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New genotypic and phenotypic analyses of clinically-relevant Gram-negative, non-fermenting bacteria: MALDI-TOF MS as a rapid, high-resolution method for identifying and typing microorganisms

Poster
Authors Liselott A Svensson
Margarita Gomila
Sashka A Mihaylova
Marcel Erhard
Edward R.B. Moore
Published in 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Vienna, Austria
Publication year 2010
Published at Institute of Biomedicine, Department of Infectious Medicine
Language en
Subject categories Molecular biology, Microbiology, Biological Systematics

Abstract

Objectives. Identification of Gram-negative, non-fermenting bacilli, using phenotypic characterization is problematic. Many of the species of this group are frequent nosocomial infectious agents and are ubiquitous in the environment. The aims of this study were to assess the resolving capacities of “house-keeping” gene sequences, including 16S rRNA, atpD, gyrB, recA, rpoB and rpoD, and to compare a multi-locus sequence analysis (MLSA) with matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry analyses for identifying and typing strains of Achromobacter, Bordetella, Burkholderia, Pseudomonas, and Stenotrophomonas species. Methods. Genotypic analyses. Type strains of the focus genera and species-complexes, other well-characterised reference strains and selected clinically-relevant strains representing a range of phenotypic and genotypic similarities were included in this study. Partial genes, 16S rRNA, atpD, recA, gyrB and rpoD recA were amplified by PCR and sequenced. DNA-DNA hybridisation analyses were carried out on selected strains for confirmation of species designations. MALDI-TOF analysis. Bacterial biomass were prepared from cultures on agar medium and analysed by MALDI-TOF MS, in the positive mode, using the SARAMIS software for analysis (1) Results. MLSA, using the respective house keeping genes were able to differentiate and identify the most closely related species of the analysed taxa and cluster analyses showed similarities of branching order between species that correlated well between different genes. However, different genes were not equally effective in differentiating species of the different genera. The MALDI-TOF analyses were effective in differentiating the most closely related species of the respective genera. Good correlation was observed between the results of MALDI-TOF MS and MLSA data. Conclusion. In most cases, clinically-relevant isolates and strains of Gram negative, non-fermenting bacilli exhibited good agreement between the methods of this study. In some cases, strains previously defined as given species were observed to be genotypically more similar to other species, as well as some strains with highly aberrant phenotypes were almost genotypically identical to the type strain. MALDI-TOF identification was very well correlated to the MLSA results, and is a much less expensive and effectively able to reduce identification times by 24-48 hours. (1) Vanlaere E et al. J. Microbiol. Meth. 75: 279-286 (2008).

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