Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting

Abstract

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

FIG. 1.

Bacteria were recovered from blood culture fluids by differential centrifugation. Washed pellets were resuspended in 300 μl H₂O, inactivated with ethanol, and prepared for subsequent mass spectrometry fingerprinting by formic acid extraction (see the text for details).

FIG. 2.

Mass spectra from blood culture samples spiked with different concentrations of S. aureus Newman. Lower inocula resulted in decreasing numbers of detected bacterial peaks and reduced concordance scores for the best-matching reference spectrum from the Biotyper database (inscribed numbers). At 10⁶ CFU/ml, spectra resembled those from sterile cultures, and species identification failed.

FIG. 3.

Effect of bacterial concentration and culture age on identification scores from direct mass spectrometry fingerprinting. Blood culture samples preincubated for 1 (filled circles) or 7 days (open circles) were spiked with different concentrations of S. aureus Newman and E. coli DH5α and subjected to mass spectrometry fingerprinting. Symbols and error bars indicate mean identification scores (in arbitrary units [a.u.]) and standard deviations from at least four independent sample preparations. Solid and dotted lines represent best fits from nonlinear regression with 95% CIs. The shaded area marks the range of bacterial concentrations at the time of culture positivity as detected in a random selection of 30 study samples. A clear association between bacterial concentration and identification scores was observed. Although advanced culture age led to reduced identification scores, the majority of clinical samples are predicted to yield sufficient scores for secure species-level identification.