Estudio molecular de poblaciones Pseudomonas ambientales

Résumé

The main goal of the present work is a thorough study of the diversity of Pseudomonas populations present in several habitats. In chapter one, the population structure and microdiversity of 53 Pseudomonas aeruginosa isolates from environmental samples and clinical specimens obtained in Mallorca (Spain) has been analyzed by a multilocus sequence typing approach (MLST). Antibiotic multiresistance to several antibiotics was only found in isolates of clinical origin. The high number of new alleles and new sequence types found in a limited area reflects the great diversity of P. aeruginosa populations and the high plasticity of a paradoxically phylogenetic conserved genome of P. aeruginosa. The calculated genetic diversity index also demonstrated the high diversity of the population under study. Clonality tests demonstrated that recombination plays a key role in the distribution of alleles. The ST-1146 was the only one found in both kind of samples, 3 environmental isolates (from the same site isolated at 2 different dates) and 1 clinical isolate, with differences in its antibiotic susceptibility profile. For this reason, the 4 genomes of newly described sequence type ST-1146 have been sequenced and analyzed. In the second chapter, the four genomes of ST-1146 were obtained and the sequences assembled de novo and compared with the CD-HIT program. Results showed that the number of isolate-specific genes was higher in the clinical isolate (SD9) than in environmental isolates (P37, P47 and P49). Some genes related to phage Pf1 and to other phages similar to bacteriophages F116 and H66 were found in isolate SD9 but not in the other isolates of ST-1146. The bacteriophage Pf1 region in isolate SD9 accumulated the highest number of mutations in comparison with the environmental isolates. Comparative genomic methods indicated that the isolates of ST-1146 are closely related, and most genes implicated in pathogenicity are highly conserved in the environmental isolates, suggesting the genetic potential for infectivity similar to that of the clinical isolate. Moreover, the four genomes were mapped against the reference genomes of P. aeruginosa PAO1-UW and UCBPP-PA14. A mutational profile was performed as a result of each comparison. The clinical isolate showed in both comparisons a number of exclusive alleles 2.5 and 3.6 times greater than the environmental isolates. These results suggest that the mutation pressure is not the same in the environmental isolates than in the clinical one. In the third chapter, the River Woluwe has been taken as a model habitat for the study of the diversity of species in the genus Pseudomonas. A water sample from a non-contaminated site at the source of the river was analyzed by culture-dependent and –independent methods. Identification of the Pseudomonas isolates was performed by sequencing and analysis of their rpoD sequence. Culture-independent methods consisted of a cloning and pyrosequencing of a specific rpoD amplicon obtained from total DNA extracted from the same sample and amplified by Pseudomonas rpoD primer set EGPsF340-EGPsR 780. It was remarkable the number of known species detected in the sample by the three different methods: 26 species distributed in 13 phylogenetic groups or subgroups within the genus. Pyrosequencing was the more powerful analysis; sequences obtained represented the 24 species with the exception of P. stutzeri and P. simiae. The predominant phylogenetic group within the Pseudomonas genus was Pseudomonas fluorescens group in the cultures and in the culture-independent analysis. In all analysis a high number of putative novel species were found indicating the enormous diversity not described yet. In the fourth chapter, several Pseudomonas strains have been isolated from environmental samples, from soil and intertidal habitats. In the identification process, some of these strains have not been assigned to known Pseudomonas species and were considered members of putative novel species. In their phylogenetic characterization by MLSA we found that strains in the culture collection of our laboratory were close-related and therefore they were also included in the taxonomic characterization of these putative novel species. MLSA demonstrated that 3 putative novel species were represented by 6, 5 and 1 strains respectively, which will be the subject of additional studies. Four other strains were deeply studied by a taxonomic polyphasic approach, including morphological, physiological, biochemical, chemotaxonomic and genomic characterizations. These studies demonstrated that the four strains cannot be assigned to any of the known Pseudomonas species and we propose the creation of two novel species, Pseudomonas aestusnigri (strain VGXO14T = CECT 8317 T = CCUG 64165 T) and Pseudomonas terricola (strain S58 T = CECT 8389 T = CCUG 64415 T).