Team 6: Jean-Marc Rolain, PU-PH
- Jean-Marc ROLAIN, PU-PH, HDR
- Martine REYNAUD-GAUBERT, PU-PH, HDR
- Jean-Christophe DUBUS, PU-PH, HDR
- Véronique ANDRIEU, MCU
- Véronique ROUX, MCU, Praticien Attaché
- Jean-Michel BRUNEL, CR1, HDR
The team aims to identify and to describe new agents as well as to describe the microbiote responsible for respiratory tract infections in cystic fibrosis patients, to optimize the treatment of these respiratory infections by understanding the mechanisms of resistance to antibiotics of pathogenic emergent, multiresistant bacteria. The team associates multi-field competences including clinicians specialized in cystic fibrosis, a chemist, a researcher specialized in drugs working and a microbiologist.
Projects for the next four-year plan:
Project 1: Evaluation of the sensitivity and the resistance to the anti-infectious agents – molecular mechanisms of resistance to antibiotics - epidemiological survey.
The life expectancy of cystic fibrosis patients increased primarily thanks to the use of antibiotics to treat bronchial exacerbations during the disease. Nevertheless, the chronic colonization of these patients as well as the iterative antibiotic cures at the origin on the one hand of an increase in resistances of pathogenic bacteria and on the other hand of the emergence of multiresistant organisms, some of which we have characterized during the preceding four-year plan (1-6). The objectives for the next four-year plan are including:
- Genetic identification and study of the molecular mechanisms of resistance to antibiotics of emergent and multiresistant pathogens during cystic fibrosis – Development of fast molecular detection of resistance by pyrosequencing.
- Study of lateral transfert of genes encoding resistance to antibiotics - Resistance to antibiotics and generalized transduction induced by Acyl Homoserine Lactones (AHL) during the cystic fibrosis. This project is currently underway and preliminary data indicate that the AHL of P. aeruginosa induces the phages of S. aureus and thus induce the transfert of genes including genes of resistance (unpublished data).
- Genomic analysis of epidemic and/or multiresistant bacterial strains by high throughput pyrosequencing. During the preceding four-year plan we sequenced and analyzed the genome of an epidemic strain of Staphylococcus aureus isolated from cystic fibrosis patients (7). We recently isolated and characterized for the first time a new multiresistant pathogen, Pandoraea pulmonicola responsible for the death of a patient after bipulmonary transplantation (unpublished data). This organism is resistant to all currently known antibiotics (except for the tigécycline), we plan to sequence its genome by high throughput pyrosequencing during the next four-year plan in order to understand and to characterize the genetic determinants associated with this multiresistant phenotype.
- Identification and biotyping of the bacterial strains during respiratory infections by MALDI-TOF. The work completed during the preceding four-year plan enabled us to use the MALDI-TOF technique to characterize toxins present in some S. aureus strains (8). We now aim to use this method to identify and characterize S. aureus and Pseudomonas aeruginosa isolates from cystic fibrosis patients, and in particular to use this technique for biotyping and epidemiologic monitoring. Some works indicate that it is possible to carry out this type of monitoring in real time as for cystic fibrosis patients. In addition we could show that this approach made it possible to detect epidemics among cystic fibrosis patients, in particular an epidemic of respiratory infections with Corynebacterium pseudodiphtheriticum in cystic fibrosis children (Bittar F et al , Emerg Infect Dis, 2010, In press).
- Phenotypic detection of antibiotic resistances and virulence factors by MALDI-TOF.
- Epidemiologic monitoring of multiresistant bacteria – development of molecular techniques for the detection of genes encoding resistance to antibiotics.
- Molecular typing of the bacterial isolates from various patients to document an "epidemic" condition or from the same patient to evaluate the stability of colonization among these patients.
Project 2: Detection and Identification of emerging pathogens in respiratory tract infection. The respiratory tract microbiote of cystic fibrosis patients.
During the preceding four-year plan we could show the extraordinary bacterial diversity of the respiratory tract microbiote in cystic fibrosis patients (4) and this was confirmed by another research team (9). This work opened a new field of research which will be developed during the next four-year plan, the characterization of the respiratory microbiote among these patients in order to better understand microbial associations possibly correlated to an unfavourable evolution among these patients in order to be able to propose innovating therapeutic strategies.
- Molecular identification and bacterial taxonomy of new pathogenic agents.
- Realisation of studies of viral and bacterial metagenomics on pulmonary tract specimens of cystic fibrosis patients – Characterization of the microbiote during the cystic fibrosis. Recent work highlighted the role of the respiratory microbiote in the clinical evolution of the cystic fibrosis patients (10).
- Realisation of metagenomic studies on pulmonary samples of patients for whom no agent could be identified (in collaboration with the team 9: L Papazian).
- By systematic sampling of expectorations, realisation of exhaustive research on emergent and/or multiresistant infectious agents responsible for colonisations and infections of the respiratory tract among cystic fibrosis patients.
- By using original techniques of isolation and culture, or of detection and molecular identification for the infectious agents during pulmonary infections.
- Bacterial colonisation and pulmonary transplantation on cystic fibrosis patient. Comparative metagenomics.
- Genotyping of highly pathogenic and highly resistant to antibiotics strains in order to be able to propose isolation measures for the patients if necessary.
- Phenotypic identification of lung cancers by MALDI-TOF (with L Papazian).
Project 3: New therapeutic approaches and development of the new galenic forms for the administration of antibiotics
- Development of new galenic vector forms of drugs for the pulmonary way and optimization of the existing forms (anti-infectious agents and anti-inflammatory drugs) – (VA-JCD)
- Design and evaluation of a new atomizer for infants (accepted ANR project)
- Effectiveness of aminosterol derivatives administrated as aerosols in a murine model of chronic Pseudomonas aeruginosa bronchial colonisation (with L Papazian and A Roch)
- Effectiveness of pomade in a murine model of Staphylococcus aureus decolonisation (with JM. Brunel)
- Development of new chemical molecules derived from the squalamine for a use as biocides for the decontamination of inhalation devices and in the local aerosol treatment of chronic bacterial colonisations in cystic fibrosis patients – Clinical trial.
- Development of new galenic forms vectors of disinfecting for the sterilization of the inhalation devices.
The team will continue to teach doctorants, on average 2 by senior that is between 6 and 8 as well as Master 2 students, 4 per year. The team benefits from platforms (genomic, proteomic, imagery, bioinformatic) and from the expertise of the engineers in charge of the platforms.
On the whole the team constituted for the next four-year plan is in perfect coherence with the URMITE and has a will to go-on developing a therapeutic department with a potential for the evaluation of innovative therapeutic strategies by approaches radically different in the field from emergent and multiresistant pathogens responsible for respiratory tract infections, in particular in cystic fibrosis patients.
(1) Bittar F, Rolain JM. Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients. Clin Microbiol Infect 2010;in press.
(2) Armougom F, Bittar F, Stremler N, Rolain JM, Robert C, Dubus JC, et al. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing. Eur J Clin Microbiol Infect Dis 2009 May 16.
(3) Bittar F, Leydier A, Bosdure E, Toro A, Boniface S, Stremler N, et al. Inquilinus limosus and cystic fibrosis. Emerg Infect Dis 2008;14(6):993-5.
(4) Bittar F, Richet H, Dubus JC, Reynaud-Gaubert M, Stremler N, Sarles J, et al. Molecular Detection of Multiple Emerging Pathogens in Sputa from Cystic Fibrosis Patients. PLOS One 2008;3(8):e2908.
(5) Bittar F, Reynaud-Gaubert M, Thomas P, Boniface S, Raoult D, Rolain JM. Acetobacter indonesiensis pneumonia after lung transplant. Emerg Infect Dis 2008 Jun;14(6):997-8.
(6) Menuet M, Bittar F, Stremler N, Dubus JC, Sarles J, Raoult D, et al. First isolation of two colistin-resistant emerging pathogens, Brevundimonas diminuta and Ochrobactrum anthropi, in a woman with cystic fibrosis: a case report. J Med Case Reports 2008;2:373.
(7) Rolain JM, Francois P, Hernandez D, Bittar F, Richet H, Fournous G, et al. Genomic Analysis of An Emerging Multiresistant Staphylococcus aureus Strain Rapidly Spreading in Cystic Fibrosis Patients Revealed the Presence of an Antibiotic Inducible Bacteriophage. Biol Direct 2009 Jan 13;4(1):1.
(8) Bittar F, Ouchenane Z, Smati F, Raoult D, Rolain JM. MALDI-TOF-MS for rapid detection of staphylococcal Panton-Valentine leukocidin. Int J Antimicrob Agents 2009 Nov;34(5):467-70.
(9) Harris JK, De Groote MA, Sagel SD, Zemanick ET, Kapsner R, Penvari C, et al. Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis. Proc Natl Acad Sci U S A 2007 Dec 18;104(51):20529-33.
(10) Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS ONE 2010;5(6):e11044.