A novel short-term high-lactose culture approach combined with a matrix-assisted laser desorption ionization-time of flight mass spectrometry assay for differentiating Escherichia coli and Shigella species using artificial neural networks.

Abstract

Escherichia coli is currently unable to be reliably differentiated from Shigella species by routine matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. In the present study, a reliable and rapid identification method was established for Escherichia coli and Shigella species based on a short-term high-lactose culture using MALDI-TOF MS and artificial neural networks (ANN). The Escherichia coli and Shigella species colonies, treated with (Condition 1)/without (Condition 2) a short-term culture with an in-house developed high-lactose fluid medium, were prepared for MALDI-TOF MS assays. The MS spectra were acquired in linear positive mode, with a mass range from 2000 to 12000 Da and were then compared to discover new biomarkers for identification. Finally, MS spectra data sets 1 and 2, extracted from the two conditions, were used for ANN training to investigate the benefit on bacterial classification produced by the new biomarkers. Twenty-seven characteristic MS peaks from the Escherichia coli and Shigella species were summarized. Seven unreported MS peaks, with m/z 2330.745, m/z 2341.299, m/z 2371.581, m/z 2401.038, m/z 3794.851, m/z 3824.839 and m/z 3852.548, were discovered in only the spectra from the E. coli strains after a short-term high-lactose culture and were identified as belonging to acid shock protein. The prediction accuracies of the ANN models, based on data set 1 and 2, were 97.71±0.16% and 74.39±0.34% (n = 5), with an extremely remarkable difference (p < 0.001), and the areas under the curve of the receiver operating characteristic curve were 0.72 and 0.99, respectively. In summary, adding a short-term high-lactose culture approach before the analysis enabled a reliable and easy differentiation of Escherichia coli from the Shigella species using MALDI-TOF MS and ANN.