Cultural, Serological, and Genetic Methods for Identification of Bacteria

Sutherland, John B.; Rafii, Fatemeh

doi:10.1002/0471748641.ch1

Cited by 3 publications

(1 citation statement)

References 74 publications

(148 reference statements)

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“…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).…”

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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

Uhlík

Strejcek

Junková

et al. 2011

Appl Environ Microbiol

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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 ...

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confidence: 99%