Whole-cell MALDI-TOF mass spectrometry and multilocus sequence analysis in the discrimination of Pseudomonas stutzeri populations: three novel genomovars.

Pseudomonas stutzeri is a widely distributed species with very high genetic diversity and metabolic capacities, occupying many diverse ecological niches. A collection of 229 P. stutzeri strains isolated from different habitats and geographical locations has been previously characterised phylogenetically by rpoD gene sequencing analysis and in the present study 172 of them phenotypically by whole-cell MALDI-TOF mass spectrometry. Fifty-five strains were further analysed by multilocus sequencing analysis to determine the phylogenetic population structure. Both methods showed coherence in strain grouping; 226 strains were allocated in the 18 genomovars known presently. The remaining three strains are proposed as references for three novel genomovars in the species. The correlation and usefulness of sequence-based phylogenetic analysis and whole-cell MALDI-TOF mass spectrometry, which are essential for autoecological studies in microbial ecology, is discussed for the differentiation of P. stutzeri populations.

Concordance between whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the genus Pseudomonas.

Multilocus sequence analysis (MLSA) is one of the most accepted methods for the phylogenetic assignation of Pseudomonas strains to their corresponding species. Furthermore, updated databases are essential for correct bacterial identification and the number of Pseudomonas species is increasing continuously. Currently, 127 species are validly described in Euzéby's List of Species with Standing in Nomenclature, and 29 novel species have been described since the publication of the last comprehensive MLSA phylogenetic study based on the sequences of the 16S rDNA, gyrB, rpoB and rpoD genes. Therefore, an update of the sequence database is presented, together with the analysis of the phylogeny of the genus Pseudomonas. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight (WC-MALDI-TOF) mass spectrometry (MS) analysis has been applied very recently to the identification of bacteria and is considered to be a fast and reliable method. A total of 133 type strains of the recognized species and subspecies in the genus Pseudomonas, together with other representative strains, were analyzed using this new technique, and the congruence between the WC-MALDI-TOF MS and MLSA techniques was assessed for the discrimination and correct species identification of the strains. The utility of both methods in the identification of environmental and clinical strains is discussed.

Environmental microbiota represents a natural reservoir for dissemination of clinically relevant metallo-beta-lactamases.

A total of 10 metallo-ß-lactamase-producing isolates of six different species, including Brevundimonas diminuta (n = 3), Rhizobium radiobacter (n = 2), Pseudomonas monteilii (n = 1), Pseudomonas aeruginosa (n = 2), Ochrobactrum anthropi (n = 1), and Enterobacter ludwigii (n = 1), were detected in the sewage water of a hospital. The presence of bla(VIM-13) associated with a Tn1721-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, which produced VIM-2), and in two of them (R. radiobacter), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-ß-lactamase genes.

Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov.

Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3(T), CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase ß-subunit and RNA polymerase ß-subunit, genomic DNA-DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3(T) (CCUG 58127(T)=CECT 7729(T)) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta.

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences.

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg(2+). Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (K(obs) = 1.2 (+/-0.3) x 10(5) M(-1)) is tighter than that of MSK (2.9 (+/-1.0) x 10(4) M(-1)) at 25 degrees C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25 degrees C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.