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Research in Microbiology 157 (2006) 5–12

www.elsevier.com/locate/resmi

Survey of environmental biocontamination on boardthe International Space Station

Natalia Novikovaa,∗, Patrick De Boeverb, Svetlana Poddubkoa, Elena Deshevayaa,Nikolai Polikarpova, Natalia Rakovaa, Ilse Coninxb, Max Mergeayb

a State Scientific Center of Russian Federation, Institute of Biomedical Problems RAS, Khoroshevskoye Shosse 76 A, Moscow 123007, Russiab Laboratory for Microbiology and Radiobiology, Belgian Nuclear Research Centre (SCK-CEN), Boeretang 200, 2400 Mol, Belgium

Received 12 April 2005; accepted 20 July 2005

Available online 1 December 2005

Abstract

The International Space Station (ISS) is an orbital living and working environment extending from the original Zarya control module1998. The expected life span of the completed station is around 10 years and during this period it will be constantly manned. It is inevthe ISS will also be home to an unknown number of microorganisms. This survey reports on microbiological contamination in potable wand on surfaces inside the ISS. The viable counts in potable water did not exceed 1.0 × 102 CFU/ml. Sphingomonas sp. andMethylobacteriumsp. were identified as the dominant genera. Molecular analysis demonstrated the presence of nucleic acids belonging to various pano viable pathogens were recovered. More than 500 samples were collected at different locations over a period of 6 years to charactesurface contamination in the ISS. Concentrations of airborne bacteria and fungi were lower than 7.1× 102 and 4.4× 101 CFU/m3, respectively.Staphylococcus sp. was by far the most dominant airborne bacterial genus, whereasAspergillus sp. andPenicillium sp. dominated the fungapopulation. The bacterial concentrations in surface samples fluctuated from 2.5× 101 to 4.3× 104 CFU/100 cm2. Staphylococcus sp. dominatedin all of these samples. The number of fungi varied between 2.5× 101 and 3.0× 105 CFU/100 cm2, with Aspergillus sp. andCladosporium sp.as the most dominant genera. Furthermore, the investigations identified the presence of several (opportunistic) pathogens and strainsthe biodegradation of structural materials. 2005 Elsevier SAS. All rights reserved.

Keywords: International Space Station; Space flight; Contamination; Bacteria; Fungi; Air and surface sampling; Pathogenicity; Biodeterioration

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1. Introduction

Man’s instinct to explore space can be exemplified bymerous manned missions (Apollo program, construction,exploitation of the Mir orbital station, etc.) and unmanned msions (Cassini–Huygens mission, Mars Express mission, eThe main goal of manned exploration is to achieve a prolonstay in space, e.g. in an orbital station or in planetary basethe moon and/or Mars. It goes without saying that such msions can only be realized if the cosmonaut’s health and wbeing are secured. The characterization of microbiological ctamination on board spacecraft and orbital stations is there

* Corresponding author.E-mail address: novikova@imbp.ru (N. Novikova).

0923-2508/$ – see front matter 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.resmic.2005.07.010

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of paramount importance. Russian reports on the exploitaof the orbital station Mir have indicated that microorganisare ubiquitously present and that they should be considereindigenous to any spacecraft environment [19,21,25]. Micbial contamination may originate from different sources aincludes the initial contamination of space flight materials ding manufacturing and assembly, the delivery of supplies toorbital station, the supplies themselves, secondary contamtion during the lifetime of the orbital station, the crew and aother biological material on board, e.g. animals, plants andcroorganisms used in scientific experiments [22,25].

The cosmonaut is probably the most important contamtion source, as his body contains a large amount of bactThese are found on the skin, on mucous membranes, inupper respiratory tract, the mouth, the nasal passage, athe gastrointestinal tract. The two major routes through wh

6 N. Novikova et al. / Research in Microbiology 157 (2006) 5–12

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the human microbiota can be spread in the environment(i) the air followed by sedimentation on surfaces; and (ii)rect transfer to a surface. Although most microorganisms dothreaten human health, it has been reported that in a conenvironment such as a space cabin, microorganisms mayduce adverse effects on the optimal performance of the screw and the integrity of the spacecraft or habitat. These efrange from infections, allergies, and toxicities to degradatioair and water supplies [24]. Biodegradation of critical matermay result in system failure and this may jeopardize the cStudies performed in Mir indicated that some equipmentstructural materials were prone to the accumulation andliferation of biodestructive bacteria and fungi [21,22]. Damato polymers and metals could be observed and this resultcases of malfunctioning and even breakage of certain unitsair conditioners, water recycling systems, etc. [10,13].

The International Space Station (ISS) is the orbital stathat is being built by the United States in collaboration wRussia, 11 nations of the European Space Agency, CanJapan and Brazil (Fig. 1). The ISS is the largest and most cplex international scientific project in history. More than fotimes as big as the Russian Mir space station, the compISS will have a mass of about 500 tons. It will measure 10across and 88 m in length, with more than 4000 m2 of solarpanels providing electrical power to six state-of-the-art laratories. The projected lifetime of the ISS after completionapproximately 10 years. During this period, the station willperience periodic visits from international spacecraft for cexchanges, resupply of food and consumables, and manyloads with scientific experiments [26].

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Space agencies attempt to avoid microbiological prlems by developing strategies to limit microbial contaminataboard the ISS (disinfection and sterilization of space flmaterials during assembly and transport to the ISS, rigocleaning procedures, etc.). Nevertheless, monitoring of bioical contamination is imperative and the results of the invtigations may trigger specific countermeasures when microconcentrations pass defined thresholds. Over the past few ya number of research groups have had access to air,and surface samples taken aboard the ISS. These samplebeen characterized using state-of-the-art microbiological ansis techniques.

Investigations into the microbial load of ISS potable wahave been mainly reported by La Duc and coworkers [15,The latter authors analyzed ISS potable water samples aferent timings (preflight, during flight and postflight) andvarious stages of purification, storage and transport. A comnation of culture-dependent and culture-independent anawas used to characterize microbial contamination. A bioctreatment in the form of iodine was responsible for the fthat the preflight potable water had bacterial concentratbelow the detection threshold (i.e. less than 1 CFU/100 ml).A water sample collected from the humidity condensate recery system yielded 5.1 × 10 CFU/100 ml and isolates of botAcinetobacter radioresistens and Acidovorax temperans wereobtained [17]. Characterization of the microbial content ofhumidity condensate is important because this water isfor generation of potable water in the ISS. La Duc and colorators performed a molecular microbial diversity analysisreveal that ISS drinking water contained DNA sequences f

Fig. 1. Overview of the main structural elements of the ISS. The information is taken from http://spaceflight.nasa.gov/station/assembly/index.html (last accessionon July 4th, 2005).

N. Novikova et al. / Research in Microbiology 157 (2006) 5–12 7

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many types of bacteria [16]. Bacterial small subunit rDNA framents were PCR-amplified with the eubacterial primers Band B512R. Purified amplicons were cloned and represetive DNA inserts were sequenced. A first sample contaiDNA sequences from N2-fixing Bradyrhizobium sp., and var-ious biofilm-producing microbes (Caulobacter sp., Hyphomi-crobium sp., etc.). The analysis also confirmed the presencDNA sequences originating fromOchrobacter sp., Propioni-bacterium sp., andBrevundimonas species. The latter organisms are catalogued as human pathogenic bacteria. A sesample contained DNA sequences ofMethylobacterium exo-torquens andDelftia acidovorans. These organisms are knowto colonize and attack a large variety of polymeric and metasurfaces. Samples originating from the ISS humidity condsate water were rich with sequences of the bacterial pathAfipia broomeae. La Duc and colleagues were not able to prothe presence of active pathogens in the ISS water samplehence they could not calculate concentrations of pathogbacteria. Their study suggests that pathogenic bacteria mapresent in the water systems of the ISS [15,16]. Future reseshould be directed towards identification of niches in whthese microorganisms may accumulate and survive. Thishelp to assess the health risk for cosmonauts. A second minformation source is the research conducted by Castro e[4]. Water samples were collected from the hot and cold pof the humidity condensate recovery system. The potableter supply that is generated by reclaiming humidity condenconsistently provided water with bacterial levels below theacceptability limit of 1.0 × 102 CFU/ml. Twenty-seven bacterial colony types were isolated within the frame of this stuThe identifications were performed by sequencing a 527fragment of the 16S rDNA gene using the eubacterial primB005F and B531R included in the Microseq 500 16S rDBacterial Sequencing kit from Applied Biosystems (USA). Thumidity condensate contained mostly Gram-negative bacand the population was predominantly made up of the geSphingomonas sp. (25%) andMethylobacterium sp. (18%).

Publicly available documents about airborne contaminaand contamination associated with the ISS interior are spWe report in this survey the results of a six-year campa

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during which airborne and surface contamination in thewas monitored. The investigations have been carried outhe Institute of Biomedical Problems (IBMP, Russia), whis responsible for monitoring biological contamination in thabitable compartments of the ISS for safety and hygienicsons, and the Belgian Nuclear Research Centre (SCK-CBelgium).

2. Materials and methods

2.1. Surface samples

Surface sampling was done by swabbing a 10× 10 cm sur-face of the interior and equipment using an in-house-madepling kit (Surface pipette kit). This kit consists of a belt wipockets containing fluoroplastic tubes with swabs impregnin a phosphate buffer containing vaseline (Fig. 2).

Upon receipt of the samples in the laboratory, aliquots winoculated on Petri dishes containing various nutrient me(tryptic soy nutrient agar, mannitol salt agar, Mac Conkey aSabouraud chloramphenicol agar, potato dextrose agar,Czapek–Dox agar). The Petri dishes were incubated at 3◦Cfor 48 h or 28◦C for five to seven days to recover bacteria afungi, respectively. Subsequently, the bacterial and fungalcentrations were determined. Morphologically different bacria were picked for Gram staining and identification by meof the Vitek system (BioMérieux, France). Fungal strains widentified microscopically by their morphological charactetics [11]. Yeasts and yeast-like fungi were also identified usthe Vitek system. The Vitek system makes use of tests cmade up of 30 or 45 microwells containing either identificatsubstrates or antibiotics. The growth pattern of axenic isolin these test cards is recorded and used for species identificusing specialized software.

2.2. Air samples

The Russian Ecosphere kit includes a spaceflight-certSAS air sampler (PBI International, Italy) that collects airbobacteria by impaction on agar medium, and sets of Petri di

Fig. 2. Surface pipette kit for sampling internal surfaces and structural materials of the ISS.

8 N. Novikova et al. / Research in Microbiology 157 (2006) 5–12

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Fig. 3. The Russian Ecosphere kit includes a spaceflight-certified SAS airpler and sets of Petri dishes containing nutrient-rich media.

containing nutrient-rich media (tryptic soy nutrient agarbacteria and Czapek–Dox agar for moldy fungi). The eqment is stored permanently onboard the ISS, whereas theof Petri dishes are delivered by cargo and transport veh(Fig. 3). Air samples of 90 l were taken one to two days priothe undocking of the cargo vehicle and return to earth. Uponceipt in the IBMP laboratory, the Petri plates were incubatedescribed above and colonies were counted afterwards. Thtained isolates were identified using the procedures descunder the section of surface samples.

Identification procedures using molecular tools startedgenerating an overnight culture of the axenic bacterial straIncubation was done in Luria broth at a temperature of 28◦C.Genomic DNA was extracted using the Wizard SV GenoDNA Purification system (Promega, USA) according to the ptocol supplied by the manufacturer. The genomic DNA wused for performing a PCR reaction. The PCR reaction mture contained per sample 9.5 µl sterile double-distilled wa1 µl forward primer, 1 µl backward primer, 2.5 µl bovine seralbumin (stock concentration of 1 mg/ml), 2.5 µl dNTPs (stocksolution of 2 mM), 2.5 µl 10× PCR buffer, 1 µl Taq DNA poly-merase and a 5 µl sample. The PCR reaction was perfoon 25 µl samples with a GeneAmp 2700 (Applied Biostems, USA) using the following protocol: denaturation (3 mat 94◦C), followed by 34 cycles of denaturation (45 s at 94◦C),annealing (45 s at 56◦C), and extension (45 s at 72◦C). Finalextension was done for 7 min at 72◦C. Amplification was per-formed using the eubacterial primers B008F (AGATTTGATCTGGCTCAG) and B926R (CCGTCAATTCCTTTRAGTTTThe positions refer to the 16S rDNA ofEscherichia coli. PCRproducts were cleaned up with a Wizard SV gel and PCR clup system (Promega, USA) using the protocol described bymanufacturer. The sequencing reaction was performed acing to the following protocol. Five to twenty ng of PCR produwas mixed with 2 µl (±3.2 pmol) F-primer or R-primer (seabove) and 8 µl of big dye mix (Applied Biosystems, USAPCR reaction conditions were 3 min at 96◦C, followed by 25cycles of 30 s at 96◦C, 30 s at 50◦C, and 4 min at 60◦C. Thereaction was stopped by putting the mixture at 4◦C. Purifica-

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tion of the reaction mixture was done using Centri-Sep colu(Princeton Separations, USA) according to the instructionsplied by the manufacturer. DNA sequences were determby performing capillary electrophoresis with fluorescent detion on an ABI310 automatic sequencer (Applied BiosysteUSA). Data were collected with the Sequencing Analysis sware v 3.7 of Applied Biosystems. The DNA sequences wcompared by BLAST analysis to all sequences in the Genbdatabase using the software tool described by Devulder andlaborators [6].

3. Results and discussion

The compositional analysis of microorganisms residingthe ISS environment has been performed over a period oyears. Samples from nine main missions and seven Soyuzflights to the ISS have been processed from the year 199the present. A total of 419 samples from air and surfaces wscreened for the presence of bacteria. Bacteria were recoin 71% of the cases (i.e. 297 samples). In addition, thegal contamination aboard the ISS was investigated by analy378 samples from air and surfaces. Fungi were obtained92 samples, which is 24% of all the samples. The large spediversity that was obtained from the environmental samplesmore pronounced in the surface samples than in the air samA total of 36 and 15 bacterial species were isolated from surand air samples, respectively. A total of 32 and 5 fungal spewere isolated from surface and air samples, respectively.

During the monitoring campaign, 243 swab samples wtaken from structural elements and internal surfaces at 33ferent locations in the Service Module (SM), Functional CaBlock (FGB), NODE-1, and LAB. The surfaces were sampone to two days before the end of each mission. Identificaof the microbial contaminants was performed as describeSection 2.1. The bacterial concentrations fluctuated withbroad range, i.e. from 2.5 × 101 to 4.3 × 104 CFU/100 cm2.A temporary increase in bacterial concentrations was registfor some locations, e.g. table surface in SM and behinderal panels of FGB. The concentrations of fungi varied betw2.5× 101 and 3.0× 105 CFU/100 cm2. The largest number oviable fungal filaments was discovered on panel 402 of theventilation screen. The data show that the maximum allowconcentrations for surface contamination established in “International Space Station Medical Operations RequiremDocument” (MORD SSP 50260) were exceeded on some osions. The latter document was enacted at the commenceof the operation of the ISS and serves as a code of standaquirements at the phase of pre-launch treatment and in-floperation [20]. In this document it is stated that surface ctamination levels should not exceed the limits of 1.0 × 104

and 1.0 × 102 CFU/100 cm2 for bacteria and fungi, respetively. Whenever the contamination threshold was exceedea specific surface, the latter was cleaned with the disinfecfungistat wipes that are routinely used in ISS. These ware impregnated with a mixture of a quaternary ammonsalt (N -alcanoylaminopropyldimethylbenzylammonium chride with urea) and a compound of hydrogen peroxide with u

N. Novikova et al. / Research in Microbiology 157 (2006) 5–12 9

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Table 1Bacterial species isolated from the ISS environment and their occurrencin the total number of samples

Number Bacterial species Environment

Surface Air

1 Acinetobacter woffii 2.8 0.82 Actinobacillus ureae 0.9 –3 Aerococcus sp. 0.5 –4 Bacillus cereus 0.5 –5 Bacillus licheniformis 2.8 –6 Bacillus pumilus 7.9 0.87 Bacillus sp. 0.5 –8 Bacillus sphaericus 12.1 –9 Bacillus subtilis 7.9 –

10 Brevibacterium vesicularis 2.3 –11 Corynebacterium sp. 8.9 0.812 Corynebacterium xerosis 0.5 –13 Eikenella corrodens 1.4 –14 Flavobacterium indologenes 1.4 –15 Gemella morbilorum 0.5 –16 Micrococcus luteus 1.4 –17 Micrococcus sp. 6.5 0.818 Pseudomonas putida 2.3 –19 Pseudomonas stutzeri 2.3 –20 Staphylococcus aureus 3.7 3.221 Staphylococcus auricularis 23.4 6.322 Staphylococcus capitis 3.3 1.623 Staphylococcus cohnii 1.4 0.824 Staphylococcus epidermidis 22.4 9.525 Staphylococcus haemolyticus 2.8 3.226 Staphylococcus hominis 9.3 5.527 Staphylococcus saprophyticus 3.3 –28 Staphylococcus simulans 6.5 1.629 Staphylococcus sp. 3.3 7.130 Staphylococcus warneri 3.7 –31 Staphylococcus xylosus 0.9 –32 Streptococcus constellatus 0.5 –33 Streptococcus intermedius 0.5 –34 Streptococcus mitis 0.5 –35 Streptococcus sp. 2.8 0.836 Xanthomonas maltophilia – 0.8

(urea peroxyhydrate). Cleaning with these wipes and mechcal moisture removal always resulted in a drop in the biologsurface contamination to below the thresholds established iMORD document.

The bacterial species that were detected in the ISS andisolation frequency are reported in Table 1. Bacteria beloing to theStaphylococcus sp. genus were isolated from 84of the surface samples. The two second most commonly itified genera wereBacillus sp. (31.7%) andCorynebacteriumsp. (9.4%). It is apparent from Table 1 that the most prelent species in surface samples wasStaphylococcus auricularis(23.4%), followed byS. epidermidis (22.4%).Bacillus sphaer-icus and S. hominis were encountered in 12.1 and 9.3%the cases, respectively. A few species that have (opporttic) pathogenic behavior were isolated, i.e.B. cereus, Eikenellacorrodens, and S. aureus. Bacterial species likeFlavobac-terium indologenes, Pseudomonas putida, and Xanthomonasmalthophila that can be involved in biodeterioration of marials were also recovered [3,8,9]. Castro and coworkers [4]characterized microbial surface contamination. They colle

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Table 2Fungal species isolated from the ISS environment and their occurrence (the total number of samples

Number Species Environment

Surface Air

1 Aspergillus candidus 0.5 –2 Aspergillus clavatus 0.5 –3 Aspergillus ficuum 0.5 –4 Aspergillus flavus – 2.55 Aspergillus janus 0.5 –6 Aspergillus nidulans 0.9 0.87 Aspergillus niger 2.7 –8 Aspergillus ochraceus 0.5 0.89 Aspergillus phoenicis 6.5 –

10 Aspergillus pulvinus 0.5 –11 Aspergillus sydowi 3.8 –12 Aspergillus ustus 0.5 –13 Aspergillus versicolor 2.3 –14 Candida sp. 0.5 –15 Candida parapsylosis 0.5 –16 Cladosporium sp. 0.9 –17 Cladosporium cladosporioides 0.5 –18 Cladosporium herbarum 0.5 –19 Cladosporium tenuissimum 0.5 –20 Cryptococcus albidus 0.9 –21 Geotrichum sp. 0.5 –22 Lipomyces sp. 0.5 –23 Penicillium aurantiogriseum 6.0 1.724 Penicillium expansum 2.3 0.825 Penicillium grabrum 0.5 –26 Penicillium italicum 0.9 –27 Penicillium lividum 0.5 –28 Phoma sp. 0.5 –29 Rhodotorula rubra 0.5 –30 Saccharomyces sp. 2.8 –31 Ulocladium botrytis 0.5 –

samples from a reusable cargo container, which is used to tport flight hardware and consumables to and from the ISSwell as from flight hardware present in NODE 1, SM, and LASurfaces of 25× 25 cm were sampled using calcium alginaswabs with a phosphate buffer as wetting agent. They rethat the contamination levels were below the acceptability1.0 × 104 CFU/100 cm2 in more than 75% of the samplintimes [24]. Our species identifications are comparable toresults mentioned by Castro and collaborators, who mainlyservedStaphylococcus sp. andCorynebacterium sp. in theirswab samples [4]. The latter authors characterized the cominants using 16S rDNA sequencing and Vitek identificatio

The list of fungi isolated from the ISS surface samplesgiven in Table 2.Aspergillus sp.,Penicillium sp., andSaccha-romyces sp. were the most dominant genera and were isolin 19.7, 10.2, and 2.8% of the samples, respectively. A divAspergillus population was recovered (13 species), whereaswas less pronounced in the case ofPenicillium (5 species) andCladosporium (4 species).

A. phoenicis andP. aurantiogriseum dominated the population with occurrence percentages of 6.5 and 6.0%, respectiSome of the samples containedA. versicolor and Cladospo-rium sp., which are known for (i) their capacity to coloninatural and synthetic polymers, and (ii) their involvement

10 N. Novikova et al. / Research in Microbiology 157 (2006) 5–12

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Table 3Number of airborne bacterial species recovered from ISS air samples

Species Frequency Species Freque

Acinetobacter radioresistens 5 Staphylococcus capitis 2Aerococcus viridans 1 Staphylococcus epidermidis 14Bacillus cereus 1 Staphylococcus haemolyticus 2Bacillus subtilis 1 Staphylococcus hominis 7Enterococcus faecalis 12 Staphylococcus lugdunensis 1Kocuria rosea 1 Staphylococcus xylosus 5Psychrobacter urativorans 2 Xanthomonas sp. 1Staphylococcus sp. 7 Unknown bacterium 2Staphylococcus aureus 2

Samples were taken within the framework of collaboration between IBMP, SCK-CEN, and ESA. Identification were performed by means of 16S rDNA sgconsidering 98% as the similarity threshold.

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the biodeterioration of electronic insulation polymers suchpolyimides [8]. Aggressive metabolites such as organic aproduced during outgrowth of species such asP. aurantiogri-seum andC. herbarum may compromise the integrity of metalic surfaces [27]. Hence, these fungi can be considered astential biocorrosive agents. These events may ultimatelyto biodegradation of space flight materials with short circand malfunctioning as a consequence [3,8,9]. Such examhave been described during the exploitation of the Mir orbstation [10,13,21]. Furthermore, pathogenic saprophytesbeen recovered. These are known to provoke mycoses andcotic intoxications in the case of an impaired immunologiresponse. Ten species out of 31 can be classified as relapathogenic and all of them can cause allergic reactions. Spthat can cause fungal infections such asCandida sp. (in partic-ular C. parapsylosis) and species that can produce toxins sAspergillus sp. (in particularA. versicolor and A. ochraceus)were encountered [2,7,14].

Over the period 1998 up to the present, the airbornetamination was characterized by analyzing 278 air samfrom 16 different sites onboard the ISS. Sampling and ansis by means of the Vitek system have been performed ascribed in Section 2.2. The results indicate that the airbomicrobial contamination was low at all sampling locatioThe highest concentration was encountered in the toiletof SM where a maximum level of 7.1 × 102 CFU/m3 wasreached. Fungal concentrations ranged between 1.1 × 101 and4.4 × 101 CFU/m3. The contamination levels measured in tair were within the limits described in MORD SSP 50260. Tthresholds for air contamination have been fixed at 1.0 × 103

and 1.0×102 CFU/m3 for bacteria and fungi, respectively [20Airborne contamination could be kept to a minimum by theof the POTOK 150MK system that efficiently filters particland microorganisms from the air [1].

Staphylococci were dominant and they were isolated38.8% of the samples (Table 1).S. epidermidis and S. au-ricularis occurred in the highest concentrations. The humpathogenS. aureus was isolated in 3.2% of the cases (TableAll other genera occurred in much lower concentrations. Fuwere recovered in only 7.4% of the air samples. Most offungi belonged to the genusAspergillus or Penicillium and onlyfive different species were identified (Table 2).A. flavus, which

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Air samples have also been analyzed at the Belgian NucResearch Center (SCK-CEN) within the framework of a colloration between IBMP and the European Space Agency (EA total of 33 samples were taken at different locations inISS (i.e. SM, NODE 1, and FGB) during three different Soytaxiflights (October 2003, April 2004, and October 2004). Tsampling was done using the Ecosphere kit with air volumranging from 30 to 240 l. After incubation of the Petri dishesa period of 7 days at 28◦C, 48% of the plates scored positifor the presence of bacteria. The highest bacterial concentrwas found in FGB and amounted to 1.1×102 CFU/m3. A totalof 66 isolates has been obtained and the bacteria weretified using 16S rDNA sequencing (see Section 2.2.). Posidentifications were made based on a 98% or better alignmwith database entries. It is apparent from Table 3 that thejority of the population consists of differentStaphylococcussp. (at least 60%) andEnterococcus faecalis (18%). Generally,the contamination levels reported by IBMP and SCK-CENcomparable to observations made by others who capturesamples (84.9 l) with a Burkard microbial air sampler [4,2Castro and coworkers [4] characterized the microbial isolby a combination of 16S rDNA sequencing and Vitek identcation.

4. Conclusion and perspectives

The environmental biocontamination of the ISS has beenlowed up since its early construction days. The main emphhas been placed on the air quality and the surface contaminof internal structures. The total number of samples analyzeIBMP and SCK-CEN amounts to 554. The microbial and funconcentrations were in most instances below the acceptity limits established in the International Space Station Mical Operations Requirements Document (MORD SSP 502An occasional rise in microbial surface colonization couldeliminated by cleaning that particular surface with wipespregnated with disinfectants. The microbiota recovered fthe ISS environment is clearly dominated by residents ofhuman mucous membranes and skin (i.e.Staphylococcus sp.andCorynebacterium sp.). Members of the rotating crews athe most likely source of the environmental contamination

N. Novikova et al. / Research in Microbiology 157 (2006) 5–12 11

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16

addition to the typical representatives of the human microbispore-formingBacillus sp., pathogenic bacteria (e.g.S. aureus),and species involved in biodeterioration have been isolatedbiocontamination experienced in ISS is to a great extent cparable to results obtained from the Mir orbital station. Bcontaminants isolated from the Mir environment were maidentified using the Vitek system. Novikova [21] reports thairborne contamination remained fairly stable during the ocpation of Mir and that the bacterial concentration was be5.0 × 102 CFU/m3 in 95% of the air samples. The most prdominant airborne bacterial genera wereStaphylococcus sp.,Corynebacterium sp., andBacillus species. The concentratioof airborne fungi was variable and fluctuated between 25.0 × 104 CFU/m3, with Penicillium and Aspergillus as thedominant genera. Contamination levels of surfaces and eqment aboard Mir were also variable, with bacterial and funconcentrations between 1.0×102 and 1.0×107 CFU/100 cm2.The dominant bacterial and fungal genera were the same athe airborne contamination.

The presence of several (opportunistic) pathogensspecies involved in biodeterioration has been confirmed inMir environment [21] as well as on board the ISS (this surveAlthough onboard cleaning procedures restrict the level of thharmful organisms to a minimum, there is always a risk ofincrease in the concentration of these potentially harmful orgisms. Continuous environmental monitoring during the lifetiof the ISS is of paramount importance to ensure (i) the cosnaut’s health and (ii) the integrity of the spatial hardware. IBMand SCK-CEN will therefore continue their efforts to motor environmental contamination in the ISS during the comyears. One disadvantage of the current strategy is that samneed to be returned to earth for analysis. This usually genea time gap of almost one week between sampling and the aability of the first results. Such an information delay maydetrimental when formulating countermeasures in the casa pathogen outbreak. In this respect, the efforts for develoon-line monitoring systems (e.g. for air and water) and on-analysis systems (e.g. for surfaces) should be intensified.by using such systems will it be possible to warn cosmonat an early stage about an increase in biological contaminaThis will give them the opportunity to take appropriate coutermeasures in time (e.g. replacing air filters, cleaning surfwith specialized surfactants and disinfectants, etc.). A secbig challenge is linked to better characterization of microbcontamination using culture-independent molecular analyUp to now, samples are grown on agar media and bacteriafungal isolates are identified afterwards. This approachlead to a biased view of environmental biocontaminationmany organisms are non-cultivable [28]. A molecular analyshould target nucleic acids in order to determine the totaldiversity pool and the biologically active microorganisms12]. Analysis protocols can be adapted fairly easily in the cof water samples and swab samples. Aliquots of the samcan be used immediately for molecular analysis. The situais somewhat more complicated in the case of air samples.fined volumes of air are being collected by impaction on Pplates containing culture media. This collection technique

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however, not suitable for direct recovery of nucleic acids. Tapplicability of collection techniques such as collection ofairborne contamination on 0.22 µm filters and filtration teniques using alginate filters should be exploited [18,23]. Thmethodologies enable isolation of contamination and conutive extraction of nucleic acids without cultivation. Howevthese methods need to be evaluated thoroughly and musta number of safety and compatibility tests before their usagallowed aboard the ISS.

In conclusion, an intensive monitoring strategy in combition with novel analysis methods and possibly new samptechniques should allow correct mapping of biocontaminaaboard the ISS. This will help to define how microbial comunities evolve in confined habitats and whether there arecrobial risks (such as pathogen outbreak, biodeterioration,functioning of hardware, etc.) during prolonged exploitationan orbital station or any envisaged moon or planetary base

Acknowledgements

Collaboration between IBMP and SCK-CEN was fundedESA (contract number 17401/03/F/DC).

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