Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA؉B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m ؊2 of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA؉B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.When vegetative cells of Bacillus species are confronted with low nutrient abundance, the bacteria can initiate the process of sporulation, in which the growing cells differentiate into dormant spores (48). Spores do not metabolize at a detectable level and are highly resistant to several perturbations, such as heat and exposure to UV and gamma radiation (33). Due to this intrinsic resistance spores are ubiquitous in the environment and have been found both above and below the surface of the Earth (33, 38).The resistance of spores has prompted agencies such as the National Aeronautics and Space Administration (NASA), the Department of Homeland Security, and others to study sporulating bacteria more closely (1,2,9,11,15,36,43,50). Recently, in several microbial diversity surveys performed over a period of 3 years, 125 aerobic microbial strains were isolated from spacecraft assembly facilities (20,24,25,52,53), and their phylogenetic affiliations were determined (23, 54). Eighty-five percent of these strains were identified as gram-positive bacteria. About 65% of the strains cultivated survived heat shock protocols used to isolate sporulating bacteria (2). Members of the genus Bacillus were the predominant microbes among the heat shock survivors (Ͼ91%). A total of 15 different Bacillus species were identified. Bacillus licheniformis ...

In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the sporeforming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus.Rapid, sensitive, and selective microbial detection and identification at the species and strain level are necessary to differentiate between viable pathogenic and nonpathogenic microbial species. The development of technology to accomplish this level of distinction for microbial species would have a significant impact on occupational and health care, homeland defense, and environmental monitoring. For over a century, microbial identification techniques have depended on conventional culture-based methods that characterize phenotypic differences and rely on biochemical and morphological tests. These methods are time-consuming and laborious, and the results are often subjective (38,41). In order to overcome the problems involved with phenotypic characterization, 16S rRNA analysis has been used for decades to more accurately define the phylogenetic affiliation of the given test microorganism (29). However, being highly conserved, the 16S rRNA molecule at times cannot differentiate closely related microbial species (41, 43). Therefore, alternative biomarkers (44) or a suite of protein profiling methods would be useful in order to effectively differentiate closely related microbial species.Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS)-based microbial detec...

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Modern taxonomy, diagnostics, and forensics of bacteria benefit from technologies that provide data for genome-based classification and identification of strains; however, full genome sequencing is still costly, lengthy, and labor intensive. Therefore, other methods are needed to estimate genomic relatedness among strains in an economical and timely manner. Although DNA-DNA hybridization and techniques based on genome fingerprinting or sequencing selected genes like 16S rDNA, gyrB, or rpoB are frequently used as phylogenetic markers, analyses of complete genome sequences showed that global measures of genome relatedness, such as the average genome conservation of shared genes, can provide better strain resolution and give phylogenies congruent with relatedness revealed by traditional phylogenetic markers. Bacterial genomes are characterized by a high gene density; therefore, we investigated the integration of mass spectrometry-based proteomic techniques with statistical methods for phylogenomic classification of bacterial strains. For this purpose, we used a set of well characterized Bacillus cereus group strains isolated from poisoned food to describe a method that relies on liquid chromatography-electrospray ionization-tandem mass spectrometry of tryptic peptides derived from whole cell digests. Peptides were identified and matched to a prototype database (DB) of reference bacteria with fully sequenced genomes to obtain their phylogenetic profiles. These profiles were processed for predicting genomic similarities with DB bacteria estimated by fractions of shared peptides (FSPs). FSPs served as descriptors for each food isolate and were jointly analyzed using hierarchical cluster analysis methods for revealing relatedness among investigated strains. The results showed that phylogenomic classification of tested food isolates was in consonance with results from established genomic methods, thus validating our findings. In conclusion, the proposed approach could be used as an alternative method for predicting relatedness among microbial genomes of B. cereus group members and potentially may circumvent the need for whole genome sequencing for phylogenomic typing of strains.