Tracking Bacillus anthracis: The Legacy of MALDI-TOF Biomarkers in Scientific Literature, a Review

Bacillus anthracis is a Gram-positive, spore-forming bacterium and the etiological agent of anthrax, a re-emerging, septicemic, hemorrhagic, and often lethal disease that affects humans as well as domestic and wild ruminants. Owing to its high lethality, spore stability, and ease of dissemination, this pathogen is recognized as a major biological-warfare agent and is classified among the Category A bioterrorism threats. Rapid and unequivocal identification is essential; however, it is hindered by the extensive similarity between B. anthracis and closely related species, particularly Bacillus cereus. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an increasingly adopted tool in diagnostic laboratories due to its cost-effectiveness, speed, and reliability in microorganism identification (Infographic 1). This comprehensive review compiles known B. anthracis biomarkers and evaluates the methodologies employed across scientific studies via systematic bibliographic analysis. We identified 87 B. anthracis biomarkers reported in the literature and classified them according to their specificity relative to those of other members of the B. cereus group. Moreover, we highlight gaps in methodological standardization and biomarker validation, emphasizing the need to develop robust protocols capable of ensuring the accurate detection of B. anthracis in biological defense contexts and surveillance. Accordingly, we propose a standardized analytical workflow aimed at improving diagnostic reliability, with recommended tools and best practices for enhancing the application of mass spectrometry in the specific identification of B. anthracis.