Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS

Abstract

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

A Combination of MALDI-TOF MS Proteomics and Species-Unique Biomarkers’ Discovery for Rapid Screening of Brucellosis

“…The considerable number of peaks observed in the present study correlates well with previous research by Ferreira1et al, Mesureur et al, and Kornspan et al, these biomarker ions are, indeed, adequate for the discrimination of Brucella at the genus and species levels. Typically, integration host factor subunit alpha (at m / z 5921), unidentified peaks at m / z 3899, 4068, and 4159, as well as RS8 (2+) (at m / z 7327), serve as species-specific biomarkers …”

“…Hence, the mapped proteins do not reflect the true subspecies biomarkers. 38 The two independent studies, SPECLUST and Mass-Up, display similar characteristic biomarker ions in genus and species-specific peak masses in Brucella. However, some highintensity peaks that were reproducibly detected in Brucella spp.…”

“…These species- and subspecies- identifying unambiguously discriminative biomarkers based on the ribosomal protein is not merely sufficient . More recently, in a study by Kornspan et al, a comparative in silico proteomics and statistical approach were applied to improve the distinction of the vaccine strain B. melitensis Rev.1 from B. melitensis field strains by describing two ribosomal marker proteins, L24 with m / z 11208 and S12 with m / z 13798. The candidate biomarker L24 was only detected in our study as a strain-specific biomarker of Brucella melitensis Rev.1.…”

“…Seven selected biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1 in 2020 by Christoforidou et al, as our findings build on existing knowledge; these mass signals (3528, 7328, 8026, 8038, 9664) could be assigned to ribosomal proteins RL30 (+2), RS8 (+2), RL19 (+2), and HU family DNA-binding protein, respectively, that serve as genes and species-specific biomarkers. Hence, the mapped proteins do not reflect the true subspecies biomarkers …”

Microbiome and resistome dynamics along a sewage-effluent-reservoir continuum underline the role of natural attenuation in effluent receiving reservoirs

“…To validate presumptive CCA colorimetric identifications, 108 randomly-selected colonies from raw sewage, MABR and the reservoir were classified on a microflex LT MALDI-TOF MS system (Bruker Daltonics GmbH, Bremen, Germany) using the Flex Control v3.4 Biotyper automation software as previously described(23). Results supported the manufacturer’s colorimetric taxonomic characterizations, as: 55 blue colonies were identified as E. coli or Shigella and 53 red colonies were identified as Klebsiella spp.…”