Applications of MALDI-TOF and SARAMIS analyses for the identification of closely related clinically-relevant bacterial species

Abstract

Identification of prokaryotes in the complexity of microbial diversity is increasingly problematic for clinical diagnoses. Among the most difficult problems for clinical diagnostics is the identification of bacteria within “complexes” of closely related species, comprising pathogenic and non-pathogenic species with limited differentiating characteristics. However, these species complexes are composed of micro-organisms with different virulence potentials and it is essential to be able to obtain and confirm reliable identifications. A “polyphasic” multi-locus sequence analysis (MLSA) strategy may be applied for the typing and identification (species- and sub-species-levels) of bacteria, including “first-phase” comparisons of partial 16S rRNA gene sequences and subsequent “second-phase” analyses of one or more conserved house-keeping genes, for identification to the species level. These genotypic data are being used to correlate with the data obtained by MALDI-TOF (matrix-assisted laser desorption-ionisation time-of-flight) IC-MS (intact-cell mass-spectrometry) and analysed with the SARAMIS (Spectral Archive And Microbial Identification System) software and database package. The high reproducibility, rapid speed and low cost of MALDI-TOF IC-MS analyses makes the methodology ideal for processing large numbers of microbiological samples sent to the clinical lab for identification. The question of the resolution able to be obtained, using MALDI-TOF IC-MS was addressed by focusing analyses on bacteria comprising clinically-relevant species-complexes. The SARAMIS analysis of the MALDI-TOF IC-MS data was observed to be able to compare spectra from bacteria, yeast and fungi with reference spectra maintained in a database, identify the most closely related bacterial species with statistically-relevant confidence values and type strains to sub-species levels. The analyses are applicable to nearly all micro-organisms with high reproducibility.