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. Key Words: Sampling—Recovery—Biomolecules—Clean room—DNA. Astrobiology 13, 189–202.
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References
1.
AmannR.I., LudwigW., SchleiferK.H.1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev, 59:143–169.
2.
ArnoldJ., BaileyG.2000. Surface finishes on stainless steel reduce bacterial attachment and early biofilm formation: scanning electron and atomic force microscopy study. Poult Sci, 79:1839–1845.
3.
BartonH.A., TaylorN.M., LubbersB.R., PembertonA.C.2006. DNA extraction from low-biomass carbonate rock: an improved method with reduced contamination and the low-biomass contaminant database. J Microbiol Methods, 66:21–31.
4.
BreitkopfC., HammelD., ScheldH.H., PetersG., BeckerK.2005. Impact of a molecular approach to improve the microbiological diagnosis of infective heart valve endocarditis. Circulation, 111:1415–1421.
5.
BrodieE.L., DeSantisT.Z., ParkerJ.P.M., ZubiettaI.X., PicenoY.M., AndersenG.L.2007. Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci USA, 104:299–304.
6.
BrownG.S., BettyR.G., BrockmannJ.E., LuceroD.A., SouzaC.A., WalshK.S., BoucherR.M., TezakM.S., WilsonM.C.2007. Evaluation of vacuum filter sock surface sample collection method for Bacillus spores from porous and non-porous surfaces. J Environ Monit, 9:666–671.
7.
BrucknerJ.C., OsmanS., ConleyC., VenkateswaranK.2008. Space microbiology: planetary protection, burden, diversity and significance of spacecraft associated microbes. Encyclopedia of Microbiology. SchaechterM.Elsevier: Oxford, 52–65.
8.
BustinS.A., BenesV., GarsonJ.A., HellemansJ., HuggettJ., KubistaM., MuellerR., NolanT., PfafflM.W., ShipleyG.L., VandesompeleJ., WittwerC.T.2009. The MIQE guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin Chem, 55:611–622.
9.
ButtnerM.P., Cruz-PerezP., StetzenbachL.D.2001. Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR. Appl Environ Microbiol, 67:2564–2570.
10.
ButtnerM.P., CruzP., StetzenbachL.D., Klima-CombaA.K., StevensV.L., CroninT.D.2004a. Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis. Appl Environ Microbiol, 70:4740–4747.
11.
ButtnerM.P., CruzP., StetzenbachL.D., Klima-CombaA.K., StevensV.L., EmanuelP.A.2004b. Evaluation of the Biological Sampling Kit (BiSKit) for large-area surface sampling. Appl Environ Microbiol, 70:7040–7045.
12.
ButtnerM.P., CruzP., StetzenbachL.D., CroninT.2007. Evaluation of two surface sampling methods for detection of Erwinia herbicola on a variety of materials by culture and quantitative PCR. Appl Environ Microbiol, 73:3505–3510.
13.
CerqueiraL., AzevedoN.F., AlmeidaC., JardimT., KeevilC.W., VieiraM.J.2008. DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH)Int J Mol Sci, 9:1944–1960.
14.
ChandlerD.P.1998. Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology. J Ind Microbiol Biotechnol, 21:128–140.
15.
CooperM., LaDucM.T., ProbstA., VaishampayanP., StamC., BenardiniJ.N., PicenoY.M., AndersenG.L., VenkateswaranK.2011. Assessing the cleanliness of surfaces: innovative molecular approaches vs. standard spore assays. Appl Environ Microbiol, 77:5438–5444.
16.
Da SilvaS.M., FillibenJ.J., MorrowJ.B.2011. Parameters affecting spore recovery from wipes used in biological surface sampling. Appl Environ Microbiol, 77:2374–2380.
17.
DalmasoG., BiniM., ParoniR., FerrariM.2008. Qualification of high-recovery, flocked swabs as compared to traditional rayon swabs for microbiological environmental monitoring of surfaces. PDA J Pharm Sci Technol, 62:191–199.
18.
DeSantisT.Z., BrodieE.L., MobergJ.P., ZubietaI.X., PicenoY.M., AndersenG.L.2007. High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone library when sampling the environment. Microb Ecol, 53:371–383.
19.
DwivediH.P., JaykusL.A.2011. Detection of pathogens in foods: the current state-of-the-art and future directions. Crit Rev Microbiol, 37:40–63.
20.
FremauxB., GritzfeldJ., BoaT., YostC.K.2009. Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed. Water Res, 43:4838–4849.
21.
GiovannoniS.2005. Crystal ball. The shape of microbial diversity. Environ Microbiol, 7:476.
GironesR., FerrusM.A., AlonsoJ.L., Rodriguez-ManzanoJ., CalguaB., Correa AdeA., HundesaA., CarratalaA., Bofill-MasS.2010. Molecular detection of pathogens in water—the pros and cons of molecular techniques. Water Res, 44:4325–4339.
24.
GrindbergR.V., IshoeyT., BrinzaD., EsquenaziE., CoatesR.C., LiuW.T., GerwickL., DorresteinP.C., PevznerP., LaskenR., GerwickW.H.2011. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage. PLoS One, 6:e18565.
25.
HauglandR.A., VarmaM., SivaganesanM., KeltyC., PeedL., ShanksO.C.2010. Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR. Syst Appl Microbiol, 33:348–357.
26.
HilscherC., VahrsonW., DittmerD.P.2005. Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability. Nucleic Acids Res, 33:e182.
27.
HolmanH.Y., WozeiE., LinZ., ComolliL.R., BallD.A., BorglinS., FieldsM.W., HazenT.C., DowningK.H.2009. Real-time molecular monitoring of chemical environment in obligate anaerobes during oxygen adaptive response. Proc Natl Acad Sci USA, 106:12599–12604.
28.
HorevajP., MilusE.A., BluhmB.H.2011. A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels. J Appl Microbiol, 111:396–406.
29.
HugenholtzP.2002. Exploring prokaryotic diversity in the genomic era. Genome Biol, 3:1–8.
KellD.B., KaprelyantsA.S., WeichartD.H., HarwoodC.R., BarerM.R.1998. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek, 73:169–187.
32.
KempfM.J., ChenF., KernR., VenkateswaranK.2005. Recurrent isolation of hydrogen peroxide-resistant spores of Bacillus pumilus from a spacecraft assembly facility. Astrobiology, 5:391–405.
33.
KirschnerL.E., PuleoJ.R.1979. Wipe-rinse technique for quantitating microbial contamination on large surfaces. Appl Environ Microbiol, 38:466–470.
34.
KwanK., CooperM., La DucM.T., VaishampayanP., StamC., BenardiniJ.N., ScalziG., Moissl-EichingerC., VenkateswaranK.2011. Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces. Appl Environ Microbiol, 77:2943–2953.
35.
La DucM.T., NicholsonW., KernR., VenkateswaranK.2003. Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility. Environ Microbiol, 5:977–985.
36.
La DucM.T., DekasA.E., OsmanS., MoisslC., NewcombeD., VenkateswaranK.2007. Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean-room environments. Appl Environ Microbiol, 73:2600–2611.
37.
La DucM.T., OsmanS., VaishampayanP., PicenoY., AndersenG., SpryJ.A., VenkateswaranK.2009a. Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods. Appl Environ Microbiol, 75:6559–6567.
38.
La DucM.T., OsmanS., VenkateswaranK.2009b. Comparative analysis of methods for the purification of DNA from low-biomass samples based on total yield and conserved microbial diversity. J Rapid Methods Autom Microbiol, 17:350–368.
39.
LeeD.S.2010. Real-time PCR machine system modeling and a systematic approach for the robust design of a real-time PCR-on-a-chip system. Sensors, 10:697–718.
40.
MonisP.T., GiglioS., SaintC.P.2005. Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem, 340:24–34.
41.
MulliganC.M., KaufmanS.R., QuarinoL.2011. The utility of polyester and cotton as swabbing substrates for the removal of cellular material from surfaces. J Forensic Sci, 56:485–490.
42.
NadkarniM.A., MartinF.E., JacquesN.A., HunterN.2002. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology, 148:257–266.
43.
NASA. 2010. Handbook for the Microbiological Examination of Space HardwareNASA-HDBK-6022National Aeronautics and Space Administration: Washington, DC.
44.
NewcombeD.A., LaDucM.T., VaishampayanP., VenkateswaranK.2008. Impact of assembly, testing, and launch operations on the airborne bacterial diversity within a spacecraft assembly facility clean-room. International Journal of Astrobiology, 7:223–236.
45.
NordgardO., KvaloyJ.T., FarmenR.K., HeikkilaR.2006. Error propagation in relative real-time reverse transcription polymerase chain reaction quantification models: the balance between accuracy and precision. Anal Biochem, 356:182–193.
46.
OliverJ.D.2010. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev, 34:415–425.
47.
PaceN.R.1997. A molecular view of microbial diversity and the biosphere. Science, 276:734–740.
48.
ProbstA., FaciusR., WirthR., Moissl-EichingerC.2010. Validation of a nylon-flocked-swab protocol for efficient recovery of bacterial spores from smooth and rough surfaces. Appl Environ Microbiol, 76:5148–5158.
RoseL., JensenB., PetersonA., BanerjeeS.N., SrduinoM.J.2004. Swab materials and Bacillus anthracis spore recovery from nonporous surfaces. Emerg Infect Dis, 10:1023–1029.
51.
RuijterJ.M., RamakersC., HoogaarsW.M., KarlenY., BakkerO., van den HoffM.J., MoormanA.F.2009. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res, 37:e45.
52.
ShendureJ., JiH.2008. Next-generation DNA sequencing. Nat Biotechnol, 26:1135–1145.
53.
SuzukiM.T., TaylorL.T., DeLongE.F.2000. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Appl Environ Microbiol, 66:4605–4614.
54.
TauscherC., SchuergerA.C., NicholsonW.L.2006. Survival and germinability of Bacillus subtilis spores exposed to simulated Mars solar radiation: implications for life detection and planetary protection. Astrobiology, 6:592–605.
55.
TsourkasA., BaoG.2003. Shedding light on health and disease using molecular beacons. Brief Funct Genomic Proteomic, 1:372–384.
56.
VaishampayanP., OsmanS., AndersenG., VenkateswaranK.2010. Molecular methods to monitor microbial community composition of the Phoenix spacecraft assembly clean room. Astrobiology, 10:499–508.
57.
VenkateswaranK., ChungS., AlltonJ., KernR.2004. Evaluation of various cleaning methods to remove bacillus spores from spacecraft hardware materials. Astrobiology, 4:377–390.
58.
VerranJ., WhiteheadK.2005. Factors affecting microbial adhesion to stainless steel and other materials used in medical devices. Int J Artif Organs, 28:1138–1145.
59.
VesleyD., KeenanK.M., HalbertM.M.1966. Effect of time and temperature in assessing microbial contamination on flat surfaces. Appl Environ Microbiol, 14:203–205.
60.
WilsonK.H., WilsonW.J., RadosevichJ.L., DeSantisT.Z., ViswanathanV.S., KuczmarskiT.A., AndersenG.L.2002. High-density microarray of small-subunit ribosomal DNA probes. Appl Environ Microbiol, 68:2535–2541.
YooS.M., LeeS.Y.2008. Diagnosis of pathogens using DNA microarray. Recent Pat Biotechnol, 2:124–129.
63.
ZainZ.M., BradburyJ.M.1995. The influence of type of swab and laboratory method on the recovery of Mycoplasma gallisepticum and Mycoplasma synoviae in broth medium. Avian Pathol, 24:707–716.
64.
ZucolF., AmmannR.A., BergerC., AebiC., AltweggM., NiggliF.K., NadalD.2006. Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. J Clin Microbiol, 44:2750–2759.
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