions between micro-organisms. Immunoproteomic and immuno-PCR will be developed in order to improve these diagnostic tools. We project to apply a first approach by immunoproteomic in order to optimize the serological diagnosis of Q fever and rickettsioses, in particular for the differential diagnosis of R. conorii and R. africae infection. The serum of patients with documented infection by C. burnetii, R. conorii and R. africae as well as control groups will be tested by two-dimensional electrophoresis coupled with the Western-blot in order to detect specific antigenic targets of each one of these micro-organisms. These detected specific peptide targets will be identified by mass spectrometry. A further step will be the production of these proteins as recombinant proteins obtained by in vitro expression. Purified, recombinant proteins will be incorporated as antigens for large scale serology by indirect immunofluorescence and ELISA. This approach will enable us to increase the specificity of serological tests. However, a more significant detection is also necessary. For example for Q fever, the diagnosis is too late. The application of techniques of ImmunoPCR would enable us to gain 4 to 5 days within the diagnostic time. It is thus a second approach by ImmunoPCR which we will apply for the diagnosis of Q fever. This technique couples ELISA technology for the immune recognition but the detection of the signal, instead of being carried out by antibodies coupled to a substrate, will be done by antibodies coupled to a fragment of DNA allowing an amplification of the signal by PCR and consequently to be more sensitive.

Finally, we will persevere in the development of serological chips. Serological chips were already set up at the unit of Rickettsies to analyse the sera according to their geographic origin. Thus, we have antigenic profiles of chips for the patients having a fever in the South-East Asia, a fever with the return of Southern Africa, or a fever after arthropod bites in Europe, or a fever in a particular context, such as endocarditis. These chips will also enable us to seek the potential pathogenic role of bacteria lately isolated to the URMITE, such as Diplorickettsia massiliensis (1) and Occidentia massiliensis. These works are based on an original and new approach and could have repercussions in the socio-economic field.

We recently started to develop fast diagnostic new approaches. We began the development of the analysis by specific Coxiella burnetii PCR in real-time from pharyngeal sampling using a cotton swab for the diagnosis of fever Q. We are developing the sampling of eschar’s inoculation using a cotton swab for the diagnosis of the rickettsioses, instead of skin biopsies, followed by an analysis by molecular biology by specific real-time PCR. These diagnostic strategies use specimens which collection does not necessitate important technicality followed by specific real-time PCR will make it possible to propose a fast and reliable diagnosis with more large scales.

Lastly, for several years, we have contributed to the discovery of a certain number of zoonotic emergent pathogens. Our objective is to continue to persevere in this axis by applying a rigorous strategy. In this aim, it is important to multiply the sources of sampling. The presence within our team of two military veterinary doctors will enable us to continue to obtain some various atypical samplings, in particular samplings of wild animals but also of ticks, lice, fleas and other ectoparasites, which allowed us these last years to discover of new micro-organisms. Some of these works will be carried out in collaboration with the team of Pr Philippe Parola. Besides, the presence within our UMR of a IRD team with Dr. Oleg Mediannikov will allow samplings human and animal specimens in Africa, in particular via our laboratory in Dakar.

 

Project 2. Therapeutic of intracellular bacteria infections (DR, FF, PYL)

 

            The management of infections caused by fastidious intracellular bacteria is often a therapeutic dead-end. Moreover, the increase in resistances of the bacteria to antibiotics poses more and more problems. Our project concerning this thematic will include two axes and will be carried out in collaboration with the team of Pr Jean-Marc Rolain.

1. The optimization of the management of infections caused by intracellular bacteria will be carried out by the molecular diagnosis of resistances. For Whipple’s disease, for example, we recently highlighted the presence of mutations of the folP gene from Tropheryma whipplei strains of patients with clinical resistance to the treatment by sulfamethoxazole-trimethoprime (2). However, this molecule is still largely prescribed in first intention among patients with Whipple’s disease. We are developing a tool which will allow among all patients at the time of the diagnosis of this infection to detect the presence of mutations that are suspected to be responsible of resistance of the bacterium to this antibiotic or to present a strong risk of development of resistance. With regard to Q fever, two complete genomes of C. burnetii, a first from a strain sensitive to doxycycline and a second from a strain resistant to doxycyline have been sequenced. Their bioinformatic analysis will make it possible to highlight mutations on target genes. In the future, the amplification coupled with the sequencing of these genes should make it possible to be able to detect the patients reached by potentially resistant strains.

2. New therapeutic approaches are currently evaluated. Recent works suggest that the statines, molecules initially prescribed against the hypercholesterolemy, would have also a beneficial effect during infections. In vitro models on cell culture showed that the statines (in particular lovastatine) had an activity against C. burnetii and R. conorii. These preliminary results, even if they require to be confirmed and widened, enable us to suppose that some statines could prevent or moderate the clinical manifestations of Q fever and rickettsioses. An animal model also recently showed the effectiveness of the statines to protect against  Yersinia pestis, thus consolidating the potential of these molecules like anti-infectious agents.

The omeprazole is a proton-pump inhibitor prescribed in the treatment of gastro-duodenal ulcers. Preliminary work suggests that this molecule has also a direct effect on the multiplication of C. burnetii and could thus be prescribed in the treatment of Q fever.

In the future, we will be able to further explore the impact of these molecules in the prevention of infectious diseases, in particular those related to intracellular bacteria, alone or possibly associated with antibiotics.

 

Project 3. Tropheryma whipplei and Whipple’s disease (FF, DR, FB)

 

In 2000, the first culture of Tropheryma whipplei, the bacterium responsible of Whipple’s disease, was carried out in our laboratory, enabling us to begin research in order to trace its natural history but also to propose a reference centre for the diagnosis and therapeutic council. Whipple’s disease was regarded for a long time as a metabolic disease then like a rare disease due to a rare bacterium. In fact, thanks to our work, we know now that T. whipplei is a frequent bacterium responsible for acute infections (gastro-enteritis, pneumopathy, bacteremia), asymptomatic carriage (stools specimens, saliva), localised chronic infections with manifestations other than the digestive tract or Whipple’s disease as described since the beginning (3;4). This last infection is serious and probably linked to genetic causes. Our project falls under the continuity of our last activity.

We will look further into the epidemiological studies on the bacterium by carrying out on the one hand family investigations among patients with infections with T. whipplei as well as the asymptomatic carriers and by making on the other hand studies of seroprevalence in France and in Senegal. The high number of specimens which we receive in our centre (13,382 samplings received since 2001, including 3,618 in 2009) allows us to be confronted with new situations which should enable us to better know the panel of infections related to T. whipplei. A study, carried out via a PHRC, on the samplings of stools specimens and saliva of children of less than 6 years consulting the emergency department Northern hospital in Marseilles will make possible to consolidate the role of T. whipplei as the agent most frequently responsible for gastro-enteritis.

Efforts must be still provided in order to optimize the diagnosis of the infections with T. whipplei. The serological tool by western-blot targeting T. whipplei allows us since recently a good discrimination between the asymptomatic carriers and the patients and it will have to be applied systematically among all patients presenting a positive PCR to T. whipplei. However, the diagnosis of certain infections to T. whipplei such as endocarditis or encephalitis still remains very difficult. The optimization of the culture techniques of T. whipplei will have to be realized in order to isolate new strains. Our challenges will be in particular to obtain isolates of T. whipplei from the saliva from patients, from the stools of patients with gastroenteritis, as well as from the stools and saliva of asymptomatic carriers of the bacterium.

One century after its description and 10 years after the culture of the bacterium that has allowed a more rational therapeutic approach, the treatment of classic Whipple’s disease remains always difficult and complex. It is necessary to underline the contrast between the spectacular effectiveness of antibiotics and the lack of effectiveness of the treatment by sulfamethoxazole-trimethoprime for a length of 18 months or even more. The evaluation of the new therapeutic protocols is currently performed thanks to a rigorous follow-up of patients. The analysis of the duodenal biopsies will have to be also performed from the qualitative and quantitative points of view. This follow-up will have to be carried out for a long time and ideally for all the life. The management of the patients will have also to be adapted, progressively, on the basis of the gained experience. One of the questions will be also the management of the subjects who are a priori asymptomatic carriers of T. whipplei. It will be indeed necessary to look systematically for the presence of an immune response by serology western-blot for all the subjects having a positive PCR detection of T. whipplei in stools, saliva or digestive biopsies without another positive sampling.

The research of the genetic factors and the immune specific deficits predisposing to Whipple’s disease is also one of our objectives. There exists currently a very strong beam of arguments showing that the occurrence of Whipple’s disease would depend to a large extent on the susceptibility/genetically given resistance of the infected host. This great variability of response to the infection to T. whipplei, and in particular the fact that the majority of the individuals can be regarded as resistant to the development of Whipple’s disease, is a major argument in favour of the role of genetic factors of the host in this infectious illness. We formulate as assumption that Whipple’s disease testifies to a genetic Mendelian predisposition, i.e. it rises of new types of hereditary immune deficits. The objective of our work will thus aim at identifying the molecular bases of the predisposition to Whipple’s disease. This work will be completed in collaboration with the department of Medical Genetics of the Infectious Diseases of Pr Jean-Laurent Casanova and Dr. Laurent Abel, Hospital Necker, Paris. The recruitment of the patients and the constitution of a bank of sampling have started. During their consultation in Marseilles, a skin biopsy and a blood test, in particular to constitute banks of DNA, sera and cells, are carried out among patients. Functional and molecular immune studies, the study of genetic epidemiology as well as the search for mutations by an approach "gene target" according to knowledge of the ways of the immune response will be realized as well as the analysis of all the data about the complete genomic sifting (according to the characteristics of the families collected). The identification of genes of predisposition to Whipple’s disease will have major implications on the immunological level (dissection of the mechanisms of the immune response) but also on the clinical level (management of the healthy carriers of the bacterium, treatment directed towards the restoration of a defective immune response, strategies of prevention for the genetically predisposed people).

 

Project 4. Infectious cardiology (GH, JPC, FT, DR)


Infectious cardiology is one of the themes headlights in our UMR (GH, JPC, FT, DR). Since 1994, we developed a diagnostic kit allowing the realization of systematic analyses for the diagnosis of infectious endocarditis and also ensuring the collection of the epidemiologic and clinic elements in a standardized way (5). The composition of this diagnostic kit was regularly evaluated and adapted according to the current knowledge. Currently, more than 3,000 patients presenting a suspicion of infectious endocarditis are included in our database. On the basis of the same approach, we have constituted a diagnostic kit for pericarditis.

Our first research orientation concerning this thematic will be to persevere in the etiologic optimization of the diagnosis and the development of diagnostic tools as well as tools for the evaluation of the prognosis. The etiologic diagnosis is capital because its absence is a factor of bad prognosis for the patient. Our first work on endocarditis allowed a better detection of the fastidious bacteria or bacteria among patients already under antibiotics as well as the description of non-infectious endocarditis. Indeed, we have recently diagnosed inflammatory endocarditis (suggesting the necessity to look systematically for the presence of rheumatoid factors and antinuclear antibodies), of allergic endocarditis (suggesting the necessity to look for the presence of anti-pig antibody) as well as endocarditis related to the reaction of the graft against the host. We recently developed a panel of specific PCRs in the blood which seems to improve the etiologic diagnosis. We aim also to apply a method of differential amplification based on the differential hybridization of infected and not infected tissues in order to detect possible infectious agents including viruses. A preliminary test of this method had enabled us to identify a posteriori the pathogenic role of Mycobacterium tuberculosis in a mortal case of endocarditis. Recently we published a work on the transcriptional profiles of cardiac valves and total blood of patients with infectious endocarditis. This study, carried out from the analysis of the whole human genome, made it possible to highlight an over-expression of certain genes, never reported before in infectious endocarditis, of which in particular those of the aquaporine-9 (AQP-9), which is a membrane protein controlling the permeability of water, mainly known to be implied in processes such as the immune response, the oedema in the inflammatory responses but also in syndromes such as the renal insufficiency and cardiac failure. Our current working hypothesis is to know if serum dosage or amplification of the gene of the AQP-9 among patients with infectious endocarditis could be a predictive factor of unfavourable evolution such as a hemodynamic failure. To test this hypothesis, we will dose AQP-9 (ELISA) and we will analyze the expression of its gene (PCR) in patients hospitalized for infectious endocarditis. The prognostic interest of the analysis of the AQP-9 will be compared with that of the other known factors such as the BNP, a marker of cardiac failure. If the results went in favour of an interest of the AQP-9, we could base part of our surgical indications on this dosage in order to further decrease mortality.

Our second research orientation aims at optimizing the therapeutic management of infectious endocarditis. Indeed, treatment of endocarditis is very heterogeneous, including within the same hospital, with in particular a low level of compliance to the recommendations. This report led our laboratory to propose since 2002, a simplified and rational therapeutic protocol using only four antibiotic molecules. This protocol was applied systematically to the patients hospitalized for infectious endocarditis. The effectiveness of this standardized therapeutic strategy, but also the compliance of the hospital services, will be evaluated. Another therapeutic project will aim at evaluating the utility of the statines to prevent the occurrence of endocarditis or to decrease their severity. Indeed, of the recent studies suggest an effectiveness of these molecules to reduce the incidence of the infections, in particular at the patients with cardiovascular disease.

Our third research orientation will more specifically target the pericarditis with the retrospective analysis of the series of the pericarditis. The latter will make it possible to establish the panel of different etiologic diagnosis and to evaluate the etiologic proportion of case without diagnosis. A project of pathovirome in collaboration with the team of Dr. Christelle Desnues will be also begun. The objective of this work will be on the one hand to catalogue the human “virome” starting from samplings of liquids or pericardial biopsies and on the other hand to identify new pathogens implied in pericarditis.

 

Project 5. Emerging digestive Pathogens in Africa (DR, FF, BD, JLM)


The inclusion of a IRD team in our UMR led interest in emergent pathogens in stools specimens in Africa (4). The first work carried out in Senegal in rural area, on human stools, enabled to highlight an important prevalence of the carriage of T. whipplei and to establish a link with diarrheal episodes. We showed that up to 44% of the children between 2 and 10 years were carrying the bacterium in the stools in Ndiop and Dielmo, 2 villages of the area of Sine-Saloum. We are also performing the repertory of the bacteria present in a sampling of stools of healthy adult coming from Senegal. We apply 3 complementary approaches: specific PCR, metagenomic approach and culture using an exhaustive panel of culture media of various compositions, under various atmospheric conditions and with pH extremely varied, coupled with the identification of the colonies by MALDI-TOF and PCR targeting the ARN 16S ribosomal sequence followed by sequencing. Currently, 88 bacteria were isolated including one new species and ten species never described in human. This triple approaches also enables us to compare the various methods. In particular, we are showing that the metagenomic approach makes it possible to detect the bacteria only when they are present in very great quantity in the sampling (108) whereas the culture, certainly more fastidious, makes it possible to detect bacteria even present at low inoculum thanks to the use of specific conditions. These approaches will be applied to more large scales in Africa. We began besides a work in collaboration with the Pasteur Institute from Dakar on 160 stools specimens of Senegalese patients with gastroenteritis versus a group controls subjects without diarrhoea. We also will apply these techniques on stool specimens collected from travellers in Africa. We will be able to thus establish the repertory of the bacteria present in the stools before the departure, at the time of a possible diarrheal episode in Africa and at return. We will search for micro-organisms in these stools specimens in collaboration with the team of Pr Bernard Scola, by inoculating these samples on amoebas, in order to isolate giant viruses. During a first test, we successfully isolated a new type of Marseilles virus from stools. Lastly, a project in relation to the team of Pr Michel Drancourt will enable us to develop DNA chips making it possible to test the whole of pathogens suspect to be responsible for diarrhoea at a large scale. We also will carry out the bacterial repertory of the stools of primates. Indeed, observation of primates is essential for the study of certain diseases which circulate between the animal and the human. Researchers already succeeded in detecting the antibodies directed against the simian HIV and sequenced also the viral ARN present in the stools. The presence of Plasmodium falciparum was also detected in the stools of gorillas. Our connection with the veterinary world and our former collaborations with the Center of Primatologie of the International Center of Medical research of Franceville (Gabon) will allow us, via convention of research to carry out the collection of stools of primates that are in captivity, but also in the natural environment. In addition to the bacterial repertory, we will search in these stools the virus of hepatitis E as well as the antibodies directed against this last in collaboration with Dr. Philippe Colson.

 

References

(1) Mediannikov O, Sekeyova Z, Birg ML, Raoult D. A novel obligate intracellular gamma-proteobacterium associated with ixodid ticks, Diplorickettsia massiliensis, Gen. Nov., Sp. Nov. PLoS One 2010;5:e11478.

(2) Fenollar F, Rolain JM, Alric L, Papo T, Chauveheid MP, van de BD, et al. Resistance to trimethoprim/sulfamethoxazole and Tropheryma whipplei. Int J Antimicrob Agents 2009;34:255-9.

(3) Lagier JC, Lepidi H, Raoult D, Fenollar F. Clinical presentation of 142 patients with systemic Tropheryma whipplei infections diagnosed or confirmed in a reference center. Medicine 2010;89:337-45.

(4) Fenollar F, Mediannikov O, Socolovschi C, Bassene H, Diatta G, Richet H, et al. Tropheryma whipplei bacteremia during fever in rural West Africa. Clin Infect Dis 2010;51:515-21.

(5) Fournier PE, Thuny F, Richet H, Lepidi H, Casalta JP, Arzouni JP, et al. Comprehensive diagnostic strategy for blood culture-negative endocarditis: a prospective study of 819 new cases. Clin Infect Dis 2010;51:131-40.


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