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

ABSTRACT The human skin microbiome is a diverse ecosystem that can help prevent infections by producing biomolecules and peptides that inhibit growth and virulence of bacterial pathogens.Staphylococcus aureusis a major human pathogen responsible for diseases that range from acute skin and soft tissue infections to life-threatening septicemia. Its ability to form biofilms is a key virulence factor contributing to its success as a pathogen as well as to its increased antimicrobial resistance. Here, we investigated the ability of bacterial skin commensals to produce molecules that inhibitS. aureusbiofilm formation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified 77 human skin microbiome bacterial isolates fromStaphylococcusandBacillusgenera. Metabolites from cell-free concentrated media (CFCM) from 26 representative isolates were evaluated for their ability to inhibit biofilm formation by both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA)S. aureusstrains. CFCM, derived from most of the isolates, inhibited biofilm formation to varying extents but did not inhibit planktonic growth ofS. aureus. Size fractionation of the CFCM of threeS.epidermidisisolates indicated that they produce different bioactive molecules. Cluster analysis, based on either MALDI-TOF mass spectra or whole-genome sequencing draft genomes, did not show clear clusters associated with levels of biofilm inhibition amongS. epidermidisstrains. Finally, similar biosynthetic gene clusters were detected in allS. epidermidisstrains analyzed. These findings indicate that several bacterial constituents of the human skin microbiome display antibiofilmin vitroactivity, warranting further investigation on their potential as novel therapeutic agents. IMPORTANCEThe skin is constantly exposed to the environment and consequently to numerous pathogens. The bacterial community that colonizes healthy skin is thought to play an important role in protecting us against infections.S. aureusis a leading cause of death worldwide and is frequently involved in several types of infections, including skin and soft tissue infections. Its ability to adhere to surfaces and produce biofilms is considered an important virulence factor. Here, we analyzed the activity of different species of bacteria isolated from healthy skin onS. aureusbiofilm formation. We found that some species ofStaphylococcusandBacilluscan reduceS. aureusbiofilm formation, although a generally lower level of inhibitory activity was observed compared toS. epidermidisisolates. AmongS. epidermidisisolates, strength of activity was dependent on the strain. Our data highlight the importance of mining the skin microbiome for isolates that could help combat skin pathogens.