ASTROBIOLOGYVolume 5, Number 3, 2005© Mary Ann Liebert, Inc.

Research Paper

Recurrent Isolation of Hydrogen Peroxide-Resistant Spores of Bacillus pumilus from a Spacecraft

Assembly Facility

MICHAEL J. KEMPF, FEI CHEN, ROGER KERN, and KASTHURI VENKATESWARAN

ABSTRACT

While the microbial diversity of a spacecraft assembly facility at the Jet Propulsion Labora-tory (Pasadena, CA) was being monitored, H2O2-resistant bacterial strains were repeatedlyisolated from various surface locations. H2O2 is a possible sterilant for spacecraft hardwarebecause it is a low-temperature process and compatible with various modern-day spacecraftmaterials, electronics, and components. Both conventional biochemical testing and molecularanalyses identified these strains as Bacillus pumilus. This Bacillus species was found in bothunclassified (entrance floors, anteroom, and air-lock) and classified (floors, cabinet tops, andair) locations. Both vegetative cells and spores of several B. pumilus isolates were exposed to5% liquid H2O2 for 60 min. Spores of each strain exhibited higher resistance than their re-spective vegetative cells to liquid H2O2. Results indicate that the H2O2 resistance observed inboth vegetative cells and spores is strain-specific, as certain B. pumilus strains were two tothree times more resistant than a standard Bacillus subtilis dosimetry strain. An example ofthis trend was observed when the type strain of B. pumilus, ATCC 7061, proved sensitive,whereas several environmental strains exhibited varying degrees of resistance, to H2O2. Re-peated isolation of H2O2-resistant strains of B. pumilus in a clean-room is a concern becausetheir persistence might potentially compromise life-detection missions, which have very strictcleanliness and sterility requirements for spacecraft hardware. Key Words: Bacillus—Spores—H2O2 resistance—Spacecraft assembly facility. Astrobiology 5, 391–405.

391

INTRODUCTION

THE SEARCH FOR LIFE on other planets will in-volve ultrasensitive technologies for the de-

tection of biomarkers. Contamination of sampleswith terrestrial cells or biomarkers (also knownas forward contamination) would seriously com-promise interpretation, particularly since mete-

orite exchange between Earth and Mars indicatesa finite possibility that martian life could have re-sembled life on Earth (Thomas-Keprta et al.,2002). Elimination of such forward contaminationwill require novel cleaning and sterilization tech-nologies that are compatible with modern-dayspacecraft (National Research Council, 2001). Inorder to develop these new technologies, it is nec-

Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology,Pasadena, California.

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essary to gain an understanding of the microbialdiversity on spacecraft and in spacecraft-associ-ated environments, and any extreme characteris-tics that naturally occurring microbes might pos-sess (Venkateswaran et al., 2001, 2003a,b; La Ducet al., 2003; Link et al., 2003).

H2O2 is a strong oxidizing agent that producesfree hydroxyl radicals, which attack essential cellcomponents, including lipids, proteins, andDNA, resulting in a biocidal effect (McDonnelland Russell, 1999). High concentrations of H2O2(10–30%) and longer contact times are requiredfor sporicidal activity (Russell, 1991). The spori-cidal effectiveness of H2O2 is significantly in-creased in the gaseous phase when comparedwith the liquid phase (McDonnell and Russell,1999). In contrast to the liquid treatment, the va-por H2O2 system creates an electromagnetic fieldin which the H2O2 vapor breaks apart and pro-duces a low-temperature plasma cloud that con-tains ultraviolet (UV) and free radicals. VaporH2O2 has been used as a decontaminating or ster-ilizing agent in the pharmaceutical and medicalindustries (Crow and Smith, 1995), and is underconsideration as a possible sterilant for flighthardware because it is a low-temperature steril-ization process that is more suitable for variousspacecraft components than the currently useddry heat methods (Rohatgi et al., 2001). Althoughthe mechanism of resistance to H2O2 is not fullyunderstood, bacterial spores are generally moreresistant to liquid H2O2 than are vegetative cellsof the same species (Imlay and Linn, 1988; Bloom-field and Arthur, 1994; Palop et al., 1998). How-ever, few reports are available about the effect ofvapor H2O2 on microbes, and invariably all mi-crobial lethality assessments and sterilizer vali-dation have been carried out using biological indicator strains of Bacillus subtilis and/orGeobacillus stearothermophilus (Klapes and Vesley,1990; Crow and Smith, 1995).

A wide variety of microorganisms can live inextreme conditions, such as low and high pH, lowand high temperatures, high salinity, and/orhigh hydrostatic pressure (Horikoshi and Grant,1991; Cavicchioli, 2002), as well as oligotrophic(low nutrient) conditions (Cavicchioli et al., 2003).Similarly, bacterial spores have been reported tosurvive in the space environment (Horneck et al.,1994) and under simulated martian UV radiationconditions (Schuerger et al., 2003). However, verylittle is known about organisms that can inhabithyperoxidative environments caused by factors

such as high concentrations of H2O2 (Ichise et al.,1999; Yumoto et al., 1999). Likewise, clean-rooms,such as those in medical operation theaters, semi-conductor assembly rooms, and spacecraft as-sembly facilities, might exert selective pressureson indigenous microbes where certain strains orspecies might adapt to the low-nutrient, con-trolled humidity, and clean conditions. Hence,clean-rooms should be considered as “extremeenvironments.”

In this study, we describe the repeated isola-tion of H2O2-resistant Bacillus pumilus sporesfrom a class 100K clean-room at the Jet Propul-sion Laboratory spacecraft assembly facility (JPL-SAF) in Pasadena, CA. Surface samples from theunclassified and classified clean locations of JPL-SAF, as well as natural fallout particulates of theclean-room, were systematically collected to elu-cidate the microbial diversity as well as to un-derstand whether any subset of a problematic mi-crobial community was abundant and resistant tovapor H2O2. Our results indicate that B. pumiluswas abundant and some spores of some B.pumilus strains were more resistant to H2O2 thanspores of other Bacillus species, which include thestandard dosimetry B. subtilis strain. These ob-servations not only have implications for thepreparation of spacecraft, specifically for life-de-tection missions, but also in the medical and phar-maceutical fields, where H2O2 is intended for usein sterilizing various sensitive equipment.

MATERIALS AND METHODS

Isolation and identification of microbes from thespacecraft assembly facility

A typical spacecraft assembly facility consistsof both classified clean-rooms and associated un-classified locations. The assembly crew enters thefacility by cleaning their footwear and air-show-ering before changing their clothes into clean-room-specified garments in an anteroom. Theserooms are not clean-room classified locations. Anunclassified air-lock room separates the anteroomfrom the classified clean-room. The dimensionsof the main assembly area of the JPL-SAF are 25m wide, 36 m long, and 15 m high. Relative hu-midity is controlled at 40 � 5% with a cap at 45%,and the average temperature is maintained at20 � 5°C. This JPL-SAF room was maintained asa Class 100K clean-room. [Clean-room classifica-

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tion is based on the cumulative distribution ofparticles �0.5 �m in one cubic foot of air (ISO14644-1 Part 1: Classification of air cleanliness)(see http://www.iest.org/iso/iso1.htm).] Althoughqualified maintenance personnel rigorously cleanedthe clean-room floors, it is the assembly crewswho regularly clean their work tables, using alcohol as a biocide, before starting the assembly(see http://dnp-lib.jpl.nasa.gov/dnp-lib/dscgi/ds.py/Get/File-1671/_Toc495979317). There arenumerous sources of microbial contaminationsuch as the accumulation of particulates on tallcabinets (�6 feet) kept in these rooms that would be unnoticed by the day-to-day assemblycrewmember.

Samples were collected at different times fromboth unclassified (entrance floors, anteroom, andair-lock) and classified (floors, cabinet tops, andair) locations of the JPL-SAF during 1999 and 2000.Swabs and fall-out witness plates were used to col-lect the samples, and details have been reportedelsewhere (Venkateswaran et al., 2001). For swabsamples, cotton (or polyester) swabs were moist-ened in 10–ml of sterile phosphate-buffered saline(PBS; pH 7.2) or sterile water. An area of 25 cm2

was swabbed to obtain particulates, microbes, andspores. The collected swab samples were sonicated(frequency of 25 kHz) for 2 min to release microbesinto the medium and either subjected to heat shock(80°C for 15 min) to kill the vegetative cells or leftuntreated. Suitable aliquots of the heat-shockedsamples were plated using pour plates, with tryp-tic soy agar (TSA) medium, and incubated at 32°Cfor 3–7 days.

Identification of B. pumilus strains

Type strain counterparts of the various Bacillusspecies isolated from the JPL-SAF were obtainedfrom the American Type Culture Collection(ATCC) (Manassas, VA). Several different bio-chemical and molecular techniques were used tocharacterize the isolated strains (Claus and Berke-ley, 1986; Venkateswaran et al., 2001, 2003a; LaDuc et al., 2003; Dickinson et al., 2004b). Chro-mosomal DNA (�10 ng) from each of the strains,extracted by standard solvent extraction andethanol precipitation protocols (Johnson, 1981),was used as the template for polymerase chainreaction amplification. Procedures developed byYamamoto and Harayama (1995) were followedfor gyrB analysis, except 35 cycles of amplifica-tion were performed. The 1.2-kb amplicons were

gel-excised, purified with Qiagen (Valencia, CA)columns, and sequenced as described elsewhere(Venkateswaran et al., 2001; La Duc et al., 2003).The phylogenetic relationships of organisms cov-ered in this study were determined by compari-son of individual gyrB sequences with other ex-isting sequences in the public databases (seewww.ncbi.nlm.nih.gov and www.mbio.co.jp).Evolutionary trees were constructed with PAUPsoftware, following maximum-parsimony para-meters (Huelsenbeck et al., 2000). The 16S rDNAand gyrB sequences of the 11 isolates and twoATCC strains have been deposited in the Gen-Bank nucleotide sequence database under the fol-lowing accession numbers: AF234851, AF234854,AF234856, AY030327, AY167879 to AY167884,and AY167886 for 16S rDNA and AY167866 toAY167878 for gyrB sequences.

Microscopy

An Olympus (Nepa, CA) phase-contrast mi-croscope (BX-60) was used to determine the re-fractile nature of the spores. A field-emission environmental scanning electron microscope(ESEM) (Philips XL30, FEI Co., Potomac, MD)was used for non-destructive examination ofspores and vegetative cells. Specimen prepara-tion procedures, which often lead to sample arti-facts, are not necessary when using the ESEM. Inaddition, a transmission electron microscope(TEM) was used to examine the surface detailsand cross-sections of the spores, respectively, perestablished methods (Cole and Popkin, 1981).Briefly, the spores were suspended in an equalvolume of 5% glutaraldehyde in aqueous 100 mMHEPES buffer (pH 6.8) containing 2 mM MgCl2as a fixative. After several washes (three washesranging from 10 min to overnight incubation) inthe HEPES buffer fixative solution, spores wereallowed to sit for 2–4 h at room temperature. Thespore pellets were then suspended in 1% osmiumtetroxide for 2–4 h at room temperature andwashed with the HEPES buffer. The fixed sporeswere embedded in agar, and the agar cubes weredehydrated using successive ethanol treatment(70–100% ethanol). Embedding and thin section-ing procedures were followed as described else-where (Cole and Popkin, 1981).

Sporulation of Bacillus isolates

A nutrient broth sporulation medium was usedto induce sporulation from the various isolates

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(Schaeffer et al., 1965; Nicholson and Setlow,1990). A single purified colony of the strain to besporulated was inoculated into liquid nutrientbroth sporulation medium. After 2–3 days of in-cubation at 32°C, the cultures were examined inwet mounts on an Olympus BX-60 phase-contrastmicroscope to determine the level of sporulation.Spores appeared as phase-bright bodies whenviewed with the phase-contrast microscope. Oncethe number of free spores in the culture wasgreater than the number of vegetative cells, theculture was harvested, and the spores were pu-rified.

Spores were purified by treatment with ly-sozyme and washing with salt and detergent(Nicholson and Setlow, 1990). Briefly, cultureswere harvested by centrifugation (5,000 rpm, 10min, 4°C). The pellets were washed with 1 MKCl/0.5 M NaCl and incubated for 60 min at 37°Cin Tris-HCl (50 mM, pH 7.5) containing lysozyme(50 �g/ml) to degrade immature spores, any re-maining vegetative cells, and cell-wall debris. Thespores were cleaned by alternate centrifugation(10,000 rpm, 10 min, 4°C) and washing sequen-tially with NaCl (1 M), deionized water, sodiumdodecyl sulfate (0.05%), and TEP buffer (50 mMTris-HCl, pH 7.5, 10 mM EDTA, and 2 mMphenylmethylsulfonyl fluoride), and finally threewashes with deionized water. The purifiedspores were resuspended in sterile deionized wa-ter, heat-shocked (80°C for 15 min), and stored at4°C in glass tubes.

Resistance to liquid H2O2

The liquid H2O2 assay used by Riesenman andNicholson (2000) was modified and used to de-termine the resistance of spores and vegetativecells to H2O2. Briefly, 933 �l of each spore sus-pension (�108 spores/ml) in PBS was placed ina 1.7-ml microfuge tube. After 100 �l was re-moved for spore titer determination, 167 �l of30% H2O2 (J.T. Baker, Phillipsburg, NJ) wasadded to the remaining spore suspension (833 �l).The final H2O2 concentration was 5%. The sus-pension was incubated at room temperature(�25°C) with continuous gentle mixing. After 60min of exposure, a 100-�l sample was removedand immediately diluted 1:10 with a solution ofbovine catalase (100 �g/ml in PBS; Sigma, St.Louis, MO) that previously had been filter-steril-ized (pore size, 0.45 �m). The catalase treatmentneutralized any remaining H2O2. Serial 1:10 di-

lutions of the catalase-treated suspension werediluted in liquid tryptic soy broth (TSB), and ap-propriate aliquots of the diluted samples wereplated onto TSA and incubated at 32°C for quan-titative measurements of the H2O2-resistantspores. Untreated samples were compared withtreated samples to determine the approximatelevel of resistance.

In addition, the most probable number (MPN)technique was used to estimate the viable sporepopulation after exposure to liquid H2O2. Themethod used for the MPN technique is similar tothe spread plate method; however, instead ofPetri plates the “effective dilution on microtiterplate” was 20 �l into a 96-well plate with eachwell filled with 180 �l of TSB. A three-tube MPNtechnique was followed with a 1:10 serial dilu-tion ranging from 10�2 to 10�10. After incubationat 32°C for 1, 2, and 7 days, turbidity was mea-sured spectrophotometrically at 600 nm ab-sorbance using a VERSAmax microtiter platereader (Molecular Devices, Sunnyvale, CA) to es-timate growth. An absorbance of �0.25 was con-sidered as positive for growth since this ab-sorbance corresponds to 105 colony-formingunits/ml. Since all dilution steps were equal andthe inoculant volume was constant, the sporepopulation estimate was obtained directly fromthe appropriate MPN table generated using MP-NES software (Woomer et al., 1990). The MPNwas calculated by computing from the number ofpositive results at six dilution levels for the10–fold dilution series with three replicates(Woomer, 1994), and the lower as well as upperconfidence limits were estimated as previouslydescribed (Cochran, 1950).

Determination of vapor H2O2 resistance

Approximately 107 spores of B. pumilus FO-033and FO-036b and B. subtilis ATCC 6633 were spot-ted onto spacecraft-qualified metal coupons (alu-minum and titanium; 1 cm � 2 cm) or glass fiberfilters (7 mm in diameter) and allowed to dry atroom temperature. The coupons and filters werewrapped in suitable envelopes and placed insidea Sterrad 100 vapor H2O2 chamber (AdvancedSterilization Products, Irvine, CA). The couponswere exposed to one to four cycles of H2O2 in-jections. Each cycle consisted of establishing a 40mPa vacuum (vacuum phase), injection of theH2O2 (final concentration of 2–6 mg/L; injectionphase), and exposure of the material to the H2O2

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for 12 min (diffusion phase). In each cycle, anelectromagnetic field is created in which the H2O2vapor breaks apart, producing a low-temperatureplasma cloud and free radicals (plasma phase; 2min). A UV-based device, an integral part of thesystem, measures the H2O2 concentration in thechamber. This device does not contribute tokilling of microbes in the system. After exposure,the coupons and filters were placed into TSB todetermine growth. In addition, to determinequantitatively the number of surviving organ-isms, coupons were placed in sterile water, soni-cated, and heat-shocked (as described above) before plating onto TSA. Likewise, the morpho-logical changes before and after H2O2 exposureswere examined using ESEM as described above.

The desiccation effect on the survival of sporesand the effect of the heat shock (80°C for 15 min)along with sonication were determined. Thesecontrols were included to determine the efficacyof the vapor H2O2 exposures.

RESULTS

Isolation of microbes from a spacecraft assembly facility

During the course of a 2-year microbial diver-sity study in the JPL-SAF, several hundred strainswere isolated. Among 124 isolates characterizedfor their phylogenetic affiliation, 85% of thestrains were identified as Gram-positive bacteria.About 65% of the total strains isolated survivedheat-shock protocols (i.e., 80°C for 15 min), withmembers of the Bacillus genus being the pre-dominant microbes among the survivors (�91%).Bacillus licheniformis (25%) and B. pumilus (16%)were the most prevalent Bacillus species isolatedfrom this facility. These results are similar tothose found for clean-room facilities at KennedySpace Center (LaDuc et al., 2003). In addition toBacillus, members of Paenibacillus, Sporosarcina,Urebacillus, Nocardiopsis, and Streptomyces alsosurvived heat-shock. All of the B. licheniformisstrains that were tested were susceptible to 5%H2O2 and were not studied further. Eleven B.pumilus strains (Table 1) were chosen for furtherstudy because of their repeated isolation from alllocations of the facility tested, whereas strains re-lated to B. licheniformis were restricted mostly tothe unclassified locations with a higher incidencein the shoe-cleaner area. Three isolates (FO-033,

FO-036b, and FO-038) were isolated from clean-room dust particles (Venkateswaran et al., 2001).Three isolates (SAFR-032, SAFN-029, and SAFN-034) were from the air-lock between the clean-room and the entrance floor. Two isolates (SAFN-036 and SAFN-037) were from the clean-roomfloors. The remaining three isolates were each re-covered from different locations: entrance floor(SAFN-001), anteroom (SAFN-027), and clean-room cabinet top (KL-052). These eleven isolates,together with two ATCC strains, were character-ized with various biochemical and moleculartechniques.

Identification of B. pumilus strains

Identification of the 13 B. pumilus strains wasachieved via 16S rDNA sequencing, gyrB analy-sis (Table 1), mass spectrometry analysis, andDNA-DNA hybridization. When these B. pumilusstrains were characterized for gyrB, three groupswere recognized (Fig. 1). About 10% variation inthe 1.2-kb gyrB sequence was observed amongthese groups. The type strain group containedtwo ATCC strains and three wild strains, all iso-lated from different samples of the same air-lockarea of the JPL-SAF during December 2000(ATCC 7061T, ATCC 27142, SAFN-029, SAFN-034, and SAFR-032). This group showed differ-ences of only two to three nucleotides in their 1.2-kb gyrB sequences among wild strains and 97%similarity with the B. pumilus type strain, ATCC7061T. The gyrB sequence similarity of the secondgroup was 91% with the type strain, and thisgroup consisted of six strains (FO-033, FO-036b,FO-038, SAFN-001, SAFN-036, and SAFN-037)isolated from different locations and samplingperiods. These second group strains were isolatedfrom air-borne particulates and floors of the 100Kclass clean-room as well as the unclassified en-trance floor area of the JPL-SAF. Similarly, strainsisolated from different locations during April andDecember 2000 shared a 99% gyrB sequence sim-ilarity and formed a third group. The SAFN-027strain was isolated from the unclassified ante-room area in April 2000, where normal clothesare exchanged with clean-room clothes, and theKL-052 strain was isolated from the particulatescollected from a cabinet top kept inside the 100Kclass clean-room facility in April 2000. This thirdgroup showed only 92% gyrB sequence similar-ity with the B. pumilus type strain ATCC 7061T.The gyrB sequence similarity of the strains be-

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tween the second and third groups was �98%. Itfollows that strains in clusters 2 and 3 are moreclosely related to each other than members ofcluster 1. Strains of cluster 1 were the most sim-ilar to the B. pumilus type strain ATCC 7061T. Themass spectrometry and DNA-DNA relatedness-based groupings of various B. pumilus strainssupported the gyrB clustering (Dickinson et al.,2004a).

Resistance of organisms to liquid H2O2

Both vegetative cells and purified spores of B.pumilus strains were exposed to 5% liquid H2O2.Vegetative cells of wild strains exhibited resis-tance to H2O2, whereas the cells of the ATCC7061T strain did not show any growth after ex-posure to liquid H2O2. However, cells of the �-radiation-resistant B. pumilus ATCC 27142 andthe B. subtilis 168 strain exhibited some resistanceto liquid H2O2. In addition, as expected, sporesof the strains were much more resistant (1–5 logreduction) than the vegetative cells (5–8 log re-duction) except with purified spores of ATCC

7061T. In three separate experiments, spores ofwild isolates exhibited a wide range of resistanceto H2O2, as measured by MPN analysis (Table 1).Some of the strains exhibited lower log reductions(1–2 log reduction for FO-033, FO-036b, SAFN-001, SAFN-027, and SAFR-032) as compared withothers (3–5 log reductions, as seen with the B. sub-tilis 168 strain).

Quantitative reduction of bacterial spores inthe presence of 5% liquid H2O2 was tested inthree B. pumilus strains, representing three cladesseen in Fig. 1, and in the B. subtilis 168 strain, abiodosimetry strain (Fig. 2). The resistance toH2O2 was not specific to the spores of any phy-logenetic cluster but was strain-specific. Thespores of SAFR-032, most closely related to theATCC 7061 type strain, showed �12% survivalwhen exposed to 5% liquid H2O2 for 60 min andwere comparable to the resistance levels docu-mented for SAFN-037 (�12%) and FO-036b (9%)spores. A third cluster representative, KL-052,which was closely related to cluster 2 (FO-036b),exhibited one order of magnitude less resistancethan the other spores tested (0.7%). Likewise,

H2O2-RESISTANT B. PUMILUS SPORES 397

FIG. 1. Phylogenetic tree of B. pumilus based on gyrB nucleotide sequences. The GenBank nucleotide accessionnumber follows the strain number. Numbers above the lines are the percent bootstrap values of 1,000 replications ofthat branch of the tree.

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there were no survivors for the B. pumilus typestrain after exposure to 5% liquid H2O2 for 60min. The higher H2O2 resistance (9–12% sur-vival), noticed in the three strains of the B. pumilusspores that were exposed to 5% liquid H2O2 com-pared with the 4% survival of B. subtilis 168spores, should be considered significant.

Vapor H2O2 resistance

Since cellulose-containing filter papers were reported to absorb H2O2 and reduce the lethal ef-fect of H2O2, glass-fiber discs were used as sug-gested by the manufacturer (Advanced Steriliza-tion Products). Among all strains tested, the onlysurvivor in one to four cycles of vapor H2O2 wasFO-036b (cells and spores). In all experiments, thebiological indicator, G. stearothermophilus, pro-vided by the manufacturer, was included to en-sure the performance of the H2O2 sterilizer. Whenthe biological indicator did not show any growthafter exposure in a given cycle, the sterilizer was

considered to be operating within limits for thatrun. The concentration of vapor H2O2 was notconstant and varied from run to run (2–6 mg/Lper cycle). Forty-five runs were carried out withcycle numbers ranging from one to four cycles ofvapor H2O2. In all experiments the biological in-dicator did not survive even after one cycle. TheFO-036b cells and/or spores survived in all 45runs.

The H2O2-treated and untreated glass-fiberdiscs of both G. stearothermophilus spores and B.pumilus FO-036b cells were observed underESEM. As the glass-fiber disc was woven withthin fibrous glass materials, visualization of thecells was difficult. Hence, spacecraft-qualifiedaluminum and titanium coupons were inoculatedwith 107 cells or spores of various strains and ex-posed for one to four cycles of vapor H2O2. Themorphological changes before and after H2O2treatment of these cells placed on aluminumcoupons are shown in Fig. 3. The vegetative cellsof B. subtilis ATCC 6633 (Fig. 3A and B), B. licheni-

KEMPF ET AL.398

FIG. 2. Quantitative results of the liquid H2O2 assay. The white bars represent the untreated spores. The blackbars represent the H2O2-treated spores. The liquid H2O2 assay was performed in triplicate, and the percentage of sur-vival shown is the average of the three assays. The number of spores was measured by the spread plate method asdescribed in Materials and Methods. B. subtilis 168 was chosen to represent a biodosimetry strain, and the three B.pumilus strains represent the three clades shown in Fig. 1. CFU, colony-forming units.

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formis FO-017b (Fig. 3C and D), and B. pumilusFO-036b (Fig. 3E and F) were clearly seen in theuntreated microcosms. The cells of B. subtilis weredamaged beyond recognition after exposure toone cycle of H2O2. When the H2O2-treated B. sub-tilis test material was placed in TSB and incubated

under the appropriate conditions, no visiblegrowth was observed. Although B. licheniformiscells were intact in some regions, the cells wereelongated and did not grow in TSB. In contrast,vegetative cells of B. pumilus FO-036b exhibitedno morphological changes, and structures preva-

H2O2-RESISTANT B. PUMILUS SPORES 399

FIG. 3. Morphological changes of various Bacillus species exposed to vapor H2O2 from ESEM analysis. Vegeta-tive cells of B. subtilis 168 (A and B), B. licheniformis FO-017b (C and D), and B. pumilus FO-036b (E and F) that weredried on aluminum but not exposed to H2O2 (A, C, and E) and after four cycles of H2O2 exposure (B, D, and F) areshown. The cells of B. subtilis 168 were damaged beyond recognition after exposure to H2O2 (B). Although B. licheni-formis FO-017b cells were intact in some regions (D), the cells were elongated and not viable as assessed by the lackof growth in liquid media. The B. pumilus FO-036b cells exhibited no morphological changes after four cycles of H2O2exposure (F).

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lent before H2O2 treatment (Fig. 3E) were intacteven following four injections of vapor H2O2 (Fig.3F). Similarly, like B. pumilus FO-036b cells,spores of this strain showed no morphologicalchanges due to H2O2 treatment (Fig. 4A and B).

Effect of vapor H2O2 on the survival of Bacillusspores deposited on spacecraft materials

The survival of the purified spores of B. pumilusFO-036b after four injections of H2O2 was quan-titatively measured by inoculating varying con-centrations (107, 106, and 105 spores) on the sur-face of spacecraft-qualified materials (Table 2). Inaddition to the effect of H2O2, the influence ofother physical factors, such as desiccation, heat-shock (80°C for 15 min), sonication (25 kHz for 2

min), and adhesion of B. pumilus FO-036b sporesonto the surfaces, was estimated. Irrespective ofsurfaces tested, a reduction of about 2 logs inspore counts were observed due to desiccation(simple drying). Heat-shock and sonication pro-tocols killed 70% of spores on the glass-fiberdiscs, 55–85% on aluminum, and 0–30% on tita-nium surfaces. After four injections of H2O2, ap-proximately 85% of the remaining spores werekilled on the glass-fiber discs, 75–99% on the alu-minum, and 99.7% on the titanium surfaces.Among the surfaces tested, H2O2 treatment killedthe majority of the spores when inoculated ontotitanium, and no spores survived when only 105

spores were initially inoculated. Spore survivalduring H2O2 treatment was similar on both alu-minum and glass-fiber materials.

KEMPF ET AL.400

FIG. 4. Morphological changes of Bacillus spores exposed to H2O2. The ESEM photomicrographs of B. pumilusFO-036b before (A) and after (B) four cycles of vapor H2O2 exposure showed no morphological changes. The crosssections examined by transmission electron microscopy of B. pumilus FO-036b spores (C) showed a highly undulatedspore coat compared with the H2O2-susceptible B. subtilis 168 spore coat (D) after 5% liquid H2O2 exposure for 60min. The arrows show the undulated spore coats of FO-036b.

5703_04_p391-405 5/27/05 1:28 PM Page 400

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5703_04_p391-405 5/27/05 1:28 PM Page 401

DISCUSSION

Robotic spacecraft destined to explore life be-yond Earth are assembled in various classes ofclean-room facilities. The air circulation, humid-ity, and cleanliness maintained in these spacecraftassembly facilities result in low particle move-ment, low humidity, and low nutrient conditions.This poses a challenge for microbial life to adaptand survive in such a niche. Microbial contami-nation in clean-room facilities might be due to thetranslocation of particles to which microbes haveadhered. Based on studies conducted at variousspacecraft assembly facilities, NASA has docu-mented that the presence of humans within theclean-rooms was the single most critical factor inincreasing the microbial bioload of the air (Faveroand Drake, 1966; Favero et al., 1968). Furthermore,microbial species recovered from air or surfacesamples in clean-rooms were nearly identical tospecies recovered from surfaces of unmannedspacecraft (Favero and Drake, 1966; Favero, 1971;Puleo et al., 1975; Vasin and Trofimov, 1995; LaDuc et al., 2003).

B. pumilus was the predominant resistant mi-crobe recovered from spacecraft (Viking in 1972to Mars Odyssey in 2001) and their assembly fa-cility surfaces (Jet Propulsion Laboratory andKennedy Space Center) (Puleo et al., 1977;Venkateswaran et al., 2001, 2003b; La Duc et al.,2003). No report is available as to how this preva-lent microbial species was transmitted into the fa-cility or how these microbes adapted to survivein the conditions of the facility. In this currentwork, there was no noticeable phenotypic differ-ence among the B. pumilus strains isolated fromvarious locations and sampling periods; how-ever, genetic fingerprinting grouped all 11 iso-lated strains into three clusters. Isolation of B.pumilus in all locations (clean-room floors, cabi-net top, and air-borne particulates) and duringseveral sampling periods (November 1999, April2000, and December 2000) substantiated the factthat B. pumilus might have adapted to the classi-fied and unclassified clean-room conditions.Higher genetic similarities were seen amongseven of the 11 B. pumilus strains (�80% DNA re-latedness; �98% gyrB sequence similarity), whilethese seven strains and the B. pumilus type strainexhibited only 55–67% DNA relatedness (�91–92% gyrB sequence similarity). This suggests thatthe seven isolates have presumably acclimatedwithin the class 100K clean-room locations. Based

on the recommendation of the International Com-mittee on Reconciliation of Approaches to Bacte-rial Systematics, the seven strains isolated in theJPL-SAF that possess less than 70% DNA-relat-edness with the type strain can be considered asa subspecies of B. pumilus (Wayne, 1988). Simi-larly, Venkateswaran et al. (1998) suggested a 95%gyrB gene sequence similarity as a threshold levelfor delineating species.

In addition to the fact that these B. pumilusstrains were genetically different, they are also re-sistant to H2O2 treatment and UV254. Recently, we(Link et al., 2003) reported the elevated UVC (us-ing a Hg lamp) resistance of these B. pumilusstrains. Since the objective of this study is the mi-crobial resistance to H2O2, further discussion onmicrobial resistance to UV is not relevant to thiscommunication. Even though various spore-for-mers showed resistance to H2O2 treatment,spores of B. pumilus isolated from the JPL-SAFsurfaces exhibited elevated resistance specificallywhen compared with other Bacillus species (LaDuc et al., 2003). The resistance characteristics ofthe B. pumilus spores and their repeated isolationfrom a spacecraft assembly facility suggest that itis a challenge to eradicate them. The 1–2 log re-duction seen after 5% liquid H2O2 treatment insome JPL-SAF B. pumilus spores is significantwhen compared with the 3–5 log reduction ob-served in a B. subtilis laboratory strain and a �-radiation-resistant B. pumilus ATCC 27142 strain,as well as the complete sensitivity seen in the B.pumilus ATCC 7061 type strain. The majority ofstudies of spore resistance, and the mechanismsof resistance, to various treatments have focusedon B. subtilis, and little is known about otherspecies (Setlow and Setlow, 1993; Popham et al.,1995; Casillas-Martinez and Setlow, 1997).

Degradation of spore DNA due to H2O2, as re-ported in previous studies (Imlay and Linn, 1988),was not tested in this study. Setlow and Setlow(1995a,b) reported that oxidizing agents mightkill spores through destruction of molecules otherthan DNA—presumably proteins and/or mem-branes. It has been reported that the most dam-aging effects of H2O2 might be the targeting ofenzymes required for germination and out-growth contained in the spore core (Raso et al.,1998). In all these previously published studiesonly liquid H2O2 was examined. However, in thevapor H2O2 tested in this study, an electromag-netic field is created in which the H2O2 vaporbreaks apart, producing a low-temperature

KEMPF ET AL.402

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plasma cloud that contains free radicals. In thisstudy, the morphology of B. pumilus spores wasnot affected by exposure to vapor H2O2 [Fig. 4A(before) and B (after)]. The vapor H2O2 results re-ported in this study lend support to the theorythat oxidizing agents may kill spores by damag-ing proteins and/or membranes (Setlow and Set-low, 1993, 1994). Additional work (e.g., electronmicroscopy, protein analysis, etc.) is necessary todetermine the mechanism of resistance to H2O2in these B. pumilus spores. Factors important inspore resistance to chemicals have been identifiedand/or suggested, including the impermeabilityof the spore core, low spore-core water content,the spore coats, and protection of spore DNA by�/�-type small acid-soluble proteins (Russell,1990; Bloomfield and Arthur, 1994; Setlow et al.,2000; Tennen et al., 2000). The exact role of thespore coat layers and the precise role(s) of indi-vidual coat proteins in the resistance to oxidizingagents are not known. The cross-sections of thespores of B. pumilus FO-036b (Fig. 4C) and B. sub-tilis 168 (Fig. 4D) after exposure to 5% liquid H2O2for 60 min suggest that the FO-036b spore coatlayer is highly undulated. Further characteriza-tion of the chemical properties of the spore coatof this strain is necessary to elucidate whetherthese structures play any role in the decreasedpermeability of H2O2. It has been postulated thatthe spore coat layers serve as a diffusion barrierto H2O2 (Driks, 1999). Alternatively, the sporecoat proteins may act as oxidation targets that de-crease the effective H2O2 concentration beforeH2O2 reaches the target(s) in the spore core(Riesenman and Nicholson, 2000). Specific en-zymes that detoxify chemicals in growing cells,such as catalase and peroxidase, do not appear tobe involved in spore resistance to oxidizing chem-icals (Casillas-Martinez and Setlow, 1997). Addi-tional characterization of these B. pumilus isolatesshould elucidate their mechanism(s) of resistanceto H2O2.

In summary, the repeated isolation of B.pumilus strains whose cells and spores show re-sistance to H2O2 presents a significant challengeto maintenance of biological cleanliness in clean-room facilities. Based on our observations, itseems logical that a combination of techniques(cleaning, sterilization, and the correct choice ofspacecraft materials) will be necessary to obtainand ensure biological cleanliness for life-detec-tion missions. Our results and observations arealso significant for the medical industry, because

vapor H2O2 is used to sterilize surgical instru-ments. Future work should address optimizationof the vapor H2O2 sterilization process to im-prove its efficacy. Further analysis of the mecha-nism(s) of H2O2 resistance in these B. pumilusstrains will also help in improving the efficacy ofthe vapor H2O2 sterilization process. Eventhough the influence of metal surfaces on the sur-vivability of spores to vapor H2O2 was explored,the protective influence of salts, sand, and dustfor spores against vapor H2O2 should be studiedwhen implementing this sterilization technology.

ACKNOWLEDGMENTS

Part of the research described in this publica-tion was carried out at the Jet Propulsion Labo-ratory, California Institute of Technology, undera contract with the National Aeronautics andSpace Administration. This research was fundedby a Director’s Research Discretionary Fund(100656-00964) awarded to K.V. We are gratefulto members of the Biotechnology and PlanetaryProtection group for technical assistance. The au-thors thank C. Echeverria for H2O2 sterilization,D. Dickinson and M.S. Quigley for metabolic pro-filing determinations, P. Koen for transmissionand scanning electron microscopy, M.T. La Ducfor 16S rDNA sequence analysis, M. Satomi forDNA-DNA hybridization, and S. Nelson, D.Newcombe, and W. Nicholson for critically read-ing the manuscript. We are thankful to K.Buxbaum, T. Luchik, and G.-S. Chen for valuableadvice and encouragement.

ABBREVIATIONS

ATCC, American Type Culture Collection;ESEM, environmental scanning electron micro-scope; JPL-SAF, Jet Propulsion Laboratory space-craft assembly facility; MPN, most probable number; PBS, phosphate-buffered saline; TEM,transmission electron microscope; TSA, trypticsoy agar; TSB, tryptic soy broth; UV, ultraviolet.

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Address reprint requests to:Kasthuri Venkateswaran

Biotechnology and Planetary Protection GroupJet Propulsion Laboratory

California Institute of Technology4800 Oak Grove Drive

Mail Stop 89Pasadena, CA 91109

E-mail: kjvenkat@jpl.nasa.gov

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