MALDI-TOF Mass Spectrometry-Based Optochin Susceptibility Testing for Differentiation of Streptococcus pneumoniae from other Streptococcus mitis Group Streptococci.

Ilka D Nix,Markus Kostrzewa,Andreas Schlattmann,Karsten Becker,Evgeny A Idelevich,Katrin Sparbier

doi:10.3390/microorganisms9102010

Abstract

Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.

Highlights

The results of the classical bile solubility and optochin susceptibility testing by disk diffusion corresponded to the molecular identification results, but with limitations in correct identification of S. pseudopneumoniae (Table S1)
Twenty-four clinical isolates of the test set were confirmed as 12 S. pneumoniae and 12 Streptococcus mitis group (SMG) isolates by molecular and biochemical identification, as well as classical bile solubility and optochin susceptibility by disk diffusion, but there were difficulties in correct identification of S
This study showed that optochin susceptibility testing by an alternative MALDI-TOF

Summary

Introduction

The genus Streptococcus comprises more than 100 different species and it is to be expected that further species will be discovered [1]. Differentiation of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species in clinical diagnostics is still challenging, but of major relevance due to their different pathogenic potential [1]. Various experimental approaches for discrimination of S. pneumoniae, e.g., bile solubility, optochin susceptibility, biochemical or molecular methods, are applied in clinical diagnostics, with their respective advantages and limitations [1]. Even the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as an accurate and rapid method has limitations in differentiating S. pneumoniae from other SMG streptococci [1,2,3,4,5,6,7,8].

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