Characterization of hydrogen peroxide-resistant Acinetobacter species isolated during the Mars Phoenix spacecraft assembly.

The microbiological inventory of spacecraft and the associated assembly facility surfaces represent the primary pool of forward contaminants that may impact the integrity of life-detection missions. Herein, we report on the characterization of several strains of hydrogen peroxide-resistant Acinetobacter, which were isolated during the Mars Phoenix lander assembly. All Phoenix-associated Acinetobacter strains possessed very high catalase specific activities, and the specific strain, A. gyllenbergii 2P01AA, displayed a survival against hydrogen peroxide (no loss in 100 mM H2O2 for 1 h) that is perhaps the highest known among Gram-negative and non-spore-forming bacteria. Proteomic characterizations reveal a survival mechanism inclusive of proteins coupled to peroxide degradation (catalase and alkyl hydroperoxide reductase), energy/redox management (dihydrolipoamide dehydrogenase), protein synthesis/folding (EF-G, EF-Ts, peptidyl-tRNA hydrolase, DnaK), membrane functions (OmpA-like protein and ABC transporter-related protein), and nucleotide metabolism (HIT family hydrolase). Together, these survivability and biochemical parameters support the hypothesis that oxidative tolerance and the related biochemical features are the measurable phenotypes or outcomes for microbial survival in the spacecraft assembly facilities, where the low-humidity (desiccation) and clean (low-nutrient) conditions may serve as selective pressures. Hence, the spacecraft-associated Acinetobacter, due to the conferred oxidative tolerances, may ultimately hinder efforts to reduce spacecraft bioburden when using chemical sterilants, thus suggesting that non-spore-forming bacteria may need to be included in the bioburden accounting for future life-detection missions.

Differential recovery of phylogenetically disparate microbes from spacecraft-qualified metal surfaces.

Universal and species-specific quantitative polymerase chain reaction-based methods were employed to compare the effectiveness of four distinct materials used to collect biological samples from metal surfaces. Known cell densities of a model microbial community (MMC) were deposited onto metal surfaces and subsequently collected with cotton and nylon-flocked swabs for small surface areas and biological sampling kits (BiSKits) and polyester wipes for large surface areas. Ribosomal RNA gene-based quantitative PCR (qPCR) analyses revealed that cotton swabs were superior to nylon-flocked swabs for recovering nucleic acids (i.e., DNA) from small surface areas. Similarly, BiSKits outperformed polyester wipes for sampling large surface areas. Species-specific qPCR results show a differential recovery of rRNA genes of the various MMC constituents, seemingly dependent on the type of sampling device employed. Both cotton swabs and BiSKits recovered the rDNA of all nine of the MMC constituent microbes assayed, whereas nylon-flocked swabs and polyester wipes recovered the rDNA of only six and four of these MMC strains, respectively. The findings of this study demonstrate the importance and proficiency of molecular techniques in gauging the effectiveness and efficiency of various modes of biological sample collection from metal surfaces.

Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces.

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.