Construction of a superspectrum for rapid identification of opportunistic human pathogens belonging to the genus Phytobacter using whole-cell MALDI-TOF MS.

BACKGROUND Members of the genus Phytobacter, part of the Enterobacteriaceae, represent an emerging clinical threat. Standard identification methodologies often fail to accurately identify members of this genus because they are absent from current clinical databases. Molecular techniques are thus pivotal for differentiating Phytobacter from similar genera such as Pantoea, Kluyvera, and Kosakonia. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) provides a rapid, reliable, and cost-effective method for identification.

METHODS A Phytobacter-specific SuperSpectrum was constructed from 18 well-characterized isolates using standardized culture conditions, protein extraction, and triplicate MALDI-TOF MS acquisition. Only conserved, high-quality peaks were retained in the final model. Its performance was evaluated using 282 retrospective isolates and 23 prospective Phytobacter-suspect isolates. All Phytobacter identifications, along with 154 non-Phytobacter identifications, were benchmarked against API 20E biochemical testing, VITEK-2 automated identification, MALDI-TOF MS current database for in vitro diagnostic analysis, 16S rRNA gene sequencing, and whole-genome sequencing (WGS).

RESULTS Spectral clustering showed consistent separation of Phytobacter at the genus level. Integration of the new SuperSpectrum into the MALDI-TOF MS SARAMIS database enabled accurate identification, leading to the identification of two retrospective Phytobacter out of 282 Enterobacterales isolates. Finally, prospective testing of 23 contemporary isolates suspected to belong to Phytobacter confirmed their identification against the new reference spectra, achieving 100% concordance with WGS identification, demonstrating the reliability of the updated database for genus-level identification.

CONCLUSION Phytobacter identification by MALDI-TOF MS provides a rapid alternative method for molecular identification of this emerging pathogen.