Resolution of MALDI-TOF compared to whole genome sequencing for identification of Bacillus species isolated from cleanrooms at NASA Johnson Space Center

• DOI: 10.3389/fmicb.2025.1499516
• Publication Date: 2025-04-09T16:43:52Z
• Authors: Farnaz Mazhari; Aaron B. Regberg; Christian L. Castro; Michael G. LaMont...
• Keywords: Identification; Cleanroom

Bacteria are frequently isolated from surfaces in cleanrooms, where astromaterials curated, at NASA's Lyndon B. Johnson Space Center (JSC). Bacillus species of particular interest because endospores can endure extreme conditions. Current monitoring programs JSC rely on culturing microbes swabs followed by identification 16S rRNA sequencing and the VITEK 2 Compact bacterial system. These methods have limited power to resolve species. Whole genome (WGS) is current standard for but expensive time-consuming. Matrix-assisted laser desorption - time flight mass spectrometry (MALDI-TOF MS), provides a rapid, low-cost, method identifying isolates has higher resolution than sequencing, particularly species; however, few studies compared this WGS cleanrooms. To address this, we selected 15 analysis with MALDI-TOF MS. Hybrid next-generation (Illumina) 3rd-generation (nanopore) were used draft genomes. Mass spectra, generated MS, processed custom scripts identify clusters closely related isolates. MS identified 13/15 9/14 level, respectively, showed good agreement, terms congruence partitioning, phylotypes WGS. Pairs strains that > 94% similar each other, average amino acid identity (AAI) predicted WGS, consistently cosine similarities spectra >0.8. The only discordance was pair classified as Paenibacillus This relatively high similarity (0.85) 85% AAI. In addition, some cleanrooms appeared spacecraft assembly Since costs less whole offers throughput hundreds per hour, approach appears offer cost-efficient option species, microbes, during routine built environments.