Up-to-date MALDI-TOF MS-based identification of the <i>Corynebacterium diphtheriae</i> species complex for improved diagnostics

ABSTRACT The Corynebacterium ( C .) diphtheriae species complex ( Cd SC) comprises the closely related pathogenic species C. diphtheriae , C. belfantii, C. rouxii, C. ulcerans, C. silvaticum, C. ramonii, and C. pseudotuberculosis according to the taxonomic revisions after 2018. Due to their close relationship, reliable species identification within the Cd SC is also challenging using MALDI-TOF mass spectrometry (MS) for fast and reliable identification of bacteria. This is relevant for human and veterinary medicine. However, the Bruker MALDI-Biotyper (MBT), a commonly used MALDI-TOF MS system, does not reflect the current Cd SC taxonomy, which includes C. belfantii , C. ramonii , C. rouxii , and C. silvaticum as additional Corynebacterium species in the Cd SC. Therefore, the objective of the present study is to expand the commercial Bruker database version K (MBT K ) in order to achieve reliable identification for all currently known Corynebacterium species within the Cd SC. Furthermore, the database expansions were verified with a systematic formal validation procedure in accordance with the German guideline for species identification by MALDI-TOF MS. For this, 328 spectra made from 321 strains including all seven valid species of the Cd SC were identified using the MBT K database and a customer-supplemented modified MBT K database. This custom database version included 33 reference spectra of the Cd SC covering all member species. The results of the formal validations for both the commercial and the extended database, combined partly with a single signal analysis, provide a substantiated basis for the identification of bacteria of the Cd SC. A key element for a fast up-to-date identification has been the exchange of user-generated sets of main spectrum profiles (MSPs) on the open access MALDI User Platform (MALDI-UP) catalog. IMPORTANCE Bacteria belonging to the Corynebacterium ( C .) diphtheriae complex are relevant pathogens for humans and animals with zoonotic potential. Identification of the novel species of this complex described since 2018, that is, C. belfantii , C. rouxii , C. ramonii , and C. silvaticum , is possible using the widely approved MALDI-TOF mass spectrometry. This is important for targeted therapies, preventive measures, and epidemiological studies. Precise species identification has been achieved by using a supplemented Bruker MALDI reference database. Comprehensive validation of database entries meets the requirements for accurate pathogen identification in accordance with recognized guidelines. This approach to community-driven database expansion and validation is certainly a model for other bacterial species of interest to react to current taxonomic revisions in a short time frame.