Methods: The culture collection (n=124) comprised PCR-typed isolates of all capsule types (a-f) and NTHi, isolated from various geographical regions and time periods. For mass spectra acquisition, isolates were prepared with ethanol-formic acid extraction, and analyzed with a Microflex LT MALDI-TOF MS using default settings and mass range (2,000-20,000 m/z). A new MALDI Biotyper library of Main Spectra (MSPs) was created with isolates (n=35) from all types, and the remaining isolates (n=89) were used as test isolates. Six spectra per test isolate were acquired. Each spectrum was classified according to the capsule type of the best match in the new MSP library. Classification of ≥5 of 6 spectra to a specific type was required for isolate typing. Spectra were also manually analyzed to identify type specific peak patterns, and spectra of capsulated isolates were examined by principal component analysis (PCA).
Results: Capsule type classification using MALDI Biotyper with the novel MSP library had 100% sensitivity for all capsulated types (a-f), and an overall specificity of 98%, using our test collection. The capsule types of H. influenzae are generally clonal, with two known clusters of types a and b. Manual analysis of mass spectra of different capsule types supported this notion. Distinct patterns within the capsule types were identified as well as type specific peaks. NTHi had more heterogeneous mass spectra profiles, as expected. PCA of mass spectra revealed discrete clustering of types b, e and f.
Conclusion: The study demonstrates the capability of regular MALDI-TOF MS analysis to discriminate different capsule types of H. influenzae. With optimization, this could be a valuable tool in clinical H. influenzae diagnostics.
G. Kahlmeter, None
M. Kilian, None
J. S. Kroll, None
K. Riesbeck, None
F. Resman, None
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