Date Published: April , 2009
Publisher: A.I. Gordeyev
Author(s): E. N. Ilina.
This study examines the features and limitations of direct Matrix-Assisted Laser Desorption-Ionisation (MALDI) mass-spectrometry profiling of bacterial cells for investigating a microbial population. The optimal laboratory protocol, including crude bacteria lyses by a solution of 50% acetonitrile, 2.5% trifluoroacetic acid, and using α-cyano-4-hydroxy cinnamic acid as the MALDI matrix, has been developed. Two different bacteria species were under investigation, and representative mass spectra from 278 strains of Neisseria gonorrhoeae and 22 strains of Helicobacter pylori have been analyzed. It’s known that both bacteria demonstrate a variable degree of polymorphism. For N. gonorrhoeae, the MALDI mass spectra that was collected possessed about 70 peaks, 20 of which were good reproducible ones. In spite of the fact that three peaks were found with differing spectra in some strains, little diversity in the N. gonorrhoeae population was revealed. This fact indicates the prospects in using direct MALDI mass-spectrometry profiling for gonococcus identification. In the case of H. pylori strains, the variety in the collected mass-spectra was shown to be essential. Only five peaks were present in more than 70% of strains, and a single mass value was common for all spectra. While these data call into question the possibility of the reliable species identification of H. pylori using this approach, the intraspecies differentiation of strains was offered. Good association between MALDI profile distributions and the region of strain isolation have been found. Thus, the suggested direct MALDI mass-spectrometry profiling strategy, coupled with special analysis software, seems promising for the species identification of N. gonorrhoeae but is assumed insufficient for H. pylori species determination. At the same time, this would create a very good chance for an epidemiological study of such variable bacteria as H. pylori.
Modern microbiology and its applied branches require the development of new rapid and precise methods for identifying clinically significant pathogens and for describing their characteristic features such as virulence, antibiotic sensitivity, and strain group. The relative tolerance of Matrix-Assisted Laser Desorption-Ionisation (MALDI) to contamination with salt and other impurities allows one to conduct a direct mass-spectrometry analysis of the microbial cell content (direct profiling) without the fractionation and purification of some components.
Bacterial strains The following laboratory strains were used in the investigation: Escherichia coli DH5α, Neisseria gonorrhoeae ATCC 49226, Helicobacter pylori J99, and Helicobacter pylori 26695; 278 N. Gonorrhoeae clinical strains from different regions of Russia (Moscow, St. Petersburg, Samara, Ekaterinburg, Murmansk, and Irkutsk); and 22 H. pylori clinical strains from Mongolia, Tuva, Yakutia, and the Moscow Region.
The protocol of direct MALDI mass-spectrometry bacteria profiling was optimized using the laboratory E. coli DH5α strain, whose protein and nucleotide sequences were studied thoroughly. The method was based on the acid lysis of bacterial cells, which causes the extraction of the major ribosomal proteins that make up 20% of the total pool of E. coli proteins .
The consolidated experience of using direct MALDI mass-spectrometry bacteria profiling gives grounds to assume that this approach may be used for the specific identification of the causative agent that was demonstrated when analyzing the voluminous group of clinical N. gonorrhoeae strains. On the contrary, intraspecies classification and typing of bacteria with this method is likely to be uninformative for species with a low intraspecies variability.
The work was supported by the Bruker Daltonics Company, Germany (grant no. 270505(#30686).