Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine diagnostics

Abstract

This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

Fig 1

Algorithm for the identification of Gram-positive cocci in routine diagnostics using MALDI-TOF MS. Recommendations are based on the results of the prospective study, including 1,619 clinical isolates. *¹, species and genera with less than five isolates were not integrated in this algorithm, as the numbers are too low to give a proper recommendation. However, all rare isolates were correctly identified by MALDI-TOF MS. It is suggested that these isolates are identified by 16S rRNA gene sequencing until sufficient data are available to update the approved lists. *², group assignment as follows: CoNS, all staphylococcal species except S. aureus and S. lugdunensis; viridans streptococci, alpha-hemolytic streptococcal species except S. pneumoniae; S. anginosus group, S. anginosus/constellatus/intermedius. *³, biochemical confirmation of S. pneumoniae by, e.g., testing of bile solubility and optochin susceptibility. *⁴, observed for the following streptococci: S. anginosus/constellatus, S. dysgalactiae/pyogenes, S. dysgalactiae/canis, S. equinus/lutetiensis, S. pneumoniae/oralis, and S. vestibularis/salivarius. *⁵, 16S rRNA gene sequencing and analysis as outlined by Bosshard et al. (1).