1 microbial diversity of anaerobes from spacecraft assembly clean

1

Microbial Diversity of Anaerobes from Spacecraft Assembly Clean Rooms

Alexander Probst1,a

, Parag Vaishampayan1, Shariff Osman

2, Christine Moissl-Eichinger

3,

Gary Andersen2 and Kasthuri Venkateswaran*

1

1Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory,

California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109 5 2Center for Environmental Biotechnology, Lawrence Berkeley National

Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 3Lehrstuhl fuer Mikrobiologie und Archaeenzentrum, Universitaet Regensburg,

Universitaetsstrasse 31, 93053 Regensburg, Germany

10

*Correspondence:

California Institute of Technology, Jet Propulsion Laboratory

Biotechnology and Planetary Protection Group; M/S 89-2

4800 Oak Grove Dr., Pasadena, CA 91109

Tel: (818) 393-1481; Fax: (818) 393-4176 15

E-mail: [email protected]

Present address: aLehrstuhl fuer Mikrobiologie und Archaeenzentrum, Universitaet

Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany

Keywords: Microbial diversity, Anaerobic bacteria, Spacecraft assembly facility, Clean 20

rooms, 16S rRNA, DNA microarray, PhyloChip, Planetary Protection

Running title: Anaerobes in clean rooms

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02167-09 AEM Accepts, published online ahead of print on 12 March 2010

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ABSTRACT

Although the cultivable and non-cultivable microbial diversity of spacecraft assembly clean

rooms has been previously documented using conventional and state-of-the art molecular 25

techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain.

Therefore, anaerobic bacterial communities of three clean room facilities were analyzed

during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on

several media, and DNA was extracted from suitable anaerobic enrichments and examined

with conventional 16S rRNA gene clone library as well as high-density phylogenetic 16S 30

rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses

predominantly showed the presence of clostridia and propionibacteria strains. The 16S rRNA

gene sequences retrieved from clone libraries revealed distinct microbial populations

associated with each clean room facility, clustered exclusively within gram-positive

organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla 35

of bacteria, and provided a deeper insight into the microbial community structure of the clean

room facilities. This study presents an integrated approach for assessing the anaerobic

microbial population within clean room facilities, using both molecular and cultivation-based

analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the

clean rooms even after imposition of rigorous maintenance programs and will pose a 40

challenge to planetary protection implementation activities.


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INTRODUCTION

As mandated by United Nations treaty, space faring nations enumerate aerobic

sporeforming bacteria on spacecraft surfaces as a proxy for measuring the cleanliness of

spacecraft intended to land in particular extraterrestrial environments (5, 27). However, 45

recent use of molecular microbial community analyses on clean room samples has revealed a

much higher biodiversity—including the presence of genetic signatures from anaerobic

sporeformers—than can be assessed by the standard procedure alone (14-16). During space

travel and after inadvertent contamination of Mars, microbes are exposed to low or

nonexistent concentrations of oxygen, challenging the survival of aerobic microorganisms. 50

The study of anaerobes is therefore particularly important in the context of space research,

since proliferation of microbes adapted to Mars-like environments would increase the risk of

contaminating target planets and would compromise sensitive life detection activities. The

Mars Science Laboratory (MSL) mission aims to explore new areas of Mars and will search

for probable life on the red planet using highly sensitive biosensors, requiring high 55

cleanliness control to prevent false positives. To overcome present limitations in

characterizing the potential threat from anaerobic bacterial diversity, the objective of this

study was to utilize both culture-dependent and culture-independent molecular analyses to

characterize the obligate anaerobic bacterial communities of the three clean room facilities

used for the MSL rover assembly. 60

Recent investigations of spacecraft facilities have retrieved 16S rRNA gene sequences from

facultative and obligate anaerobic microorganisms from environmental samples (17, 25).

Some facultative anaerobes of the genus Paenibacillus and Staphylococcus have been

isolated in the course of describing the cultivable diversity of extremotolerant microbes in

clean room facilities (14). A microbial survey of European clean room facilities has reported 65

the isolation of facultative and strict anaerobes from spacecraft-associated surfaces (36).


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Typically, 20 to 50% of all isolates from different sampling events and different locations

exhibited growth under anaerobic conditions, with a small subset of isolates being strict

anaerobes (36). Nevertheless, these analyses have been based only on cultivation, and it has

been reported that only ~1% of microorganisms in environmental samples are cultivable in 70

defined media under laboratory conditions (1).

Current molecular cloning techniques targeting the 16S rRNA genes can capture a wide

spectrum of bacterial diversity and facilitate the construction of a comprehensive microbial

inventory (17, 29). Investigations utilizing these methods have revealed a much higher

biodiversity in clean rooms than is detected by the NASA standard assay procedures (15, 16), 75

with approximately 0 to 8% of retrieved sequences belonging to obligate anaerobes (24, 25).

This low percentage might be due to the fact that clone library construction is generally

limited to hundreds or thousands of sequences, which has proven to be insufficient for

detecting low-abundance organisms, including anaerobes (17).

To overcome this limitation in the current study, clone libraries were generated from the 80

samples that were pre-enriched for obligate or facultative anaerobes. The enrichment

approach suppressed aerobes and increased the efficiency of retrieving 16S rRNA gene

sequences of facultative and strict anaerobes. PhyloChip DNA microarray analysis (17) was

simultaneously carried out to detect organisms present in amounts below 10−4

abundance of

the total sample (3). In addition to molecular analyses, established culture methods were also 85

used to successfully isolate obligate and facultative anaerobes.

MATERIAL AND METHODS

Sampling locations. The Jet Propulsion Laboratory (JPL) clean room facilities used for

assembly of the MSL spacecraft components were sampled. In four sampling events [JPL

Spacecraft Assembly Facility (SAF)-A, Building-233-B, JPL-SAF-C, Biomolecule Detection 90


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Laboratory (BDL)-D], 53 surface samples from three different clean room facilities were

examined. For each sampling event, ten surface samples were taken using Biological

Sampling Kits (BiSKit, see below). In addition, Ground Support Equipment (GSE) of the

spacecraft was sampled when present (JPL-SAF-A and JPL-SAF-C) using wipes and

BiSKits, respectively. Samples were taken from the JPL-SAF clean room on two occasions 95

(at exactly the same locations), since it hosts the majority of the MSL assembly and activities.

During sampling, several MSL spacecraft assemblies and GSE structures were present in the

JPL-SAF (cruise stage, spacecraft assembly and rotation fixture tower, and cradle) and a high

level of human activity was observed (20 to 30 people). Clean room facility Building 233

(Bldg-233) harbored the pressure tanks and heater assemblies of MSL prior to their transfer 100

to the JPL-SAF but also had a high human activity (~10 people). However, this clean room is

smaller than JPL-SAF. The BDL, where the last sampling event took place, has low human

activity (2 people) because it hosted no ongoing assembly. The BDL served as a storage and

test location to keep mission components at the same cleanliness level and was therefore

studied for comparison. An overview of the specific sampling locations from each facility, 105

physical characteristics, and their corresponding clean room class designations is given in

Supplementary Table 1.

Prior to entering clean rooms, staff take appropriate actions to minimize the influx of

particulate matter. The air of all facilities is filtered through HEPA filters. EMS sensor sets

are located in appropriate locations, and each set contains a temperature sensor, a relative 110

humidity sensor (Vaisala Model 260EX), and a laser particle counter that measures airborne

particle concentration (Met One model 237A). Air is volumetrically exchanged a minimum

of four times per hour, with positive pressure maintained at all times. These facilities are all

operated as Class 100K (100,000 particles >0.5 µm ft³ air) or ISO 8 (3,520,000 particles

>0.5 µm m³) clean rooms. 115


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Sample collection. Surface sampling of the clean room floors (1 m2) was performed using

Biological Sampling Kits (BiSKits, QuickSilver Analytics, Abingdon, Maryland) that were

pre-moistened with the manufacturer-provided sterile buffer as previously described (4, 17).

GSE samples of JPL-SAF-C were also taken using BiSKits, whose sterile phosphate-buffered

saline was replaced with the same amount of sterile pure water prior to sampling to avoid 120

exposing spacecraft components to salts and other chemicals. Some GSE surfaces were

sampled using wipes pre-moistened with water (one wipe per square meter) according to the

NASA standard assays procedures (26). Since BiSKits were not flight-qualified due to

electrostatic discharge–related issues, wipes were used to collect samples from any GSE in

direct contact with the spacecraft (JPL-SAF-A). The wipes were then resuspended in 40 ml 125

sterile phosphate-buffered saline (pH 7.2) and mixed for 15 seconds. The resulting sample

(15 ml for BiSKit and 40 ml for wipe samples) was concentrated to a volume of 500 µl via

centrifugation (Amicon 50, Millipore, Billerica, Massachusetts) and carried forward to the

isolation strategies (see below). In general, samples were processed and used for inoculation

within two hours after collection. All of these sample collections were carried out in the 130

presence of oxygen since alternative methods to minimize the exposure of samples to oxygen

were not available (36).

Media preparation. All media preparation, inoculation, and appropriate incubation were

carried out under suitable conditions using an anaerobic chamber (Coy Laboratory Products,

Grass Lake, Michigan) when necessary. All reagents and organic compounds were purchased 135

from Difco (Franklin Lakes, New Jersey) unless otherwise stated. Three different anaerobic

media were used for isolation of anaerobic microorganisms (the recipes are given for 1 liter

medium to be prepared with distilled water): (a) anaerobic trypticase soy liquid medium (TS,

gas phase N2) (36); (b) Modified American Type Culture Collection (ATCC) medium 591

(pH = 8, gas phase CO2) (2), with 1 ml Wolfe’s vitamin and 1 ml Wolfe’s mineral solution in 140


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place of the default vitamin and trace element solutions (41); and (c) thioglycolate liquid

medium (TG, gas phase N2) (36). Each medium contained L-cysteine (1.0 g) and sodium

thioglycolate (0.5 g, Sigma-Aldrich, St. Louis, Missouri) as reduction reagent as well as

sodium resazurin (1 mg, Serva Electrophoresis, Heidelberg, Germany) as an oxygen

indicator. Media preparation and portioning into serum bottles (Pharmapack Stute, 145

Rheinbreitenbach, Germany) were achieved as described elsewhere (36). Solid media were

prepared by adding agar (15 g/l) to liquid medium. Either sterile NaOH (1 M) or HCl (1 M)

was used to adjust pH after autoclaving.

Inoculation and enrichment. Appropriate aliquots (200 µl of concentrated samples; two-

fifths of original collection fluid) of each sample collected from ten various locations per 150

sampling event were individually placed into 20 ml of liquid TS medium. The same volumes

of all ten samples were pooled before inoculating 200 µl in duplicates into several liquid

media (TS, ATCC 591, and TG). Furthermore, 200 µl of the pooled samples were spread

plated in duplicate onto anaerobic solid TS medium. Samples taken from GSE structures

were pooled and processed similarly to the pooled samples taken from clean room surfaces. 155

Cultivation and purification. Samples spread plated on anaerobic TS agar medium were

incubated at 32°C. Growth of the colonies on plates was observed daily for 14 days.

Enrichments in liquid media were achieved by shaking at 200 rpm at 32°C for 14 days.

Growth from enrichment cultures was regularly observed via phase contrast light microscopy

(Olympus microscope BX-90) by removing 0.1 ml of the culture with a disposable syringe 160

and hypodermic needle. Appropriate aliquots of the cultures (50 µl to 200 µl) that exhibited

positive growth were also spread plated on corresponding anaerobic solid medium and

incubated at 32°C, while the remaining cultures were stored at 4°C for later molecular

investigations (see below). To obtain pure cultures, colonies of different morphologies were

picked and re-streaked. To focus on isolation of obligate anaerobes, each purified strain was 165


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additionally incubated under aerobic conditions after streaking onto oxygen-enriched TSA

plates. Based on the ability to grow under anaerobic conditions as well as the absence of

growth under aerobic conditions, obligate anaerobic strains were transferred to anaerobic TS

medium by picking a single purified colony, which was used for further characterization.

Since several studies have already documented the presence of the facultative anaerobes in 170

clean rooms, these isolates were not further characterized (10, 14, 25).

Identification. DNA of each strict anaerobic culture was extracted using an Autolyser A-2

automated DNA extraction instrument (Axcyte Genomics, Menlo Park, California) as

described elsewhere (18). In order to identify the strains, approximately 10 ng purified DNA

was used as a template for 16S rRNA gene polymerase chain reaction (PCR) amplification 175

using MJ Research (Waltham, Massachusetts). Bacterial 16S rRNA gene was PCR-amplified

using a suitable primer set (B27F; 5′-AGA GTT TGA TCM TGG CTC AG-3′ and 1492R; 5′-

GGT TAC CTT GTT ACG ACT T -3′) (25). PCR amplification was performed according to

the following conditions: 95ºC for 4 min; 35 cycles at 95ºC for 60 s, 55ºC for 60 s, 72ºC for 1

min 30 s, and a final incubation of 72ºC for 10 min (25). The purified PCR product 180

(QIAquick PCR purification kit; Qiagen, Chatsworth, California) was sequenced by bi-

directional sequencing analysis (Agencourt, Beverly, Massachusetts). The identity and

phylogenetic relationships of the strict anaerobic strains were determined by comparing the

individual 16S rRNA gene sequences to existing sequences in a publicly available database

(http://blast.ncbi.nlm.nih.gov/). Nucleotide sequence accession numbers are given in Table 1. 185

Molecular analysis of enrichment cultures.

(i) Construction of clone libraries. For each sampling event, two clone libraries were

constructed using standard and gradient PCR (17), respectively. Irrespective of visible

growth, 1-ml samples of the enrichment cultures from each sample event location were

pooled. This pooled suspension (~10 ml) was aseptically transferred into an Amicon Ultra-15 190


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centrifugal filter tube (Millipore, Jaffrey, New Hampshire, Ultracel-50 membrane Cat

#UFC905096) and concentrated to a volume of 500 µl via centrifugation. DNA was extracted

using bead beating (6.5 M/s for 60s) (FastPrep-24, MP) followed by AutoLyser processing as

described elsewhere (18). The bacterial 16S rRNA genes were then PCR-amplified using the

same primers as mentioned above for phylogenetic analysis of the cultivated strains. 195

Additionally, a gradient PCR using the same primer set but varying the temperature for

primer annealing (48°C, 50.1°C, 54.4°C, and 57.5°C) was performed to increase the detected

biodiversity. Eight clone libraries were generated by ligating 16S amplicons into the pCR4-

TOPO cloning vector and transforming chemically competent E. coli cells (TOPO 10), which

were then grown on agar plates according to the manufacturer’s instructions (Invitrogen). 200

Colonies (96 clones from each library) were robotically picked, and inserts were sequenced

bi-directionally using M13F and M13R primers (Agencourt Corp., Massachusetts).

(ii) Sequencing and phylogenetic analysis. Out of 96 sequences generated from each clone

library, only high-quality (PHRED 20, www.phrap.com) sequences were retained (~66% of

288 clones for standard PCR and 62% of 384 clones for gradient PCR) for further analysis. 205

The nearly full-length (~1400 bp) 16S rRNA sequence of each clone was classified to the

genus level using the Ribosomal Database Project (RDP) II classifier tool

(http://rdp.cme.msu.edu/). Phylogenetic positions of clones were determined by comparison

of cloned sequences with quality checked type strain 16S rRNA gene sequences (31, 42)

using a BLAST function. The 16S rRNA gene sequences collected in this study were 210

deposited in the National Center for Biotechnology Information (NCBI) nucleotide sequence

database; their accession numbers are FJ957454 to FJ957851.

(iii) Statistical analysis. Sequences were aligned using ClustalW (19), and a Jukes-Cantor–

corrected distance matrix was constructed using the DNADIST program from PHYLIP (8).

The DOTUR-1.53 program (35) was applied to all clone libraries generated in this study to 215


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cluster sequences into operational taxonomic units (OTUs) based on 97.5% sequence

similarity (11, 20, 33). Rarefaction curves were produced in DOTUR by plotting the number

of OTUs observed against the number of clones screened. The coverage of clone libraries

was calculated according to Good (12) using the equation C= [1−(n1/N)]*100, where C is the

homologous coverage, n1 is the number of OTUs appearing only once in the library, and N is 220

the total number of clones examined. UniFrac significance and principal coordinates analysis

were performed using the UniFrac software tool (22, 23) which allowed comparison of the

clone libraries based on phylogenetic information.

(iv) DNA microarray-based microbial community analyses. Sampling, enrichment for

anaerobic condition, DNA extraction, PCR conditions, etc., were carried out as detailed 225

above. Gradient PCR amplicons from each of the four samplings were hybridized on the

DNA microarray G2 PhyloChip. A detailed explanation of the processing of the PhyloChip

assay is published elsewhere (17, 40). Briefly, the pooled PCR product from each sampling

event was spiked with known amounts (5.02 × 108 to 7.29 × 10

10 molecules) of synthetic 16S

rRNA gene fragments and non-16S rRNA gene fragments (total, 200 ng). Fluorescence 230

intensities from these controls were used as standards for normalization among samples.

Target fragmentation, biotin labeling, PhyloChip hybridization, scanning, and staining, as

well as background subtraction, noise calculation and detection, and quantification criteria

were performed as reported elsewhere (9). An OTU was considered present in the sample

when 90% or more of its assigned probe pairs for its corresponding probe set were positive 235

(positive fraction of >0.90).

(v) Controls. To ensure the sampling and processing procedures were correctly performed,

several negative controls were included in every step described herein. For each sampling

event, one negative control (field blank) was carried through processing. While sampling, one

BiSKit was exposed to the clean room air for the same period of time without active 240


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sampling. These negative controls were obtained for every sampling event and included for

culture-dependent and molecular analyses. None of the four negative controls showed the

amplification of the targeted gene. Cultivation and isolation controls similarly revealed no

positive results.

RESULTS 245

Sensitivity of the methods employed. A comparison of wet and dry sampling with the

BiSKit indicated that dry sampling was more efficient (efficiency 18.4%) than wet sampling

(efficiency 11.3%) (4). However, it has been reported that, although the sampling efficiency

was lower for the wet sampling method, the wet samples were more concentrated due to the

low sample volume and therefore had greater detection sensitivity when they were used 250

directly for biological detection assessment (4). With reference to sample recovery, BiSKit

samplers are superior (~10 to 20%) to the wipe samples (less than 10%) because the BiSKit

samplers are compact and smaller volumes of sample buffer are required to recover the

impacted bacteria. Since cultivation is based on the enrichment methods, theoretically, even

if only one anaerobic cell is present in the sample, it should be enriched. However, it is not 255

possible that all known anaerobes will grow under the conditions employed here.

Furthermore, there is a consensus that PCR-dependent techniques require ca. 103 initial

copies of the target to reproducibly assess microbial diversity for standard PCR and ca. 102

initial copies for gradient PCR (17). Therefore, although theoretical calculations posit the

need for as few as one copy of a given target molecule, signal-to-noise ratios demand that 260

target molecules exist in excess of 102–3

copy numbers (due to indigenous DNA in reagents

and sampling materials). In this study, since initial enrichment was employed before

extracting DNA and the sample was analyzed via conventional cloning or PhyloChip

analyses, sensitivity is not a problem for these assays but collection of the target organism

from the environment needs to be resolved. 265


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Isolation of strict anaerobes. A total of 113 anaerobic strains were isolated during this

study, including 21 strains isolated exclusively from GSE present within the JPL-SAF. The

TS/TG enrichments followed by plating on anaerobic TS agar medium (88 strains) promoted

greater isolation of anaerobes compared to the direct agar plating on TS agar (20 strains)

without enrichment. However, enrichment in the modified ATCC 591 medium under CO2 gas 270

phase yielded only six anaerobic strains and did not result in the abundant growth observed

with TS enrichment. The majority of isolates (93 of total 113 strains) exhibited growth in the

presence of oxygen and were not further characterized. Obligate anaerobes were successfully

isolated from each clean room facility (Table 1). All 20 strict anaerobic strains (17.7% of all

isolates in this study) were gram-positive bacteria belonging to the genera Clostridium (four 275

species) or Propionibacterium (one species). Three different species (C. colicanis, C.

sporogenes, and P. acnes) were retrieved from clean room floors, while four species were

obtained from JPL-SAF GSE (C. perfringens, C. saccharolyticum, C. sporogenes, and P.

acnes). Since analysis of the 16S rRNA gene cannot distinguish C. sporogenes from C.

botulinum, the presence of the C. botulinum Type A toxin gene was also tested using 280

established procedure (21). The strains in question did not yield Type A toxin gene

amplicons, confirming that they did not belong to C. botulinum (data not shown). Sporulation

was observed in all strains of the genus Clostridium (except C. colicanis) after anaerobic

incubation for an appropriate duration in TS medium. Some of these clostridial strains were

able to grow on modified ATCC 591 medium with CO2 gas phase as well as on TS medium 285

with N2 gas phase (Table 1).

Molecular microbial community analyses:

(i) Conventional clone library analysis. All four samples tested via gradient PCR showed

16S rRNA gene amplification products. By comparison, only three were successful (the

JPL-SAF-A sample did not yield appreciable 16S amplicons) when standard PCR 290


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amplification conditions were employed. Retrieval of high-quality 16S rRNA gene sequences

was similar when different PCR conditions were compared (~66% of 300 clones for standard

PCR and 62% of 400 clones for gradient PCR). Although clones were robotically picked and

sequenced from each library, after quality control (chimera check, elimination of short

sequence, etc.) only 63.5% (JPL-SAF-A), 67.2% (Bdg-233-B), 57.3% (JPL-SAF-C), and 295

66.1% (BDL-D) of clones yielded high-quality sequences, which were used for subsequent

phylogenetic and comparative analysis. The enrichment followed by cloning and sequencing

approach revealed sequences related to strict anaerobes (27%; 114 clones) and facultative

anaerobes (77%). The sequences retrieved spanned 10 families, 11 genera, and 70 distinct

OTUs when analyzed by DOTUR analysis (Table 2). All 70 OTUs detected in this study 300

clustered with 20 known bacterial species. Only members of the phyla Actinobacteria (13

OTUs; 4 species) and Firmicutes (57 OTUs; 16 species) were observed. Sequences of two

strictly anaerobic microorganisms (P. propionicum and Sarcina ventriculi) retrieved via

molecular analysis were not isolated using the cultivation methods employed. Likewise, only

two species of strict anaerobes (C. colicanis and P. acnes) isolated from enrichment cultures 305

during this study were present in the corresponding clone library, while C. sporogenes could

not be detected in the clone library even though it was successfully isolated. All other strict

anaerobes isolated from GSE samples were not observed in the clone libraries. About 33% of

the sequences (142 out of 427 clones) shared lower than 97.5% 16S rRNA gene sequence

similarity with closest type strains and may belong to novel species. In addition to the 310

obligate anaerobes, 70 (16%) sequences were related to species of Actinobacteria, 176 (41%)

sequences to sporeformers (Paenibacillus, Bacillus, Clostridium, Sarcina), and 178 (42%)

sequences to Staphylococcus species. About 70% of sequences were affiliated to the

microbes that are human commensals and/or human pathogens (e.g. Staphylococcus,

Propionibacterium, and Dermabacter). 315


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The coverage values of the clone libraries ranged from ~73% to ~97%, and the rarefaction

curves for seven clone libraries are shown in Fig. 1. Coverage values for JPL-SAF-A,

JPL-SAF-C, and BDL-D were >88% and reached a plateau in rarefaction analysis, indicating

that the methods used to pick clones were quite sufficient. Samples from Bdg-233-B

exhibited the highest number of OTUs (Table 2) and lowest coverage value (73%) plus a 320

rarefaction curve with a steep slope (Fig. 1) indicating incomplete sampling of clones for

sequencing. When standard PCR conditions were used to construct clone libraries, only 36

different OTUs were observed, while use of gradient PCR revealed 47 OTUs based on

DOTUR analysis (Table 2). Furthermore, the standard PCR conditions exhibited lower

coverage values (1 to 5% less than gradient PCR) and slightly steeper rarefaction curve 325

slopes compared to libraries constructed from gradient PCR products (Fig. 1). Although a

slight increase in the OTUs incidence (one to five OTUs more) was observed in the libraries

constructed after gradient PCR, UniFrac analysis indicated marginal to no significant

statistical differences in bacterial diversity between standard PCR and gradient PCR clone

libraries from all samples. Regardless of PCR protocol, however, UniFrac significant analysis 330

(p=<0.001) and principal coordinates analysis (Fig. 2) revealed significant differences in

bacterial population among the four sampling events, indicating distinct bacterial populations

within each clean room location (Fig. 2).

Among the clean rooms tested, only the JPL-SAF-C locations revealed no 16S rRNA gene

sequences of strictly anaerobic microorganisms, but sequences of the facultative anaerobe S. 335

epidermidis were retrieved from all facility floors. S. caprae and S. capitis subsp. capitis

sequences were found to be present in three of the sampling locations. Interestingly, despite

the fact that all clean rooms are in the same geographical/climate location, they did not share

similar bacterial populations. While members of Bacillus and Staphylococcus were common

at the genus level, JPL-SAF-A and JPL-SAF-C shared no common species except S. 340


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epidermidis.

(ii) DNA microarray-based microbial community analysis. Figure 3 demonstrates the

distribution of bacterial diversity detected by DNA microarray technology. Additional raw

data based on family-level incidence are given (see Supplementary Table 2). Comparisons of

the microbial diversity in each sampling event indicate that JPL-SAF-A possessed the highest 345

diversity (719 OTUs), but when the same facility was sampled again within a span of a

month (JPL-SAF-C), the lowest diversity was observed (422 OTUs). Compared to the clone

libraries, where only gram-positive bacterial sequences were retrieved, DNA microarray

analysis showed that only two-thirds of the total diversity were gram-positives in these

facility locations. All families presented in clone libraries were also detected via microarray, 350

with the exception of Carnobateriaceae (Granulicatella), which was sequenced from the

JPL-SAF-A sample and exhibited 98% similarity with the described type strain. Unlike the

results from the cloning-sequencing methods, DNA microarray technology detected strict

aerobes such as members of the family Micrococcaceae.

At the family level, Bacillaceae dominated each sampling, which is consistent with previous 355

cultivation-based studies (32). Interestingly, microarray analysis showed higher diversity of

Clostridiaceae in Bldg-233-B compared to the other samples. Furthermore, the diversity of

Lachnospiraceae and Peptostreptococcaceae, both clustering within the Clostridiales and

capable of growing only under strict anaerobic conditions, were present in all facilities.

Several families detected—such as Helicobateriaceae, Enterococcaceae, Mycobacteriaceae, 360

or Spirochaetaceae—include opportunistic pathogens. Though sequences of the members of

Staphylococcaceae and Propionibacteriaceae dominated in clone libraries, only a few OTUs

were observed as hybridization signals in DNA microarray. In addition, the diversity of

Propionibacteriaceae was low using either molecular method but still dominant in clone

library analysis if present (JPL-SAF-A). 365


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DISCUSSION

Previous microbial surveys of clean room environments have been analyzed either via a

culture-based approach or molecular methods (16, 17, 25, 36, 37). In contrast, this study

combined a primary enrichment suitable for anaerobes in oxygen-free media and

subsequently analyzed them to elucidate a more complete view on anaerobic microbial 370

diversity using the state-of-the art molecular methods (13, 38). Although used for anaerobic

cultivation, samples were aerobically collected and processed, since on site handling of

anaerobic equipment was not possible due to the ongoing spacecraft assembly. When

anaerobic and aerobic sampling procedures were used in parallel for sample collection, there

were no significant differences in detection between the two sampling methods (36). In 375

addition, the isolation and retrieval of 16S rRNA gene sequences of non-sporeforming

obligate anaerobes in the samples collected under aerobic conditions suggested that when

samples are analyzed within an hour of sampling, elaborate use of anaerobic sample handling

instruments can be avoided.

The enrichment approach was selected to suppress the growth of aerobes not able to thrive 380

under the anaerobic conditions provided. Clone libraries constructed from enrichment

cultures revealed only anaerobes and suggested that all sequences retrieved were of microbes

able to grow under these defined enriched culturing conditions. Moreover, the broad

spectrum of strict anaerobes detected in clone libraries compared to cultivation studies

indicated the advantage of this technique. Certainly, the enrichment conditions employed 385

(media, temperature, gas phase, etc.) might preferentially support growth for only a subset of

all known anaerobes; hence, the approach taken during this study does not cover all

anaerobes that might possibly be present in a sample. Because universal primer set(s) to

selectively amplify anaerobes do not currently exist, and since the previous molecular studies

suggested that only 0 to 8% of the total rRNA gene sequences retrieved belong to anaerobes, 390


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the approach used in this study is a step forward in identifying the extent of anaerobic

bacterial diversity compared with approaches reported in other studies (25, 36, 37).

Compared to the cultivation approach (two microbial genera), the amplification and cloning

of 16S rRNA genes from enrichments revealed a broader diversity of microorganisms (11

microbial genera). Interestingly, the two different amplification methods performed (standard 395

PCR and gradient PCR) resulted in no significant diversity differences. Despite their

similarities in geographic location and environmental controls, UniFrac analysis found

distinct bacterial populations associated with each clean room facility. More research is

necessary to confirm whether the microbial diversity in the clean rooms is influenced by the

environment as has been suggested by previous studies (14, 25). In addition, the analysis 400

presented here indicates that the presence of spacecraft hardware in the clean room and high

human activity has a strong influence on the transport of microbes into the clean room. The

change in microbial diversity between JPL-SAF-A (before spacecraft components arrival)

and JPL-SAF-C (only four weeks between the samplings; during spacecraft assembly) might

be attributed to the fact that selective pressure can cause the bacterial population to change 405

very fast; but more sampling points are needed to validate this assumption.

The findings of the microarray analysis were consistent with those of the clone libraries

concerning the distribution of the detected diversities. In general, the microbial population of

the sampled clean rooms seemed to be very similar at the phylum level (Fig. 3), but analysis

of bacterial families revealed major differences. The advantages of PhyloChip over clone 410

libraries has already been reported (6, 17); and similar to previous investigations (9 to 70-fold

increase), this study found an approximate 10-fold increase in number of different OTUs

when PhyloChips were used. The high number of detected gram-positive bacterial families

indicated that their presence is abundant in all clean rooms analyzed. The results also suggest

the presence of a wide range of low-abundance organisms, as one-third of the bacterial types 415


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found by the PhyloChip belonged to phyla not observed by clone libraries. Likewise, the

diversity of strict anaerobes, especially within the order Clostridiales was found to be more

diverse when PhyloChip was employed. These results are in contrast to those of a published

study (36) that may have underestimated the obligate anaerobes in clean room facilities due

to the use of a cultivation-based analysis only. 420

In summary, the two-step approach (initial enrichment followed by the molecular analysis)

implemented during this study to survey anaerobic bacterial diversity, yielded two

advantages. Initially, the dilution of the inocula causes attenuation of aerobic bacterial DNA

that might have been available for clone library analysis. Secondly, anaerobic bacteria were

preferentially enriched under the cultural conditions employed, making anaerobic bacterial 425

DNA more readily available for PCR amplification. These predictions were reflected in the

results of this study: aerobic microbial diversity was absent in clone libraries due to cloning

biases (for abundant DNAs in ligation and transformation), whereas the PhyloChip detected

low abundance aerobic DNA (e.g., Micrococcaceae) due to its high sensitivity, as reported

elsewhere (3, 17). However, the low abundance of Propionibacteriaceae signatures in 430

PhyloChip compared to their dominance in both clone libraries (Table 2) and isolation

procedures (Table 1) suggest that more scrutiny may be required in the microarray probe

design for this bacterial group.

Results of this study indicate that anaerobic sporeformers such as clostridia may have access

to the MSL components. Since many of these anaerobes were isolated from the GSE used to 435

contain and support MSL subsystems during assembly, it is recommended that the

quantitative measurement of anaerobic population (and appropriate cleaning protocols) be

considered in strategic planning to preserve the scientific integrity of high-sensitivity

missions. Though clostridia may not be able to germinate or thrive on Mars due to their

fermentation-based metabolism, the high level of resistance of their spores to heat, chemicals 440


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and UV irradiation (particularly noted in C. perfringens) may allow survival and proliferation

during space flight; this could potentially cause forward contamination significant enough to

confound the results of a life detection mission (28, 30, 34).

The composition of the Martian atmosphere is dominated by CO2 with traces of N2, O2, CO

and H2O, and the aerobic microbes are unlikely to grow under these conditions. Furthermore, 445

no organic compounds have yet been detected on the Martian surface; the absence of organic

compounds limits the potential number of microbes that could thrive on the planet’s surface

(39). The isolation of several strict anaerobes that use nitrogen (such as P. borealis) and CO2

to synthesize organic compounds in European Space Agency clean rooms (7, 36) is a concern

since their transport to the exploring planets via human made satellites is possible. However, 450

it is important to note that chemolithotrophs, which could act as pioneers in colonizing Mars,

were not detected by either clone libraries or PhyloChips in the anaerobic enrichment cultures

prepared over the course of this study. Apparently, an appropriate culture condition enriching

chemolithotrophs could be used to confirm this assumption (36). Future studies should

consider characterizing resistance of these anaerobes to space conditions and isolating 455

anaerobic chemolithotrophs from spacecraft and associated surfaces. Although the results of

this study are of particular interest to space faring nations, the study will also benefit a wide

range of scientific, electronic, homeland security, medical, and pharmaceutical ventures that

produce sensitive products or assemble products in clean rooms.

ACKNOWLEDGMENTS 460

The research described in this publication was carried out at the Jet Propulsion Laboratory,

California Institute of Technology, under a contract with the National Aeronautics and Space

Administration. This research was funded by the Mars Program Office at JPL. We are

grateful to K. Buxbaum for funding, J. A. Spry for proofreading and critique of the

manuscript, S. Foster for editing, and to all of the members of the JPL Biotechnology and 465


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Planetary Protection Group for technical assistance. Especially acknowledged are Wayne

Schubert for assistance and maintenance of the anaerobic glove box and J. Nick Benardini for

sampling MSL components and assembly facilities. The authors are particularly grateful to

Todd DeSantis at Lawrence Berkeley National Laboratory for his input on PhyloChip

analysis and for use of the greengenes suite of molecular analysis tools (greengenes.lbl.gov). 470

Copyright 2009. All rights reserved.


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Figure legends.

Figure 1. Rarefaction analysis of anaerobic bacterial diversity from several spacecraft

assembly facilities. Coverage values (C) for each clone library are given in parentheses. 605

Rarefaction curves of standard and gradient PCR samples are depicted with asterisks and

in italics, respectively.

Figure 2. Environmental cluster analyses showing the relationship between samples.

Figure 3. Overview of phyla retrieved from the DNA microarray analysis. The number in

parentheses given in X axis after each sampling event are the number of OTU counts 610

retrieved. Families having a minimum of 10 OTU counts are given in parentheses.

Underlined families were also detected in conventional clone libraries of related

samplings. The “others” category denotes the phyla having fewer than 10 OTU counts as

well as unclassified members.

615


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TABLE 1. Obligate anaerobes isolated from various spacecraft-associated surfaces.

Phylum/Family Genus and species (of closest relative type strain), strain, accession number

% of 16S rRNA gene sequence

similarity

Number of

isolatesa

Media used to isolate

Sample Facility and sampling

event

Accession numbers (FJ957-)

Firmicutes / Clostridiaceae:

Clostridium colicanis DSM 13634 (AJ420008)

99.6 8 TS, TGA AA-019 Bldg-233-B 863-870

Clostridium perfringens ATCC 13124 (M59103)

99.6 1b TS GSE JPL-SAF-C 872

Clostridium saccharolyticum DSM 2544 (Y18185)

98.3 3 modified ATCC 591, TS

GSE JPL-SAF-C 873-875

Clostridium sporogenes ATCC3584 (X68189)

100.0 1 modified ATTC 591, TS

GSE JPL-SAF-A 860


100.0 1b

modified ATTC 591, TS

GSE JPL-SAF-C 871


100.0 1 modified ATTC 591, TS

AA-036 BDL-D 876

Actinobacteria / Propionibacteriaceae:

Propionibacterium acnes DSM 1897 (X53218)

99.0 1 TS, TGA AA-003 JPL-SAF-A 857


99.0 2 TS, TGA pooled JPL-

SAF-A JPL-SAF-A 858, 859


99.0 2 TS, TGA GSE JPL-SAF-A 861, 862

aSequences were assigned to OTUs using DOTUR analysis, and a representative sequence from each OTU was compared against RDP-type

strain database using BLAST function. bIsolates were retrieved from direct culturing (spread plating). Others were isolated from enrichment cultures.


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TABLE 2. Anaerobic bacteria diversity–based clone libraries of enrichments of various spacecraft facilities.

Number of clones retrieved fromb:

JPL-SAF-A

Bldg-233-B JPL-SAF-C BDL-D Phylum/Family (# of OTUs)

Genus and species (of closest relative type strain), strain, accession number

OTU#a

gradient PCR (10)

standard PCR (24)

gradient PCR (27)

standard PCR (6)

gradient PCR (7)

standard PCR (10)

gradient PCR (15)

Actinobacteria (13)

Cellulomonadaceae (1)

Oerskovia paurometabola; DSM 14281; AJ314851

1 1 1

Dermabacteraceae (2)

Dermabacter hominis; DSM 7083; X91034 2 5 5

Propionibacteriaceae

(10) Propionibacterium acnes; DSM 1897; X53218 7 24 18 12

Propionibacterium propionicum; DSM 43307; AJ003058

3 4

Firmicutes (57)

Bacillaceae (13) Bacillus licheniformis; DSM 13; X68416 8 3 41 29

Bacillus aerius MTCC 7303; AJ831843 1 1

Bacillus thuringiensis; ATCC 10792; AF290545 2 24 14

Bacillus weihenstephanensis; DSM 11821; AB021199

2 1 3

Carnobacteriaceae (1)

Granulicatella adiacens; ATCC 49175; D50540 1 1

Clostridiaceae (19)

Clostridium colicanis; DSM 13634; AJ420008 8 11 10

Sarcina ventriculi; DSM 286; X76649 11 19 16

Leuconostocaceae (1)

Leuconostoc lactis; DSM 8581; AF175403 1 1

Paenibacillaceae (3)

Paenibacillus odorifer; ATCC BAA-93; AJ223990 1 1

Paenibacillus borealis; DSM 13188; AJ011322 2 1 2


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Staphylococcaceae (19)

Staphylococcus capitis subsp. capitis; ATCC 49326T; AB009937

1 1

Staphylococcus caprae; ATCC 35538; AB009935

2 1 2 4 2 5

Staphylococcus epidermidis; ATCC 14990; D83363

14 30 18 14 31 29 9 30

Staphylococcus saccharolyticus; ATCC 14953; L37602

1 1

Staphylococcus warneri; ATCC 27836; L37603 1 1

Streptococcaceae (1)

Streptococcus parasanguinis; ATCC 15912; AF003933

1 1

aEven though some of the clone sequences retrieved show similarities with the nearest neighbor, the DOTUR analysis considered these sequence as different OTU.

bNumbers of OTUs are given in brackets. Clones were assigned to OTUs using DOTUR analysis, and a representative clone from each OTU was compared against

RDP-type strain database using BLAST function.


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Fig 1

Bldg-233-B* (C = 78.38%)

Bldg-233-B (C = 72.73%)

BDL-D* (C = 94.83%)

BDL-D (C = 94.20%)

JPL-SAF-A (C = 88.52 %)

JPL-SAF-C* (C = 96.61%)

JPL-SAF-C (C = 96.08 %)

Number of sequences

0 20 40 60 80

Nu

mb

er

of

OT

Us

0

5

10

15

20

25

30


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Fig 2

JPL-SAF-A

Bldg-233-B

BDL-D

JPL-SAF-C

P2

–P

erc

en

t va

ria

tion

exp

lain

ed

21

.36

%

P1-percent variation explained 39.82%

PCA-P1 vs P2


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Fig 3

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

JPL-SAF-A (719) BLDG-233-B (626) JPL-SAF-C (422) BDL-D (524)

Others*

Verrucomicrobia

Spirochaetes

Chloroflexi

Acidobacteria

Bacteroidetes

Proteobacteria

Actinobacteria

Firmicutes

Acidobacteriaceae (13)

Streptomycetaceae (16)

Micromonosporaceae (14)

Nocardiaceae (11)

Cellulomonadaceae (10)

Microbacteriaceae (10)

Micrococcaceae (10)

Bacillaceae (45)

Lachnospiraceae (39)

Clostridiaceae (37)

Peptostreptococcaceae (18)


Streptococcaceae (15)

Enterococcaceae (11)

Helicobacteraceae (19)

Mycobacteriaceae (10)

Bacillaceae (59)

Clostridiaceae (54)




Lactobacillaceae (13)


Bacillaceae (61)








Spirochaetaceae (16)


Helicobacteraceae (10)



Microbacteriaceae (12)

Mycobacteriaceae (11)

Micrococcaceae (10)

Bacillaceae (56)


Clostridiaceae (23)





Firmicutes

Others

Verrucomicrobia

Spirochaetes

Chloroflexi

Acidobacteria

Bacteroidetes

Proteobacteria

Actinobacteria


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1 microbial diversity of anaerobes from spacecraft assembly clean

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