Abstract
Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000–20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories.
Highlights
MALDI-TOF MS is an emerging technology that has been increasingly used in clinical laboratories for rapid bacterial identification[6]
The capsular types were previously determined for all 416 strains by the Quellung reaction with antisera kindly provided by the Streptococcus Laboratory at the Centers for Disease Control and Prevention (CDC, GA, USA), and by multiplex PCR for a fraction of the isolates[12,13,14,15,16,17]
Isolates belonging to certain capsular types, such as 9N and 9V, showed a more random distribution across the tree, while strains belonging to other serotypes, such as 6B and 14, showed a tendency to a more homogeneous clustering
Summary
A total of 416 Streptococcus pneumoniae isolates were analyzed, including 141 strains of serotype 14, 49 strains of serotype 6A, 143 strains of serotype 6B, 38 strains of serotype 6C, 24 strains of serotype 9N and 21 strains of serotype 9V. Isolates from carriage studies, including both children and adults, were recovered from clinical specimens as approved by the ethics committees of the institutions involved. All biomarkers automatically detected by BioNumerics were exported to a spreadsheet containing all strains (see Supplementary Spreadsheet S1). This spreadsheet was visually analyzed by counting the number and calculating the percentage of strains containing a certain biomarker within each serotype. Reference or internal control S. pneumoniae strains (strain ATCC700902 of serotype 14, strain Sp1019 of serotype 9N, strain ATCC700671 of serotype 9V, strain ATCCBAA659 of serotype 6A, strain ATCC700675 of serotype 6B and strain 2008008385 of serotype 6C) were analyzed
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