Submitted to Microbiology and Immunology · Submitted to Microbiology and Immunology Title of the...

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Original Article

Submitted to Microbiology and Immunology

Title of the paper: Characterization of Fungi Isolated from the Equipment Used in

the International Space Station or Space Shuttle

Full names of the authors: Kazuo Satoh1,2,3, Takashi Yamazaki1,2,3,4, Takako

Nakayama2, Yoshiko Umeda1,2, Mohamed Mahdi Alshahni1,2, Miho Makimura1, Koichi

Makimura1,2,3,*

Institutions: 1General Medical Education and Research Center, Teikyo University, 2Lab. Space and Environmental Medicine, Graduate School of Medicine/Medical

Technology Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605; 3Teikyo

University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo, 192-0395; and 4JEM Utilization Center, Japan Aerospace Exploration Agency (JAXA) 2-1-1 Sengen,

Tsukuba, Ibaraki, Japan, 305-8505.

*Correspondence: General Medical Education and Research Center, Teikyo University,

2-11-1 Kaga, Itabashi, Tokyo, Japan, 173-8605. TEL. +81-3-3964-8367, FAX.

+81-3-3964-8413, E-mail: [email protected]

Short running title: Fungal Characterization of KIBO, ISS

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12375.

© 2016 The Societies and John Wiley & Sons Australia, Ltd

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List of abbreviations

CBEF: Cell Biology Experiment Facility

CLSI: Clinical Laboratory Standards Institute

HEPA: High Efficiency Particulate Air

ISS: International Space Station

ITS1: internal transcribed spacer 1

JAXA: Japan Aerospace Exploration Agency

JEM: Japanese Experiment Module

MDS: Microbe Detection Sheet

MEC: Minimum Effective Concentration

MIC: Minimum Inhibition Concentration

MPC: Multi-Protocol Converter

NASA: National Aeronautics and Space Administration

Pgs1: CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase

PSU: Power Supply Unit

Rev3: DNA polymerase zeta catalytic subunit

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STS: Space Transportation System

T-CBEF: Turntable of Cell Biology Experiment Facility

TKSC: Tsukuba Space Center

Tma22: Translation machinery associated protein

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1 Abstract

As a part of a series of studies regarding the microbial biota in manned space

environments, we isolated fungi from six pieces of equipment recovered from the

Japanese Experimental Module “KIBO” of the International Space Station (ISS) and

from a Space Shuttle. Thirty-seven strains of fungi were isolated and identified, and

were investigated with regard to morphological phenotypes and antifungal

susceptibilities. The variety of fungi isolated in this study was similar to several

previous reports. The dominant species belonged to the genera Penicillium, Aspergillus,

and Cladosporium, which are potential causative agents of allergy and opportunistic

infections. The morphological phenotypes and antifungal susceptibilities of the strains

isolated from space environments were not significantly different from those of

reference strains on Earth.

Keywords: Aspergillus, Cladosporium, fungal biota, International Space Station,

Penicillium,

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Introduction

There have been several reports regarding microbial contamination of manned space

vehicle environments (1, 2, 3, 4). Some fungi (mainly Penicillium, Aspergillus, and

Cladosporium) were detected within the USA space station Skylab and Russian space

station Mir (2, 3). These fungal species present on structural materials (metal or rubber

plates) are well-known potential biodegraders of polymers (substrates of electronic

equipment), and are therefore considered to possibly produce biointerference, damage to

structural materials, and malfunctions, and induce failure of various space systems and

equipment (4). Indeed, some electronic equipment was shown to have been decomposed

by fungi on Mir (5). The fungi isolated from these space vehicles are common

saprophytic fungi present in the normal living environment, and are opportunistic

pathogens and allergens (6). Our previous study showed that the Japanese Experiment

Module (JEM), “KIBO” of ISS, was clean and no fungal strains were isolated

approximately 460 days after it was launched to the ISS (7). However, after the repeated

use of the vehicle, the fungal biota would gradually become mature (4). To prevent

health disorders and equipment problems in long-term space life, including Mars

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exploration, it is necessary to continue monitoring fungal biota in closed space

environments. However, in practice it is difficult to periodically obtain samples from

space vehicles. The crews of the ISS and other space vehicles have many missions and

sometimes encounter irregular problems. The schedules to carry out each mission are

very tight, so some missions are skipped.

In this study, we obtained samples from six pieces of equipment recovered from ISS

and other space vehicles. We cultured the isolated fungi from the equipment and

identified them by PCR and direct sequencing. All isolates were investigated for

morphological phenotype and antifungal agent susceptibilities to monitor the microbial

biota in manned space environments.

2 Materials and methods 2.1 Sampled equ ipment as funga l hab i ta ts

Table 1 shows the sampled equipment and sampling dates.

The Multi-Protocol Converter (MPC) (Japan Manned Space Systems Corporation

(JAMSS), Tsukuba, Japan in cooperation with B.U.G., Inc., Sapporo, Japan) (Fig. 1A)

is an image transfer and processing system that was developed to facilitate downlinking

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of high-definition television and Ethernet data (8). MPC and DC Power Supply Unit for

MPC (PSU) (Fig. 1B) have cooling fans in their interior. MPC1 and MPC2 were used in

the ISS, MPC3 was used in STS-125 (Space Shuttle Atlantis), and MPC4 was used in a

NASA mission, the detailed contents of which are unclear. MPC had been used for

approximately 1 hour per day in the ISS (9). The total operating times of the other

equipment were not known.

The Cell Biology Experiment Facility (CBEF) is used for various life science

experiments, such as cultivating cells, plants, etc., in the JEM Pressurized Module (10).

One G compartment has a turntable (T-CBEF) (Fig. 1C) that can control gravity from

0.1 to 2.0 G for gravity contrast experiments. The CBEF has humidification equipment

for the biological experiments.

2.2 Sampl ing

The surface samples were collected with swabs (ITW Texwipe®; ITW Texwipe,

Kernersville, NC) and Microbe Detection Sheets (MDS) (Sanita-kun® for

YEAST/MOLD; JNC Corp., Tokyo, Japan), which are culture sheets for rapid

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enumeration of fungi (11) and sampling surfaces; the MDS were stuck on and peeled

off three times. Inside of housings or substrates of MPC and PSU were swabbed 7 × 7

cm, 49 cm2. Only surfaces were sampled in the T-CBEF. Sampling was performed at

Tsukuba Space Center (TKSC) (Ibaraki, Japan) or Meisei Electric Co., Ltd. (Gunma,

Japan) (Table 1). MPC2 was hardly used, and no dust could be observed with the naked

eye. Dust and thin and short hair were observed in the other three MPCs and PSU (Fig.

1 D).

2.3 Cu l tu re

Swabs were streaked onto DG-18 (Oxoid, Cambridge, UK) plates. All of plates and

MDS were incubated at 28°C. The culture period was usually 3 – 4 days, with 1 month

as the longest.

2.4 Mo lecu la r iden t i f i ca t ion fo r funga l i so la tes

Fungal DNA was extracted from isolates as described previously (12). The DNA

fragments covering the nuclear 28S rDNA D1/D2 domain and the ITS region were

amplified using the primers 28SF1 and 635 (13), ITS1 and ITS4 (14). For genus

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Penicillium and Aspergillus, beta-tubulin gene was amplified using the primers Bt2a

and Bt2b (15), or btub1 (benA) and btub2 (benA) (16). For Alternaria tenuissima,

translation machinery associated protein (Tma22) gene,

CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (Pgs1), and DNA

polymerase zeta catalytic subunit (Rev3) gene were amplified using the primers

MS432-A-F and MS432-A-R, MS550-A-F and MS550-A-R,and MS578-A-F and

MS578-A-R (17). DNA sequencing was performed with an Applied Biosystems model

310 sequencer (Applied Biosystems, Foster City, CA). The isolated strains were

identified by comparison with DNA sequences registered in GenBank/EMBL/DDBJ as

described previously (12).

2.5 Morpho log ica l tes t ing

Microscopic morphology was investigated on slide cultures using the methods

described by Reiss et al. (18).

2.6 An t i funga l suscep t ib i l i t y tes t ing

Antifungal susceptibility testing can be performed only for the strains that produce

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sufficient numbers of conidia and 17 strains were used, as shown in Table 2. Antifungal

susceptibility tests were performed with seven antifungal drugs (micafungin,

amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and miconazole)

based on the Clinical Laboratory Standards Institute microdilution method (19).

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3 Results 3.1 Cu l tu re , i den t i f i ca t ion and morpho log ica l

f i nd ings o f funga l i so la tes

Molecular identification indicated that 37 strains of fungi were isolated from MPC1,

MPC3, MPC4, and T-CBEF (Fig. 1, Table 1). No fungi were isolated from MPC2 or

PSU. Each isolate showed 100% similarity to the DDBJ/EMBL/GenBank Accession

numbers at the DNA sequence level as unique species (Table 1). Morphological

observation of the slide cultures of these isolates showed no significant differences from

the corresponding reference strain (data not shown). The dominant species among the

isolates belonged to the genera Penicillium, Aspergillus, and Cladosporium. In T-CBEF,

the ISS equipment had formed a richer fungal biota than MPC. The MPC3 (used in the

space shuttle) and MPC4 (used in the NASA mission) had greater varieties of fungi than

MPC1 and 2 (used in the ISS).

3.2 An t i funga l suscep t ib i l i t y tes t

The results of antifungal susceptibility tests of isolates are shown in Table 2. Most of

the isolates showed sensitivity or intermediate sensitivity to micafungin, amphotericin B,

itraconazole, and voriconazole. For Trametes elegans, the MEC of micafungin was 8

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µg/ml indicating resistance. Penicillium paxilli was resistant to amphotericin B,

flucytosine, and voriconazole, Alternaria tenuissima and Paecilomyces variotii showed

resistance to voriconazole.

4 Discussion

Previously, we performed fungal biota analysis of ISS “KIBO” (Opnum; Microbe-I, II,

and III) and reported the results of Microbe-I mission (August – September 2009) (7).

No fungi were isolated from swabs obtained from the surfaces of equipment in the

KIBO area in this previous study. In the present study, however, several fungi were

isolated from dust collected from the inside and outside of equipment recovered from

the KIBO area of the ISS.

Our results indicated that the environment of the ISS was cleaner than that of the Space

Shuttle, by comparing the visual appearance of equipment and amount of isolated fungi.

However, these results also suggested that fungal contamination of the KIBO area was

progressing gradually.

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We did not detect fungi from MPC2 and PSU. MPC2 was hardly used in the ISS (JAXA

Associate Senior Engineer, personal communication). Dust had accumulated in the PSU,

but the viability of fungi would have been decreased by heat in operation. In contrast,

large amounts of fungi were collected from T-CBEF in which a humidifier was installed.

To maintain hygiene, it is important to clean areas with high humidity.

The variation of isolated fungi was similar to those in previous reports (3,4,20,21). The

dominant species of isolates belonged to the genera Penicillium, Aspergillus, and

Cladosporium. These are the most common components of the airborne fungal biota

that spread with spores (20), and are known as causative agents of allergy and

opportunistic infections (Table 1). In addition, Sordariomycetes fungi such as

endolichenic fungi were isolated, which can grow independently because they formed

spores on DG18 or PDA (data not shown). Their life cycle in such artificial

environment is obscure yet, but Sordariomycetes were isolated or detected from

spacecraft (Mir, Skylab and ISS) (2, 4, 7, 22). Therefore, they are minority, but are

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members of the fungal biota of the spaceship.

The morphological phenotypes of each isolate determined by macro- and microscopic

examination were the same as those of the corresponding reference strains. The results

of antifungal agent susceptibility tests indicated no significant differences between the

fungal isolates and reported reference strains on Earth (21). Previously, we reported that

the morphological phenotypes and antifungal susceptibilities of Aspergillus niger and

Candida albicans did not change under conditions of simulated microgravity (23). Our

results suggested that fungi will not change their morphological or biological

characteristics easily under the stress of gravity and radiation during space flight for a

few years.

There have been no reports of fungal isolates causing disorders among astronauts and/or

equipment on the ISS. However, in this study we detected the progress of fungal

contamination in the ISS “KIBO” area by analysis of samples from equipment.

Sampling from equipment seems to be a useful and practical method for monitoring of

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the microbial condition of manned space environments. Further monitoring will be

necessary to protect the crew from health disorders and equipment problems in the ISS.

5 Acknowledgments

This study was supported in part by a grant from JAXA and a grant for the KIBO 2nd

Stage Research Program for Space Utilization from the Japan Space Forum (K.M.).

This experiment was supported by was funded in part by JSPS KAKENHI Grant

Numbers 15H05946 (T.Y. and K.M.). The authors are grateful to NASA, JAXA, and

Meisei Electric Co., Ltd., staff for sample collection.

6 Disclosure

K.M. has received research grants from Japan Space Forum and World Geno Matrix.

The authors alone are responsible for the content and writing of the paper and declare

no conflicts of interest.

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8 Figure legend

Fig. 1. Sampled equipment. (A) Multi-Protocol Converter (MPC) 20 × 19 × 4 cm. (B)

Power Supply Unit (PSU) 15 × 15 × 4 cm. (C) Substrates of MPC. (D) Turntable of Cell

Biology Experiment Facility (T-CBEF).

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Table 1. Culture results.

Launching date Recovery date Sampling date Sampling site ISS/Space Shuttle Equipment Isolates (No.) Strain No. DDBJ/EMBL/GenBank accesion No.

examples of 100% similarity (Target)

Medical significance for human

Penicillium chrysogenum (6) 958, 959, 960,

961, 962, 964

KT151602 (ITS), JF922035 (D1/D2),

KT253242 (β-tubulin)

Central nervous system infectionb) Oct., 24, 2007 Sep., 12, 2009 Nov. 9, 2009 TKSC ISS MPC 1

Aspergillus cibarius (3) 963, 965, 966 KJ528497 (β-tubulin), FR848828 (ITS,

D1/D2)

Unkwown

Oct., 24, 2007 Nov., 27, 2009 Feb. 10, 2010 TKSC ISS MPC 2 0

Penicillium chrysogenum (1) 1253 KT151602 (ITS), JF922035 (D1/D2),


Central nervous system infectionb)

Penicillium corylophilum (1) 1256 KJ775120 (β-tubulin), AF034456 (ITS,

D1/D2)

Non-atopic asthma and upper respiratory diseasec)

Alternaria tenuissima (1) 1267 KP267518 (ITS), FJ755192 (D1/D2),

KP276098 (Tma22), KP275923 (Pgs1),

KP276005 (Rev3)

Plurifocal cutaneous infectiond)

May, 11, 2009 May, 24, 2009 Oct. 7, 2010 TKSC Space Shuttle

(STS-125,

Atlantis)

MPC 3

Cladosporium cladosporioides (2) 1252, 1257 KP701913 (ITS1), KC585410 (D1/D2) Phaeohyphomycosise)

Penicillium chrysogenum (1) 1254 KT151602 (ITS), JF922035 (D1/D2),



Penicillium corylophilum (3) 1255, 1258, 1259 KJ775120 (β-tubulin), AF034456 (ITS,

D1/D2)

Non-atopic asthma and upper respiratory diseasec)

Penicillium paxilli (1) 1268 LC120832 (β-tubulin)a), JN617687 (ITS),

EU427293 (D1/D2)

Mycotoxin-producerf)

Aspergillus penicillioides (1) 1289 LC127195 (β-tubulin)a), EF652036 (ITS,

D1/D2)

Allergic rhinitis and rhinosinusitisg)

Aspergillus sydowii (1) 1261 KC795921 (β-tubulin), LC105690 (ITS),

AM883159 (D1/D2)

Aspergillosish)

Cladosporium sphaerospermum (1) 1265 KT151594 (ITS), KM458639 (D1/D2) Hay feverg)

Toxicocladosporium irritans (2) 1263, 1264 LN834443 (ITS), EU040243 (D1/D2) Skin irritationi)

Leptosphaerulina chartarum(1) 1260 HQ607815 (ITS1), LC071452 (D1/D2)a) Unkwown

ND ND Oct. 7, 2010 TKSC ND (Used by

NASA mission)

MPC 4

Sordariomycetes sp. (1)l) 1266 LC120831 (ITS)a), LC071453 (D1/D2)a) Unkwown

ND ND Jul. 10, 2011 TKSC ISS T-CBEF Penicillium chrysogenum (1) 1495 KT151602 (ITS), JF922035 (D1/D2),



1

Penicillium daleae (1) 1555 LC120833 (β-tubulin)a), KM458834 (ITS),

LC071454 (D1/D2)a)

Mycotoxin-producerj)

Aspergillus sydowii (3) 1493, 1554, 1575 KC795921 (β-tubulin), LC105690 (ITS),

AM883159 (D1/D2)

Aspergillosish)

Cladosporium cladosporioides (1) 1556 KP701913 (ITS1), KC585410 (D1/D2) Phaeohyphomycosise)

Phaeophleospora hymenocallidicola (2) 1583, 1589 KR476739 (ITS), LC071455 (D1/D2)a) Unkwown

Paecilomyces variotii (1) 1496 JQ796880 (ITS), FJ345354 (D1/D2) Pneumoniak)

Trametes elegans (1) 1494 LC120834 (ITS)a), LN774884 (D1/D2) Unkwown

Sordariomycetes sp. (1)l) 1588 LC127196 (ITS)a), JQ761840 (D1/D2) Unkwown

May, 15, 2008 Jun., 1, 2011 Jan. 20, 2012 Meisei ISS Power Supply 0

TKSC; Tsukuba Space Center, Japan Aerospace Exploration Agency (Ibaraki, Japan), Meisei; Meisei Electric Co., LTD (Gunma, Japan.), ISS; International Spase Sutation, STS; Space Transportation System, MPC; Multi-Protocol Converter, T-CBEF; Turntable of Cell Biology Experiment Facility,

ND; No Data, ITS; internal transcribed spacer, D1/D2; D1/D2 region of 28S ribosomal RNA gene, Tma22; translation machinery associated protein, Pgs1; CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase, Rev3; DNA polymerase zeta catalytic subunit.

a) This study, b) Kantarcioğlu A.S., et al (24), c) McMullin D.R., et al (25), d) Robertshaw H., Higgins E. (26), e) Vieira M.R., et al (27), f) Cockrum P.A., et al (28), g) Hamilos D.L. (29), h) de Hoog G.S. et al (30), i) Crous P.W., et al (31), j) Lugauskas A., Stakeniene J (32), k) Byrd R.P., et al (33), l) Endolichenic

fungus, U'Ren J.M, et al (34).

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Table 2. Antifungal susceptibilities of fungal isolates.

MEC MIC

Name Strain No. Habitat

Micafungin Amphotericin B Flucytosine Fluconazole Itraconazole Voriconazole Miconazole

Penicillium chrysogenum 958 MPC 1 <0.015 1 16 >64 0.25 0.25 1

Reference dataa) ND 2 0.125 8 1 0.1 ND

Penicillium corylophilum 1255 MPC 4 <0.015 0.12 4 >64 0.06 0.12 0.06

Penicillium daleae 1555 T-CBEF <0.015 1 2 >64 1 1 1

Penicillium paxilli 1268 MPC 4 <0.015 4 8 >64 2 >8 1

Aspergillus cibarius 966 MPC1 <0.015 0.25 4 4 0.06 0.12 0.25

Aspergillus penicillioides 1289 MPC 4 NG NG NG NG NG NG NG

Aspergillus sydowii 1261 MPC 4 <0.015 1 32 >64 0.25 0.12 4

Reference datab) ND 1 ND ND 0.5 2 ND

Reference datac) ND 4 ND >128 >128 ND ND

Cladosporium cladosporioides 1252 MPC 3 0.25 2 8 64 0.5 2 1

Reference datad) ND 0.32 0.5 25.39 0.25 0.08 0.63

Cladosporium silenes 1556 T-CBEF <0.015 0.5 1 >64 0.25 1 0.5

Cladosporium sphaerospermum 1265 MPC 4 <0.015 1 32 >64 >8 1 >16

1

Toxicocladosporium irritans 1263 MPC 4 0.12 0.25 0.25 16 0.03 0.5 0.12

Phaeophleospora hymenocallidicola 1583 T-CBEF 0.3 2 8 >64 1 0.25 8

Alternaria tenuissima 1267 MPC 3 0.12 1 >64 32 2 4 2

Reference datae) ND 1.54 256 58.6 0.7 ND 4

Paecilomyces variotii 1496 T-CBEF <0.015 0.12 <0.12 >64 0.12 8 4

Leptosphaerulina chartarum 1260 MPC 4 0.03 0.12 2 32 2 0.5 0.25

Trametes elegans 1494 T-CBEF 8 0.06 >64 >64 0.25 0.25 2

Sordariomycetes sp. 1588 T-CBEF 0.003 0.5 32 >64 2 2 2

Sordariomycetes sp. 1266 MPC 4 NG NG NG NG NG NG NG

Concentration: µg/mL [AMPH-B; µg(titer)/mL]. MEC; Minimun effective concentration. MIC; Minimum inhibition concentration. NG; Negative growth in RPMI medium. ND; No data. a)Kantarcioğlu A.S., et al (24). b)Balajee S.A., et al (35). c)Gajjar D.U., et al (21). d)de Hoog G.S., et al (36),

e)de Hoog G.S., et al (37). f)Endolichenic fungus, U'Ren J.M, et al (34).

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