Development of multilocus sequence analysis (MLSA) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for Alteromonas species classification

Abstract

Identification and classification of novel bacterial species is traditionally based on the polyphasic approach relying on phenotypic (e.g., growth and morphology), chemotaxonomic (e.g. mol% G + C composition, lipid and fatty acid composition), genotypic (e.g., chromosomal DNA - DNA hybridization (DDH)), and phylogenetic (e.g., 16S rRNA gene sequence analysis) characteristics. With fast growing numbers of newly described bacteria and correspondent sequencing data it has become apparent that the 16S rRNA gene sequence analysis may not be sufficient for species discrimination; the commonly used threshold value of 97% 16S rRNA gene sequence similarity was found to be not satisfactory as some distinct species share up to 99.9% of their 16S rRNA gene sequence similarity. Other molecular approaches which may be adopted in bacterial systematics are currently under intensive exploration in an attempt to improve efficiency and accuracy of the identification of novel species. Recently, multilocus sequence analysis (MLSA) has been introduced into bacterial systematics, in which multiple housekeeping genes (usually 5 to 7) are assessed as a group for discrimination of phylogenetically close bacteria. MLSA has been shown to have better taxonomic resolution for classification of closely related bacteria on the species level. Another technique, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS), has been shown to be reliable, easy to perform, cost effective, can be used across different conditions, and may even be used for subspecies discrimination. This study aimed to take the advantages of MLSA and MALDI-TOF MS for the identification of Alteromonas-related bacteria. A collection of marine bacteria belonging to the genus Alteromonas, as confirmed by the 16S rRNA gene sequence analysis, was isolated from sea water samples collected in Port Philip Bay (the Tasman Sea, Pacific Ocean). Bacterial identification based on the polyphasic approach indicated that some of these bacteria may be novel species. In order to evaluate the applicability of MLSA and MALDI-TOF MS for Alteromonas-related bacteria identification, a comparative study of newly isolated bacteria and other validly described species from phylogenetically close genera was performed. In the MLSA study, a group of housekeeping genes (gyrB, rpoB, dnaK, sucC, glyA, pmg, and metG) were amplified using previously and newly designed oligonucleotide primers. For mass spectral analysis, individual colonies were extracted and transferred directly onto the MALDI target plate. Samples were then introduced into the mass spectrometer and results were generated by the MALDI Biotyper software. The results obtained in this study indicated that both molecular techniques generated reliable data useful for Alteromonas species identification as alternative to DDH