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

<i>Bacillus anthracis</i> 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 <i>B. anthracis</i> and closely related species, particularly <i>Bacillus cereus</i>. 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 <i>B. anthracis</i> biomarkers and evaluates the methodologies employed across scientific studies via systematic bibliographic analysis. We identified 87 <i>B. anthracis</i> biomarkers reported in the literature and classified them according to their specificity relative to those of other members of the <i>B. cereus</i> 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 <i>B. anthracis</i> 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 <i>B. anthracis.</i>