Abstract
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.
Highlights
The Gram-negative, aerobic, non-fermenting bacteria are ubiquitously present in various ecosystems, important for environmental and biotechnological applications and many of these microorganisms have become problematic in hospital settings
All strains used in this study were obtained from the Culture Collection University of Gothenburg (CCUG; www.ccug.se), including the type strains of eleven validly published species of the genus Achromobacter: A. xylosoxidans CCUG 56438T; A. ruhlandii CCUG 57103T; A. piechaudii CCUG 724T; A. denitrificans CCUG 407T; A. insolitus CCUG 47057T; A. spanius CCUG 47062T; A. marplatensis CCUG 56371T; A. animicus CCUG 61966T; A. mucicolens CCUG 61961T; A. pulmonis CCUG 61972T; A. spiritinus CCUG 61968T; and 46 wellcharacterized strains of Achromobacter species of clinical and environmental origin
In the course of this study, two different research groups, in independent studies, described multi-locus sequence typing (MLST) and multi-locus sequence analysis (MLSA) approaches for high-resolution discrimination of strains of individual species and the differentiation of Achromobacter species [25, 29]
Summary
The Gram-negative, aerobic, non-fermenting bacteria are ubiquitously present in various ecosystems, important for environmental and biotechnological applications and many of these microorganisms have become problematic in hospital settings. Species of Pseudomonas, Burkholderia, Acinetobacter and Stenotrophomonas are the leading nosocomial pathogens in this expanding group [1, 2] and genera of the family Alcaligenaceae, i.e., Alcaligenes, Ralstonia, Achromobacter, etc., are emerging, as well, as significant pathogens in notable patient populations [3], those suffering from cystic fibrosis (CF). The ability to detect and correctly identify Achromobacter species, A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is increasingly important. The growing number of species and increasing complexity of bacterial taxonomy and the expansion of virulence and antibiotic resistance present significant challenges, requiring new development and periodic optimisation of identification protocols for new, as well as already described taxa
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