title: Quantification of encapsulated bioburden in spacecraft polymer materials by cultivation-dependent and molecular methods.
abstract: Bioburden encapsulated in spacecraft polymers (such as adhesives and coatings) poses a potential risk to jeopardize scientific exploration of other celestial bodies. This is particularly critical for spacecraft components intended for hard landing. So far, it remained unclear if polymers are indeed a source of microbial contamination. In addition, data with respect to survival of microbes during the embedding/polymerization process are sparse. In this study we developed testing strategies to quantitatively examine encapsulated bioburden in five different polymers used frequently and in large quantities on spaceflight hardware. As quantitative extraction of the bioburden from polymerized (solid) materials did not prove feasible, contaminants were extracted from uncured precursors. Cultivation-based analyses revealed <0.1-2.5 colony forming units (cfu) per cm3 polymer, whereas quantitative PCR-based detection of contaminants indicated considerably higher values, despite low DNA extraction efficiency. Results obtained from this approach reflect the most conservative proxy for encapsulated bioburden, as they give the maximum bioburden of the polymers irrespective of any additional physical and chemical stress occurring during polymerization. To address the latter issue, we deployed an embedding model to elucidate and monitor the physiological status of embedded Bacillus safensis spores in a cured polymer. Staining approaches using AlexaFluor succinimidyl ester 488 (AF488), propidium monoazide (PMA), CTC (5-cyano-2,3-diotolyl tetrazolium chloride) demonstrated that embedded spores retained integrity, germination and cultivation ability even after polymerization of the adhesive Scotch-Weld 2216 B/A. Using the methods presented here, we were able to estimate the worst case contribution of encapsulated bioburden in different polymers to the bioburden of spacecraft. We demonstrated that spores were not affected by polymerization processes. Besides Planetary Protection considerations, our results could prove useful for the manufacturing of food packaging, pharmacy industry and implant technology.
authors.affiliation: Compliance Advice and Services in Microbiology GmbH, Cologne, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Bauermeister
authors.affiliation: Department for Microbiology and Archaea Centre, University of Regensburg, Regensburg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Mahnert
authors.affiliation: Department for Microbiology and Archaea Centre, University of Regensburg, Regensburg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Auerbach
authors.affiliation: Lehrstuhl für Makromolekulare Materialien und Oberflächen (Macromolecular Materials and Surfaces), DWI at the RWTH Aachen e.V., RWTH Aachen University, Aachen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Böker
authors.affiliation: Compliance Advice and Services in Microbiology GmbH, Cologne, Germany.
authors.forename: Niwin
authors.initials: N
authors.lastname: Flier
authors.affiliation: Compliance Advice and Services in Microbiology GmbH, Cologne, Germany; Lehrstuhl für Makromolekulare Materialien und Oberflächen (Macromolecular Materials and Surfaces), DWI at the RWTH Aachen e.V., RWTH Aachen University, Aachen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Weber
authors.affiliation: Department for Microbiology and Archaea Centre, University of Regensburg, Regensburg, Germany.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Probst
authors.affiliation: Department for Microbiology and Archaea Centre, University of Regensburg, Regensburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Moissl-Eichinger
authors.affiliation: Compliance Advice and Services in Microbiology GmbH, Cologne, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Haberer
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D050296:Microbial Viability; D011108:Polymers; D012997:Solvents / Q000737:chemistry; D018531:Spacecraft; D013171:Spores, Bacterial / Q000502:physiology
chemical_list: D011108:Polymers; D012997:Solvents
references.citation: Radiat Environ Biophys. 1991;30(2):87-103
references.pmid: 1907012
references.citation: Int J Syst Evol Microbiol. 2006 Aug;56(Pt 8):1735-1740
references.pmid: 16902000
references.citation: J Microbiol Methods. 2006 Nov;67(2):310-20
references.pmid: 16753236
references.citation: Int J Food Microbiol. 2004 May 1;92(3):265-74
references.pmid: 15145585
references.citation: Microb Ecol. 2004 Feb;47(2):150-8
references.pmid: 14749906
references.citation: Microbiol Immunol. 2012 Jan;56(1):40-7
references.pmid: 22145981
references.citation: J Gen Microbiol. 1980 Feb;116(2):511-4
references.pmid: 6768839
references.citation: Astrobiology. 2008 Feb;8(1):17-44
references.pmid: 18237257
references.citation: ISME J. 2013 Mar;7(3):635-51
references.pmid: 23178669
references.citation: Nat Rev Microbiol. 2005 Oct;3(10):777-88
references.pmid: 16205711
references.citation: Microbiol Mol Biol Rev. 2000 Sep;64(3):548-72
references.pmid: 10974126
references.citation: Appl Environ Microbiol. 2011 May;77(9):2943-53
references.pmid: 21398492
references.citation: Icarus. 2000 Jun;145(2):391-427
references.pmid: 11543506
references.citation: Appl Environ Microbiol. 2010 Aug;76(15):5148-58
references.pmid: 20543054
references.citation: J Microbiol Methods. 2004 Mar;56(3):383-94
references.pmid: 14967230
references.citation: ISME J. 2013 Feb;7(2):312-24
references.pmid: 23051695
references.citation: Res Microbiol. 2003 Sep;154(7):466-73
references.pmid: 14499932
references.citation: Syst Appl Microbiol. 1992 Aug;15(3):352-6
references.pmid: 11540077
references.citation: Astrobiology. 2012 Jun;12(6):572-85
references.pmid: 22794299
references.citation: Appl Environ Microbiol. 2011 Aug;77(15):5438-44
references.pmid: 21652744
references.citation: J Bacteriol. 2000 May;182(10):2793-801
references.pmid: 10781548
references.citation: Appl Environ Microbiol. 2009 Oct;75(20):6559-67
references.pmid: 19700540
references.citation: Adv Microb Physiol. 1999;41:93-137
references.pmid: 10500845
references.citation: Nat Rev Microbiol. 2004 Feb;2(2):141-50
references.pmid: 15040261
references.citation: Nature. 1990 May 3;345(6270):63-5
references.pmid: 1691827
references.citation: Adv Space Res. 2004;33(8):1294-301
references.pmid: 15803617
references.citation: Adv Space Res. 1989;9(6):181-4
references.pmid: 11537370
references.citation: FEMS Microbiol Ecol. 2007 Sep;61(3):509-21
references.pmid: 17655710
references.citation: Appl Environ Microbiol. 1977 Feb;33(2):379-84
references.pmid: 848957
references.citation: Syst Appl Microbiol. 2001 Jul;24(2):311-20
references.pmid: 11518337
references.citation: Nucleic Acids Res. 2001 Jan 1;29(1):181-4
references.pmid: 11125085
references.citation: Infect Control Hosp Epidemiol. 2008 Nov;29(11):996-1011
references.pmid: 18947320
references.citation: Adv Space Res. 1995 Mar;15(3):273-6
references.pmid: 11539237
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D489-93
references.pmid: 18948294
references.citation: Adv Space Res. 2005;35(9):1648-53
references.pmid: 16175730
references.citation: Clin Microbiol Rev. 2002 Apr;15(2):167-93
references.pmid: 11932229
references.citation: Appl Environ Microbiol. 1995 Mar;61(3):1104-9
references.pmid: 7793912
references.citation: Appl Environ Microbiol. 2011 Mar;77(5):1628-37
references.pmid: 21216908
references.citation: J Bacteriol. 2009 Mar;191(6):1787-97
references.pmid: 19136594
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Appl Environ Microbiol. 1990 May;56(5):1476-9
references.pmid: 2339896
references.citation: Environ Sci Technol. 2013 Jul 2;47(13):7137-46
references.pmid: 23745679