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

Introduction. Enterococcus faecium clade B has recently been re-classified as Enterococcus lactis. Although E. lactis 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 E. lactis 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 E. lactis causing bacteraemia from E. faecium. Hypothesis/Gap statement. The re-classification of E. faecium clade B as E. lactis warrants the development of rapid and accurate identification methods to distinguish these species, particularly in clinical settings where E. lactis may be misidentified as E. faecium. Aim. The aim of this study was to construct a MALDI Biotyper® custom database to rapidly identify and differentiate E. lactis causing bacteraemia from E. faecium. Methodology. A total of 97 enterococcal isolates, including 38 E. lactis, 51 E. faecium and 8 non-E. faecium non-E. lactis enterococci (E. avium, E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E. lactis, E. mundtii and E. raffinosus) 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 E. lactis 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. Results. Our in-house database correctly identified all isolates at the species level, including the E. lactis isolates, all of which were misidentified as E. faecium by the BioTyper® MBT Compass reference library (2022). Of the 38 E. lactis isolates, 84.2% (n=32) were identified at the high probable species level (score ≥2.300), while the remaining 15.8% (n=6) were identified at the probable species level (score 2.000–2.299). Similarly, all E. faecium isolates (n=51) were accurately identified, including 84.3% (n=43/51) identified at the high probable species level and 15.7% (n=8/51) identified at the probable species level. Conclusion. Our study provides a ready-to- use custom MALDI spectral database that can be implemented in clinical diagnostic and research laboratories to accurately identify E. lactis, which is currently misidentified as E. faecium by the standard spectrum database available on commercial platforms.