Genotypic and phenotypic applications for the differentiation and species-level identification of achromobacter for clinical diagnoses.

The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected 'house-keeping' genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data

Dendogram of relatedness between the <i>Achromobacter</i> species strains analyzed based on MALDI-TOF MS analysis.

<p>Dendogram of relatedness between the <i>Achromobacter</i> species strains analyzed based on MALDI-TOF MS analysis.</p

Genotypic analyses and individual <i>recA</i>- and concatenated MLSA-based identifications of clinical and environmental strains of <i>Achromobacter</i> species.

<p>*indicates novel species, <i>i.e</i>., with <i>recA</i> similarities <97.6% to a recognized species.</p>#<p>determined from the concatenated sequences of <i>atpD</i>, <i>gyrB</i>, <i>recA</i> and <i>rpoB</i>.</p>I<p>Vandamme <i>et al.</i>, 2013a described <i>A. spiritinus</i> as a species, distinct from <i>A. marplatensis</i>.</p><p>Genotypic analyses and individual <i>recA</i>- and concatenated MLSA-based identifications of clinical and environmental strains of <i>Achromobacter</i> species.</p

Primers used for PCR-amplification and sequencing in this study.

<p>*This primer has been modified from the original primer.</p>#<p>For <i>recA</i> gene, the primer combination recAF126 and recAR928 was used. When the PCR failed, recAF126 and recAR1015 combination was used.</p><p>Primers used for PCR-amplification and sequencing in this study.</p

The variation in <i>recA</i> and <i>nrdA</i>-765 sequences among <i>Achromobacter</i> species.

<p>The y-axis shows the number of nucleotide positions, within 50-nucleotide position intervals (x-axis), in the respective sequences that exhibit variation between the type strains of the species of <i>Achromobacter</i>.</p

Phylogenetic tree of the strains of <i>Achromobacter</i> used in this study based on the phylogenetic analysis of <i>recA</i> gene.

<p>Distance matrix was calculated by the Jukes-Cantor method. Dendrogram was generated by neighbor-joining. <i>Bordetella pertussis</i> CCUG 30873^T was used as an outgroup. The bar indicates sequence divergence. Bootstrap values of more than 500 (from 1000 replicates) are indicated at the nodes.</p