Evaluation of Procedures for the Collection, Processing, and Analysis of Biomolecules from Low-Biomass Surfaces

ABSTRACT 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.

[1]  W. Nicholson,et al.  Survival and germinability of Bacillus subtilis spores exposed to simulated Mars solar radiation: implications for life detection and planetary protection. , 2006, Astrobiology.

[2]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[3]  L. J. Rose,et al.  Evaluation of a Macrofoam Swab Protocol for the Recovery of Bacillus anthracis Spores from a Steel Surface , 2006, Applied and Environmental Microbiology.

[4]  Kasthuri Venkateswaran,et al.  Diversity of Anaerobic Microbes in Spacecraft Assembly Clean Rooms , 2010, Applied and Environmental Microbiology.

[5]  K. Venkateswaran,et al.  Evaluation of various cleaning methods to remove bacillus spores from spacecraft hardware materials. , 2004, Astrobiology.

[6]  Moselio Schaechter,et al.  Encyclopedia of microbiology , 2009 .

[7]  Paul A. Baron,et al.  Recovery Efficiency and Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface Samples , 2009, Applied and Environmental Microbiology.

[8]  N J Petersen,et al.  Use of Ultrasonic Energy in Assessing Microbial Contamination on Surfaces , 1967, Applied microbiology.

[9]  Christine Moissl-Eichinger,et al.  Archaea in artificial environments: Their presence in global spacecraft clean rooms and impact on planetary protection , 2011, The ISME Journal.

[10]  J. Costerton,et al.  Optical sectioning of microbial biofilms , 1991, Journal of bacteriology.

[11]  Alexander J. Probst,et al.  Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces , 2010, Applied and Environmental Microbiology.

[12]  K. Venkateswaran,et al.  COMPARATIVE ANALYSIS OF METHODS FOR THE PURIFICATION OF DNA FROM LOW‐BIOMASS SAMPLES BASED ON TOTAL YIELD AND CONSERVED MICROBIAL DIVERSITY , 2009 .

[13]  Kasthuri Venkateswaran,et al.  High-density 16S microarray and clone library-based microbial community composition of the Phoenix spacecraft assembly clean room. , 2010, Astrobiology.

[14]  Rafał Lewandowski,et al.  Use of a Foam Spatula for Sampling Surfaces after Bioaerosol Deposition , 2009, Applied and Environmental Microbiology.

[15]  K. Venkateswaran,et al.  Space Microbiology: Planetary Protection, Burden, Diversity and Significance of Spacecraft Associated Microbes , 2009 .

[16]  S. Ricke,et al.  Comparison of cryoprotectants for Escherichia coli lysine bioavailability assay culture , 2004 .

[17]  K. Jarman,et al.  Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces , 2008, Journal of applied microbiology.

[18]  Misty J. Hein,et al.  Surface Sampling Methods for Bacillus anthracis Spore Contamination , 2002, Emerging infectious diseases.

[19]  Gary L. Andersen,et al.  High-Density Microarray of Small-Subunit Ribosomal DNA Probes , 2002, Applied and Environmental Microbiology.

[20]  Kasthuri Venkateswaran,et al.  Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility. , 2003, Environmental microbiology.

[21]  Bette Jensen,et al.  Swab Materials and Bacillus anthracis Spore Recovery from Nonporous Surfaces , 2004, Emerging infectious diseases.

[22]  K. Venkateswaran,et al.  Impact of assembly, testing and launch operations on the airborne bacterial diversity within a spacecraft assembly facility clean-room , 2008, International Journal of Astrobiology.

[23]  John E. Brockmann,et al.  Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces , 2006, Applied and Environmental Microbiology.

[24]  L J Kricka,et al.  Evaluation of DNA fragment sizing and quantification by the agilent 2100 bioanalyzer. , 2000, Clinical chemistry.

[25]  Fei Chen,et al.  Recurrent isolation of hydrogen peroxide-resistant spores of Bacillus pumilus from a spacecraft assembly facility. , 2005, Astrobiology.

[26]  E. Delong,et al.  Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays , 2000, Applied and Environmental Microbiology.

[27]  Erica R. Valdes,et al.  Surface Sampling of Spores in Dry-Deposition Aerosols , 2008, Applied and Environmental Microbiology.

[28]  K. Venkateswaran,et al.  Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments , 2007, Applied and Environmental Microbiology.

[29]  Kasthuri Venkateswaran,et al.  Archaeal diversity analysis of spacecraft assembly clean rooms , 2008, The ISME Journal.

[30]  R. Paroni,et al.  Qualification of high-recovery, flocked swabs as compared to traditional rayon swabs for microbiological environmental monitoring of surfaces. , 2008, PDA journal of pharmaceutical science and technology.

[31]  M. Stieglmeier,et al.  Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms , 2009, Applied and Environmental Microbiology.

[32]  J R Puleo,et al.  Wipe-rinse technique for quantitating microbial contamination on large surfaces , 1979, Applied and environmental microbiology.

[33]  Kasthuri Venkateswaran,et al.  Molecular bacterial community analysis of clean rooms where spacecraft are assembled. , 2007, FEMS microbiology ecology.

[34]  Gary S Brown,et al.  Evaluation of vacuum filter sock surface sample collection method for Bacillus spores from porous and non-porous surfaces. , 2007, Journal of environmental monitoring : JEM.

[35]  K. Venkateswaran,et al.  Recurrent isolation of extremotolerant bacteria from the clean room where Phoenix spacecraft components were assembled. , 2010, Astrobiology.

[36]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[37]  Linda D. Stetzenbach,et al.  Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR , 2001, Applied and Environmental Microbiology.

[38]  Heather O'Connell,et al.  National validation study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces. , 2010, Journal of microbiological methods.