Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation

• Published in: European journal of clinical microbiology & infectious diseases 2017-02, Vol.36 (2), p.351-359
• Main Authors: Mulet, M., Gomila, M., Ramírez, A., Cardew, S., Moore, E. R. B., Lalucat, J., García-Valdés, E.
• Format: Article
• Language: English
• Subjects: Antibiotic resistance; Antimicrobial agents; Biomedical and Life Sciences; Biomedicine; Cluster Analysis; Deoxyribonucleic acid; DNA; DNA Gyrase - genetics; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; DNA-Directed RNA Polymerases - genetics; Genes; Genotype; Humans; Internal Medicine; Ionization; Mass spectrometry; Medical laboratories; Medical Microbiology; Microbiology; Mikrobiologi; Multilocus Sequence Typing; Nosocomial infections; Original Article; Pathogens; Phenotype; Phylogenetics; Phylogeny; Pseudomonas; Pseudomonas - chemistry; Pseudomonas - classification; Pseudomonas - genetics; Pseudomonas - isolation & purification; Pseudomonas aeruginosa; Pseudomonas Infections - microbiology; RNA polymerase; RNA, Ribosomal, 16S - genetics; Scientific imaging; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0934-9723; 1435-4373; 1435-4373
• DOI: 10.1007/s10096-016-2808-4

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene ( rpoD) . Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B ( gyrB ) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.