ANR-05-SEST-0009

The aims of this research project are: - to estimate the effect of various practices like composting, the use of wastewater treatment muds in agriculture, and the effect of natural lagooning of sewages, on the spread, growth and evolution of human bacterial pathogens (Listeria monocytogenes, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aeromonas hydrophila) ; - to study the genetic diversity of environmental populations of selected bacterial pathogens found in various composts, wastewater treatment muds, natural lagooning wastewater treatment station, and agricultural soils amended by rejects from wastewater lagoons or treatment station and other type of composts ; - to compare the genetic diversity of (the above cited) environmental strains with the one of strains recovered from community-acquired and hospital infections ; - to investigate the virulence and antibiotic resistances of environmental strains of selected groups of bacterial pathogens (Listeria monocytogenes, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aeromonas hydrophila), and to compare these properties with those of clinical isolates. The selected pathogens will be isolated from the various environmental samples (agricultural soils, sewages, composts, etc) by direct screenings on selective media. Identifications will be performed using either biochemical tests or molecular approaches (PCR screenings of virulence genes and other molecular markers). The recovered and properly identified strains will be analysed by pulsed field gel electrophoresis (PFGE). PFGE analyses will allow to estimate the genetic diversity among each population of pathogens identified, and will allow to identify and define some clonal complexes (electrophoretic types –ET complexes) that might be favored in some of the samples analysed. MLST (multi locus sequence typing) analyses of selected P. aeruginosa and B. cenocepacia environmental strains will be performed. The MLST data set obtained will be compared with those available for clinical strains. MLST will allow to clearly determine the relationships between environmental and epidemic clones recovered from major disease outbreaks. The virulence properties of the environmental strains recovered in this study will be investigated using alternative host models (primarily alfalfa, Dictyostelium amoebae, chicken eggs and BALB/C mice). PCR screenings of virulence genes will also be performed. Some virulence factors will be detected by biochemical tests. Some epidemic strains will be introduced in soil microcosms (flasks) to investigate the properties involved in adaptation to niche changes (using a proteomic approach). Expected results: - snapshots of the genetic diversity of strains of major pathogens found in french agricultural soils amended with various composts, and found in wastewater treatment stations/lagoons (that can spread these pathogens in natural water streams) ; - data on the relationships between PFGE genotypes - ET complexes (clonal complexes) of the environmental strains of these pathogens and their virulence / antibiotic properties; - MLST-PFGE data on the genetic proximity of the environmental strains of selected pathogens with strains involved in major epidemic outbreaks ; - MLST data allowing to conclude on the frequency of genetic transfers between environmental and clinical strains ; - data on the survival and growth of the various PFGE genotypes identified in the field situations selected for this study – with data accumulated for more than 3 yrs in some instances ; - data on the genetic structure of the environmental populations identified ; - data on the physiology and activities performed by these pathogens in soils