Taxonomic resolution

Though especially the cultural parameters of the studied cells have a huge impact on their signature variability it seems that - a decent Raman signature library provided - the majority of cultivation differences can be accommodated and thus no extreme standardization of culturing conditions is required for identification via this method. But for the sake of taxonomic resolution care has to be taken in finding appropriate culturing conditions to affect the implementation possibilities of Raman spectroscopy in microbial analysis positively [43]. 

Currently, advanced molecular methods represent an invaluable tool in the study of food ecosystems and the strains responsible of fermentation processes. At the same time, especially in industrial or applied microbiology, phenotypic tests, which were used widely in the past for microbial identification, are still being considered for the characterization of strain metabolic properties, growth performance, resistance to industrial processes, and shelf life. However, due to their poor reproducibility and low discriminatory power, phenotypic methods have been almost abandoned for identification purposes. Their low taxonomic resolution often leads to differentiation only at the genus level, and they require a labor-intensive approach. On the contrary, genotypic techniques provide a more robust classification and identification, and their costs, over the years, have been decreasing. 

Methods to enhance the taxonomic resolution of MALDI TOP MS to characterize bacteria to the subspecies and strain levels are reviewed in Chap. 6 by Zhang and Sandlin. They focus on several experimental factors that will improve strain-level... 

Lasch and cowoikers describe in Chap. 8 their group s efforts to improve taxonomic resolution without compromising the simplicity and the speed of MALDI TOF MS. Such improvements may be achieved by signature database expansion with novel and diverse strains, optimization, and standardization of sample preparation and data-acquisition protocols. Further enhancement in data analysis pipelines including more advanced spectral preprocessing, feature selection, and supervised methods of multivariate classification analysis also contribute to taxonomic resolution enhancements. Strains of Staphylococcus aureus. Enterococcus faecium, and Bacillus cereus are selected to illustrate aspects of that strategy. 

Maximizing the Taxonomic Resolution of MALDI-TOF-MS-Based Approaches to Bacterial Characterization From Culture Conditions Through Data Analysis... 

Fig. 6.1 Increasing taxonomic resolution is required to reliably characterize bacteria when moving from the genus level (e.g., Escherichia) to the subspecies (e.g., K-12) level. Well-described and effective methods exist for many applications at the genus and species levels, while strain-level applications require additional methodological development and optimization...

Fig. 6.2 A generie workflow from cultivation through data analysis to characterize bacteria using MALDI-TOF MS. At each step of the workflow, different approaches have been employed and can be optimized to maximize taxonomic resolution...

Several studies have suggested that protein extraction-based methods afford higher taxonomic resolution than intact cell-based methods. For example, Schitl-thess et al. (2014) compared three sample preparation methods for identification of 190 Gram-positive rods including 64 species from 21 genera. Results showed that species-level identification rates were higher with a plate-based formic acid extraction and an ethanol-formic acid extraction than with an intact cell-based direct smear method. Rates of correct identification were 68.4% with plate-based formic acid extraction, 71.6% with ethanol-formic acid extraction, and 63.7% with a direct smear method. 

Overall, MALDI-TOF profiling of bacteria has shown remarkable pronrise at the genus, species, and strain levels for various bacteria however, the limits of taxonomic resolution of this techiuque may impede its broader implemeutatioiL Additional efforts to ma ximize the taxonomic resolution of this method by optimizing experimental conditions— fiom culture condition through data analysis— are warranted. 

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