Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic <i>Escherichia coli</i>

<div><p>Background</p><p>In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic <i>Escherichia coli</i> O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.</p><p>Methods</p><p>Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 <i>E. coli</i> isolates from clinical samples collected during the outbreak.</p><p>Results</p><p>Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced <i>E. coli</i> strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.</p><p>Conclusions</p><p>MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic <i>E. coli</i>. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.</p></div

Effect of spectrum processing parameters on peak reproducibility.

<p>Number and proportion of reproducible peaks in TY-2482 formic acid extraction replicate spectra as a function of spectrum processing parameters (A). Half window sizes for SNIP baseline correction and signal to noise ratio thresholds for peak detection are represented by symbol and fill colour, respectively. For each combination, 16 variants representing different half window sizes for smoothing (2, 4, 8, 12) and peak detection (4, 8, 12, 16) are shown. Dashed lines mark the parameter combination employed for all subsequent analyses. Representative spectra from extreme positions of the parameter space (arrows) are shown in detail (B).</p

Variants of marker peak protein genes found among non outbreak study isolates by PCR and sequencing.

a<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101924#pone-0101924-t001" target="_blank">table 1</a>.</p>b<p>Outbreak strain variant.</p>c<p>No full length gene sequences for molecular weight prediction could be obtained from the respective isolates. Available partial sequences differ from the outbreak strain variant.</p><p>Abbreviations: MW Molecular weight.</p

Correct classification rates (%) of MALDI-TOF typing.

<p>Abbreviations: OREC outbreak related <i>E. coli</i> isolates, NOREC non outbreak related <i>E. coli</i> isolates, FAE formic acid extraction, DSD direct sample deposition.</p

Isolate classification with whole spectrum similarity measures.

a<p>Significant difference to the Jaccard distance (p<0.05).</p>b<p>Significant difference to FAE sample preparation (p<0.05).</p><p>Abbreviations: b binary, m metric, c correlation, FAE formic acid extraction, DSD direct sample deposition, AUC area under the ROC curve.</p

Distribution (%) of peak detection rates in triplicate spectra of outbreak isolates.

<p>Abbreviations: FAE formic acid extraction, DSD direct sample deposition.</p

Performance of isolate classification by whole spectrum similarity to reference spectra.

<p>Accuracy, sensitivity and specificity for the classification of study isolates by Jaccard’s distance to TY-2482 reference spectra as a function of the selected threshold. Grey areas represent bootstrap estimates of 95% confidence intervals for thresholds derived from the distribution of distance values among outbreak isolate triplicate spectra.</p

Oligonucleotides for biomarker gene detection and sequencing.

a<p>In <i>E. coli</i> TY-2482.</p

MALDI-TOF mass spectrum of <i>E. coli</i> outbreak isolate TY-2482.

<p>Representative whole cell MALDI-TOF mass spectrum of the Shiga-Toxigenic <i>E. coli</i> outbreak isolate TY-2482 acquired after formic acid extraction. Inlays show enlarged views of outbreak strain specific marker peaks and the amino acid sequence of the corresponding proteins. Peptides identified by LC-MS/MS are indicated by a gray background. The tick mark interval in the enlarged peak views is set to 100.</p

Whole spectrum similarity among non outbreak study isolates.

<p>Distribution of Jaccard distance values from pairwise spectrum comparisons among non outbreak study isolates (dark grey, n = 17955) and single isolate replicate spectra (light grey, n = 189). The dashed line represents a threshold for spectral identity derived from the replicate spectrum distribution (mean+2×SD). The dotted line represents a less conservative threshold that would correctly classify 95% of all isolate pairs that were found spectrally identical upon manual spectrum comparison.</p