Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil

Abstract

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.Bacterial identification has always been a major challenge in all microbiological fields. Originally, all bacteria were identified according to their phenotypic characteristics after they had been isolated in pure culture. Only in the 1980s did scientists discover a huge discrepancy in the bacterial contents in aquatic and terrestrial habitats expressed as CFU after cultivation and direct microscopic enumeration (62). This discovery contributed highly to the boom in molecular biological methods of bacterial identification, which are independent of cultivation. These techniques have enabled scientists to discover and study actual bacterial diversity, which in most environments is orders of magnitude larger than culture-based techniques are able to reveal (40). However, characterization of bacteria after isolation still remains the most effective way of identifying bacterial properties.The necessity of fast and accurate identification of bacteria has been driven mostly by the needs of clinical and food microbiology, as these fields are directly connected with human health. Whereas tests based on biochemical traits are still important for the identification of pathogenic bacteria (64), they mainly fail with microbes isolated from environmental samples, as the diversity of microbes in these habitats is enormous (70). Environmental isolates are usually classified based on the primary structures of 16S rRNA genes (9). The process of DNA isolation, amplification of target genes, and sequencing is, however, rather time-consuming. Therefore, the introduction of mass spectrometry (MS) for the purposes of bacterial identification (13,24) represented an important ...

Applicability of MALDI mass spectrometry for diagnostics of phase variants in bacterial populations

“…Currently, MALDI TOF MS (matrix assisted laser desorption/ionization time of flight mass spec trometry) is increasingly applied to a large number of issues of microbial systematics, such as differentiation between the organisms belonging to closely related genomic species, preliminarily identification of new phenotypically close isolates, and identification and classification of bacteria at the species and the subspe cies levels [1][2][3][4][5][6]. This technique is based upon the mass spectrum analysis of the mass dependent quan titative distribution of the splinters of protein mole cules (m/z).…”

“…Ribosomal proteins account for ~20% of the total cell protein weight and for approximately 3% of the total cell weight. They are specific to organisms which develop under different conditions and, therefore, they can be used as biolog ical markers for phylogenetic analysis [4,5]. Impor tantly, the efficiency of the application of cell protein profiles, similar to that of other chemodiagnostic pro cedures, depends on the standardization of analytical conditions (microbial growth conditions, sample treatment, matrices in use, etc.)…”

Plant–microorganism interactions in bioremediation of polychlorinated biphenyl-contaminated soil

“…Interestingly, when the contaminated soil from the same site was vegetated by horseradish (Armoracia rusticana), among 54 isolates only one was identified as Pseudomonas spp. [18]. This is the other evidence that different bacterial genera are favoured in rhizospheres of different plants.…”

“…After direct extraction or liquid enrichment cultivation with biphenyl, obtained extracts were used for the preparation of serial decimal dilutions in 0.85% NaCl. In three replicates, 100 ml of the third and fourth dilutions were spread on PCA (Plate Count Agar, Oxoid, UK) and mineral medium [18] with a few crystals of biphenyl as a sole source of carbon. Isolates were cultivated at 288C until countable colonies emerged.…”

Studies on endophytic Burkholderia sp. from sugarcane and its screening for plant growth promoting potential

“…Samples for the analysis were prepared according to manufacturers" recommendations: after 24-48 h of cultivation of an isolate on LB medium at 28°C, a single colony was transferred with a sterile tip onto the MALDI target in triplicates, drizzled with 1μL of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in organic solution (50% acetonitrile, 2.5% trifluoroacetic acid), and directly screened. The measurement of the spectra was performed as described by Uhlik et al (2011). The matching of unknown spectra to the reference database is based on dedicated score (point) values.…”