Assessment of the Pseudomonas species diversity along the Danube River by rpoD gene sequence analyses of cultivated strains. Danubio River

A collection of 611 Pseudomonas strains isolated from 14 sampling sites along the Danube River were identified by rpoD gene sequencing. Strains have been identified previously by MALDI-TOF MS with the VITEK MS system. A dendrogram was constructed with the protein profiles that grouped the strains in 53 clusters. Partial sequences of the rpoD gene of 190 strains representatives of all clusters were analyzed. Strains were grouped in the same phylospecies when they shared a minimum 95% similarity in their rpoD sequences. The sequenced strains were assigned to 37 known species (129 strains, 68%) and to 28 possible new species (61 strains, 32%). All strains in the same MALDI-TOF cluster were assigned to the same phylospecies when the rpoD sequences of the selected strains in the cluster showed similarities equal or higher than 95%. A total of 420 strains were identified at the phylospecies level combining both methods. The rest of strains were assigned to known Pseudomonas phylogenetic groups or subgroups. Most strains were assigned to phylospecies in the Pseudomonas putida phylogenetic group of species: 107 strains distributed in 31 different phylospecies. Special attention was given to multidrug resistant strains for their potential as environmental reservoir of resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50 % of the Pseudomonas species in the Danube River have been identified with the methodologies used. This study highlights the Pseudomonas species diversity, as well as the usefulness of rpoD gene sequences for identifications at the species level.