Accurate identification of Enterococcus lactis causing bacteraemia by matrix-assisted laser desorption ionization-time of flight mass spectrometry

<b>Introduction.</b> <i>Enterococcus faecium</i> clade B has recently been re-classified as <i>Enterococcus lactis</i>. Although <i>E. lactis</i> was previously associated with food products and probiotics, the recent re-classification has prompted the need for the accurate identification of this species and re-interpretation of its disease-causing ability. Since the re-classified <i>E. lactis</i> can currently only be identified by molecular techniques such as whole-genome sequencing, we constructed a MALDI Biotyper^® custom database to rapidly identify and differentiate <i>E. lactis</i> causing bacteraemia from <i>E. faecium</i>.<b>Hypothesis/Gap statement.</b> The re-classification of <i>E. faecium</i> clade B as <i>E. lactis</i> warrants the development of rapid and accurate identification methods to distinguish these species, particularly in clinical settings where <i>E. lactis</i> may be misidentified as <i>E. faecium</i>.<b>Aim.</b> The aim of this study was to construct a MALDI Biotyper^® custom database to rapidly identify and differentiate <i>E. lactis</i> causing bacteraemia from <i>E. faecium</i>.<b>Methodology.</b> A total of 97 enterococcal isolates, including 38 <i>E. lactis</i>, 51 <i>E. faecium</i> and 8 non-<i>E. faecium</i> non-<i>E. lactis</i> enterococci (<i>E. avium</i>, <i>E. casseliflavus</i>, <i>E. cecorum</i>, <i>E. durans</i>, <i>E. faecalis</i>, <i>E. faecium</i>, <i>E. gallinarum</i>, <i>E. lactis</i>, <i>E. mundtii</i> and <i>E. raffinosus</i>) were investigated. Whole-genome sequence analysis was used to confirm the species of each isolate. A MALDI Biotyper^® in-house database was constructed using 29 <i>E. lactis</i> isolates and the ethanol/formic acid/acetonitrile preparation protocol. The in-house database was validated using the 97 enterococcal isolates and the extended direct transfer preparation protocol.<b>Results.</b> Our in-house database correctly identified all isolates at the species level, including the <i>E. lactis</i> isolates, all of which were misidentified as <i>E. faecium</i> by the BioTyper^® MBT Compass reference library (2022). Of the 38 <i>E. lactis</i> isolates, 84.2% (<i>n</i>=32) were identified at the high probable species level (score ≥2.300), while the remaining 15.8% (<i>n</i>=6) were identified at the probable species level (score 2.000-2.299). Similarly, all <i>E. faecium</i> isolates (<i>n</i>=51) were accurately identified, including 84.3% (<i>n</i>=43/51) identified at the high probable species level and 15.7% (<i>n</i>=8/51) identified at the probable species level.<b>Conclusion.</b> Our study provides a ready-to-use custom MALDI spectral database that can be implemented in clinical diagnostic and research laboratories to accurately identify <i>E. lactis,</i> which is currently misidentified as <i>E. faecium</i> by the standard spectrum database available on commercial platforms.