Microbiological Research 170 (2015) 6168

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Use of genome sequencing to assess nucleotide structure variation of

Staphylococcus aureus strains cultured in spaceight on Shenzhou-X,
under simulated microgravity and on the ground
Jun Guo a,1 , Na Han b,c,1 , Yuanyuan Zhang b,c,1 , Haiyin Wang b,c , Xuelin Zhang a ,
Longxiang Su a , Chao Liu a , Jia Li a , Chen Chen b,c,d , Changting Liu a,
a

Nanlou Respiratory Diseases Department, Chinese PLA General Hospital, Medical School of Chinese PLA, Beijing 100853, China
Key Laboratory of Surveillance and Early-warning on Infectious Disease, Division of Infectious Disease, Chinese Center for Disease Control and Prevention,
Beijing 102206, China
c
State Key Laboratory for Infectious Disease Prevention and Control, and National Institute for Communicable Disease Control and Prevention,
Chinese Center for Disease Control and Prevention, Beijing 102206, China
d
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China
b

a r t i c l e

i n f o

Article history:
Received 12 June 2014
Received in revised form 31 August 2014
Accepted 2 September 2014
Available online 16 September 2014
Keywords:
Staphylococcus aureus
Spaceight
Genome
High-throughput
SNP

a b s t r a c t
The extreme environment of space could affect microbial behavior and may increase the risk of infectious
disease during spaceight. However, the molecular genetic changes of methicillin-resistant Staphylococcus aureus (MRSA) in response to the spaceight environment have not been fully claried. In the present
study, we determined the draft genome sequences for an ancestral S. aureus strain (LCT-SAO) isolated
from a clinical sample and three derivative strains, LCT-SAS, LCT-SAM and LCT-SAG, cultured in parallel
during the spaceight Shenzhou-X, under simulated microgravity and on the ground, respectively. To
evaluate the impact of short-term spaceight on the MRSA strains, comparative genomic analysis was
implemented. Genome-based mapping of toxin genes and antibiotic resistance genes conrmed that
these strains have the conventional pathogenicity and resistance to drugs, as none of the strains showed
signicant changes in these regions after culturing in the three different environments; this result suggests that spaceight may not change bacterial virulence or drug resistance. Thirty-nine strain-specic
sequence variants (SVs) were identied throughout the genomes, and the three derivatives exhibited
almost the same mutation rates. Fifty-nine percent of SVs were located in the intergenic regions of the
genomes, indicating that S. aureus may have an extremely robust repair mechanism responsible for recognizing and repairing DNA replication mismatches. It is noteworthy that strain LCT-SAS, cultured in
space, presented the most unique SVs (n = 9) and shared the fewest SVs with LCT-SAM (n = 5) and LCTSAG (n = 4). Furthermore, we identied 10 potential deletion regions and 2 potential insertion regions,
with LCT-SAS appearing more fragile than other strains by this measure. These results suggest that the
environment of space is inherently complicated, with multiple variables, and cannot be simulated in
a simple manner. Our results represent the rst analysis of nucleotide structure variation of S. aureus
strains in a spaceight environment and also provide a valuable insight for understanding the mutation
strategies of MRSA on earth.
2014 Elsevier GmbH. All rights reserved.

1. Introduction

Abbreviations: PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; SVs, sequence variants; MRSA, methicillin-resistant Staphylococcus aureus;
IRs, insert regions; DRs, deletion regions.
Corresponding author at: 28th Fuxing Road, Haidian District, Beijing 100853,
China. Tel.: +86 10 66876272.
E-mail address: changtingliu301@163.com (C. Liu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.micres.2014.09.001
0944-5013/ 2014 Elsevier GmbH. All rights reserved.

Following NASAs vision for space exploration, a variety of

research studies have been conducted to explore potential travel or
settlement in outer space. One of the signicant issues is whether
extreme environmental factors, such as microgravity, radiation
and vacuum, will impact the development of living organisms.
The impact of exposure to microgravity is of particular interest
(Mermel, 2013; Edsall and Franz-Odendaal, 2014). Microbe alteration during long-term space trips is one of the major challenges

62

J. Guo et al. / Microbiological Research 170 (2015) 6168

for the investigation of potential infectious diseases in space, especially during the mission to Mars in the near future (Taylor and
Sommer, 2005). It has been established that these environmental
variables play a role in dysregulation of the human immune system
and in microbial variation, which will greatly increase the risk of
infectious diseases to humans in long-term spaceight (Klaus and
Howard, 2006). Moreover, many previous studies have shown that
the extreme environment of space could increase microbial proliferation and micro-ora exchange, alter virulence and decrease
antibiotic effectiveness (Leys et al., 2004; Wilson et al., 2007;
Rosenzweig et al., 2010; Schiwon et al., 2013). Therefore, elucidation of the mechanisms of microbial alteration during and after
spaceight would be benecial to both astronauts and the general population on the earth. New treatment methods for infectious
diseases in extreme environments can then be developed.
Staphylococcus aureus is one of the leading causes of hospitalacquired infections with high-level antibiotic resistance and has
reportedly given rise to community-acquired infections for decades
(Klevens et al., 2007; Arias and Murray, 2009). Notably, researchers
have identied a greater abundance of S. aureus on the epithelium
of the skin and the upper airway, which may cause opportunistic infections (Kluytmans et al., 1997). S. aureus is well known
for its capacity to acquired antibiotic resistance and methicillinresistant S. aureus (MRSA) exhibits resistance to methicillin. A series
of recent reports have indicated that more than 60% of S. aureus
isolates are resistant to methicillin globally (Arias and Murray,
2009). Furthermore, several strains have developed resistance to
more than 20 different antimicrobial agents. It has been proven
that the lack of air in space could increase the growth of anaerobic ora versus aerobic clones (Taylor, 1974; Ilyin, 2005). S. aureus,
as a facultative anaerobic microorganism, has a higher chance to
breed and spread than the aerobic bacteria, which might raise the
potential risk for infectious diseases. With longer and increasingly
complex space missions, it is urgent to understand the nature of
the changes S. aureus acquires under conditions of microgravity
and lack of air. However, there is no clear evidence about how
S. aureus evolves during spaceight. The Shenzhou-X spacecraft,
which was launched in China on June 11, 2013 and was retrieved
on June 26, carried a clinically isolated S. aureus strain, providing an
opportunity to understand the molecular mechanisms of the natural alterations of this strain, which will be helpful for conducting
longer space travel missions in the near future.
In this study, we performed comparative genome analysis to
explore the impact of extreme environmental conditions, particularly microgravity during spaceight, on S. aureus.

2. Methods
The study protocol, including written informed consent, was
approved by the Medical Ethical Committee of Chinese PLA General Hospital. Written informed consent was obtained from the
patient from whom the bacterial strain was isolated at the time
of enrollment.

2.1. Strain isolation, culture and spaceight mission

The ancestral strain of MRSA was isolated from a purulent sputum sample of an elderly patient with severe pneumonia in Chinese
PLA General Hospital. The VITEK-2 Compact (bioMrieux, Hazelwood, MO, USA) and classical drug susceptibility test revealed that
this strain is resistant to clindamycin, ciprooxacin, erythromycin,
levooxacin, moxioxacin, oxacillin, penicillin G, rifampin and
tetracycline following the CLSI M100-S23 document (CLSI, 2013).
The original strain was named LCT-SAO and was used as a reference.

Prior to the departure of the Shenzhou-X spacecraft, the

LCT-SAO strain was inoculated into six plastic containers lled
with semi-solid Luria-Bertani (LB) medium consisting of tryptone
(10 g/l), yeast extract (5 g/l), NaCl (10 g/l) and low-melting-point
agar (5 g/l). The pH of the medium was adjusted to 7.07.2. Four
of these plastic containers (designated LCT-SAS) were launched
into space aboard the Shenzhou-X spacecraft on June 11, 2013 and
were retrieved on June 26, 2013. One plastic container was simultaneously put into an incubator that simulated the temperature
exposure of the space environment as a static control (designated LCT-SAG). Another container was cultivated under low-shear
modeled microgravity (designated LCT-SAM) as a ground-based
control (Fig. 1). The temperature in the spacecraft compartment
was reported each hour and ranged from 19 C to 23 C. After
the return module of the spacecraft landed on earth, the LCT-SAS
strain was transferred from the original containers (the contents of
four containers are mixed together primarily) to eppendorf tubes
and immediately placed in liquid nitrogen. The other strains were
stored in the same way. All strains were recovered on solid agar
plates with nutrients. One hundred clones were randomly selected
from each strain for further investigation.
2.2. Genome sequencing and mapping
The strains were sequenced at BGI (BGI, Shenzhen, China) using
a HiSeq 2000 (Illumina, San Diego, CA). Genomic DNA extracted
from the ancestral strain was used to construct 500-bp, 2000-bp
and 6000-bp insert size random sequencing libraries. Nine hundred
sixty-eight million ltered high-quality reads of the small fragment library were assembled into 8 contigs and 6 scaffolds based
on paired-end information using SOAPdenovo (Xie et al., 2014).
The potential coding regions in the draft genome of the ancestral strain were identied using Glimmer (Delcher et al., 1999) and
were annotated with Gene Ontology (GO), Kyoto Encyclopedia of
Genes and Genomes (KEGG), Swiss-Prot and Clusters of Orthologous Groups (COG) databases. Genes shorter than 100 bp and some
of those with overlaps were eliminated. RNAmmer and tRNAscan
were used for the identication of rRNA and tRNA genes, respectively (Schattner et al., 2005; Lagesen et al., 2007). The sequences
of tandem repeats were predicted using Tandem Repeats Finder
(Benson, 1999). For the other three strains, DNA with 500 bp insert
size was used to construct a library, which was then sequenced.
In total, we generated 339 MB, 344 MB and 342 MB of data for the
LCT-SAS, LCT-SAM and LCT-SAG strains, respectively. High-quality
reads were used for mapping to the ancestral strain to identify
single-nucleotide polymorphisms (SNPs) and other genomic variations. We also attempted to assemble these reads into contigs and
scaffolds; the details are described in Table 1.
2.3. Detection of SNPs and other genomic variations
To evaluate the impact of short-term spaceight on the MRSA
strains, we identied strain-specic SNPs by aligning the reads of
the space-exposed strain LCT-SAS, the microgravity-exposed strain
LCT-SAM and the ground strain LCT-SAG to the genome of the control strain LCT-SAO using SOAPsnp (Li et al., 2009). All low-quality
reads (sequences were trimmed with quality score of Q20) were
removed before SNP calling. Trimmed reads less than 30 bp were
ignored. SNPs were called if they met the default criteria using
SOAPsnp. All SNPs were mapped to the LCT-SAO reference genome
and were assigned coordinates. We performed functional annotation on genes containing or adjacent to the SNPs. The sequencing
depths of reads of LCT-SAO were quantied, and their normalized
ratios relative to the read depths of the ancestral strain were calculated to estimate the number of potentially different regions. We
assumed that the coverage obeyed a normal distribution (Fig. S1)

J. Guo et al. / Microbiological Research 170 (2015) 6168

63

Fig. 1. Schematic drawing showing clinical specimen collection and experimental owchart of the three parallel Staphylococcus aureus cultures. The ancestral strain of MRSA
(LCT-SAO) was isolated from sputum sample of an elderly patient with severe pneumonia. All three derivative strains under different experimental conditions were compared
to the ancestral strain LCT-SAO. The ancestral strain of MRSA (LCT-SAO) was isolated from clinical sputum sample of an elderly patient with severe pneumonia. Prior to the
departure of the Shenzhou-X spacecraft, the LCT-SAO strains were inoculated into six plastic containers. Four containers were launched into space aboard the spacecraft
(LCT-SAS), one was cultivated under simulated microgravity (LCT-SAM) and the other as a static control (LCT-SAG). All three derivative strains under different experimental
conditions were compared to the ancestral strain LCT-SAO. All the sequencing data of each strain were mapped to the strain LCT-SAO, and the SNPs of each were marked
above the dash line.
Table 1
Summary of genome features.

Genome
Length (bp)
GC content (%)
Coding percentage (%)
Gene number
# of tRNA

LCT-SAO

LCT-SAG

LCT-SAM

LCT-SAS

2,865,348
32.78
82.94
2699
58

2,775,578
31.85
82.03
3001
5

2,796,689
32.56
83.26
2624
42

2,773,958
31.85
82.11
2989
5

and set the cutoff ratio at 0.2. Cases where at least one strain had a
consecutive region larger than 90 bp (the length of the reads) were
considered to be deletion regions (DRs). In contrast, cases of ratios
greater than 2 where at least one strain had a consecutive region
larger than 90 bp were assigned as potential insert regions (IRs).

2.6. Nucleotide sequence accession numbers

This Whole Genome Shotgun project for the LCT-SAO, LCT-SAS,
LCT-SAM and LCT-SAG strains of S. aureus has been deposited
at GenBank under the accession numbers JANM00000000,
JANN00000000, JANO00000000 and JANP00000000, respectively.

2.4. Toxin genes and antibiotic resistance genes

3. Results
To investigate the impact of microgravity on the biological characteristics of S. aureus, toxin and antibiotic resistance genes were
identied based on previous studies (Warsa et al., 1996; AubryDamon et al., 1998; Kuroda et al., 2001; Hiramatsu et al., 2002;
Ito et al., 2003; Deurenberg and Stobberingh, 2008; Vali et al.,
2008; Grumann et al., 2014; Marasa et al., 2014). The corresponding
sequences were downloaded from GenBank, and nucleotide similarity was used to search the potential toxin genes and antibiotic
resistance genes on the ancestral S. aureus with identity >90% and
coverage >80%. The reads from the LCT-SAS, LCT-SAM and LCT-SAG
strains were mapped to the LCT-SAO genome to identify whether
there were specic mutations in the toxin and antibiotic resistance
genes.

3.1. Overview of reference MRSA isolations

The draft genome of the ancestral strain LCT-SAO was estimated
to be approximately 2.86 Mbp, with a GC content of 32.8%, which
is consistent with previously reported S. aureus genomes (Carroll
et al., 2013; Harro et al., 2013). The largest scaffold is approximately
2.18 Mbp, covering almost 76.2% of the genome, and is anked by
repeat sequences of transposase. The genomic features of this strain
are presented in Table S1. This strain was isolated from a clinical
specimen and retained the major pathogenic genomic islands and
virulence factors, such as enterotoxin, toxic shock syndrome toxin
and hemolysin (Fig. 2).

2.5. Polymerase chain reaction (PCR) validation of SNPs and

variable regions

3.2. Diversity of SNPs

Five loci containing candidate SNPs and potential DRs and IRs
were randomly selected for PCR amplication from the genomic
DNA of LCT-SAS, LCT-SAM and LCT-SAG. The selected SNPs and PCR
primers used here are available upon request.

In total, 6,084,457 reads from the LCT-SAS, LCT-SAM and LCTSAG strains were mapped to the reference genome to evaluate
genomic alteration during the period of spaceight. Relatively few
changes were observed among these strains. In total, 35, 38 and 33
SNPs were identied in the LCT-SAS, LCT-SAM and LCT-SAG strains,

64

J. Guo et al. / Microbiological Research 170 (2015) 6168

Fig. 2. Circular representation of the genomic features of Staphylococcus aureus. Each concentric circle is numbered from the outermost circle to the innermost circle. The
bars in the rst and sixth outermost circles show the positions of the putative genomic islands and SVs in the LCT-SAM, LCT-SAG and LCT-SAS strains. The IRs are depicted
in purple, and the DRs are depicted in green. The SVs located in intergenic regions are shown in blue, and those in the gene regions are shown in red. The second, third and
fourth circles show the read coverages as the same sort of genomic islands and SVs. The rth circle shows the scaffolds composing the draft genome sequence of the LCT-SAO
strain. The 6 assembled scaffolds represented most of the genome, while the remainder consists of numerous short scaffolds. The scale in the seventh circle indicates location
in Mbp. The bars in the innermost circle indicate the positions of predicted virulence genes and antibiotic resistance genes according to sequence similarity to well annotated
genes from previous published reports. Blue represents the virulence genes, and red indicates the antibiotic resistance genes. (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of this article.)

respectively. The mutation rates were estimated at 3.04 104 ,

3.3 104 and 2.9 104 per base pair per year, which seemed
likely to be an overestimate (Wielgoss et al., 2011). Continuous
SNPs are considered to be one-time mutations and are treated as
single sequence variants (SVs). In total, 39 SVs were identied for
all three strains compared to the reference genome. Details of the
SVs for individual strains are listed in Table S2. We observed that
these sites were not randomly distributed throughout the genome.
Twenty-three (59.0%) of the sites fell into intergenic regions, which
only cover 17% of the genome (data not shown). Sixteen (41.0%) of
the sites are within protein coding regions, occurring on a total
of 9 genes, showing clustering to some extent. Four genes contain
at least two SVs each, including one gene, F4L001784, which possesses 5 SVs and putatively functions as a transposase. Two of the

other three genes were predicted to be a DNA methylase and a Mur

ligase, suggesting that these mutation sites may play important
roles in bacterial processes such as mismatch repair, the timing of
DNA replication and the biosynthesis of bacterial cell-wall peptidoglycan, all relevant for adapting to different external environments.
The SVs falling into intergenic regions presented relatively little
clustering, with 23 sites distributed in 18 intergenic regions and
only 4 (22%) regions that included two or more SVs. Several sites
were in proximity to neighboring genes and may be involved with
the upstream or downstream regulatory elements.
Compared to the original strain LCT-SAO, there are 29, 33
and 32 SVs for LCT-SAG, LCT-SAM and LCT-SAS, respectively
(Fig. 3a). No signicant differences were recognized between these
three derivative strains. Among the SVs, 21 SNPs were shared by

J. Guo et al. / Microbiological Research 170 (2015) 6168

65

Fig. 3. Overview of SVs in the three derivative strains LCT-SAS, LCT-SAM and LCT-SAG. The black represents the total number of SVs, and gray and dark gray indicate SVs
located in the intergenic and gene regions, respectively. (a) Numbers of SVs from all three derivative strains compared to the original LCT-SAO strain. (b) Unique SVs distributed
in the three strains. (c) SVs shared by any two of the three strains.

LCT-SAS, LCT-SAM and LCT-SAG strains but were different than

the original LCT-SAO strain. In addition to this nding, individual strains had unique SNPs, and some sites were present in any
two pairs. The spaceight strain LCT-SAS showed the most variation, with 9 strain-specic SNPs, while LCT-SAM and LCT-SAG had
7 and 6 strain-specic SNPs, respectively (Fig. 3b). Furthermore, we
observed that LCT-SAS shared fewer SVs compared to the other two
strains, whereas LCT-SAG and LCT-SAM shared more SVs (Fig. 3c).
All of these data indicate that the environment of space may be
quite distinct from the modeled and ground synchronization environments and may have a special effect on S. aureus.

be more fragile compared to the other strains. Two short DRs, DR1
and DR4, were only identied in LCT-SAS. Two DRs, DR6 and DR9,
occurred only in LCT-SAM and LCT-SAS. By carefully scanning of
the sequences of these 4 regions, it was determined that they only
contain hypothetical genes with unknown functions. DR10 only
occurred in LCT-SAG. In addition, the length of the potential DRs
region in LCT-SAS and LCT-SAM is much longer than that of LCTSAG. Thus, after detecting the potential DRs in the genome, we
found that LCT-SAM and LCT-SAS shared more common sites compared to LCT-SAG, which indicated that microgravity could impact
genome stability. Moreover, we identied 2 IRs in the genome,
which could be caused by repeat sequences.

3.3. Large deletions and insertions (InDels)

3.4. Virulence genes and antibiotic resistance genes
The mean coverages of the genomes ranged from 118 to 120 for
LCT-SAS, LCT-SAM and LCT-SAG. The low coverage at the end of the
chromosome was anked by continuous short contigs. However,
we still identied 12 abnormal regions (Table 2). Specically, we
found 10 potential DRs and 2 potential IRs. These abnormal regions
differ from the parent strains, and their absence has been validated
across the chromosome (data not shown). The largest DR is 293 bp.
Most of these regions were identied within a single quarter or half
of the circular genome. These regions could be hotspots for insertions and deletions, which is consistent with our previous ndings
(Du et al., 2011).
Among 10 DRs, 9, 7 and 6 DRs were identied in LCT-SAS, LCTSAM and LCT-SAG, respectively. The LCT-SAS genome appeared to

The LCT-SAO ancestral strain encoded the common virulence

genes (Fig. 2, Table S3). To explore the effect of external environment on the virulence characteristics of S. aureus, we carefully
scanned the variations in these virulence genes in the 3 parallel cultured strains and did not nd any mutations occurring in
any virulence genes or their operon regions (Fig. 2), indicating
that the different environments in which the parallelly cultured
strains were kept may not have affected their virulence. We also
scanned major antibiotic resistance genes (i.e., mrsA, ermA, blaZ,
and ant(3 )-I). These genes may be directly responsible for the resistance to almost all antibiotics except for vancomycin. We did not
identify any mutations in these genes (Fig. 2, Table S4).

66

Table 2
Detection of deletion and insertion regions.
Name

LCT-SAM

LCT-SAG
Start

End

Length

DR2

794037

794126

90

DR3

794213

794302

DR4
DR5

1307391

DR6
DR7
DR8
DR9

DR10

Ratio

Length

Ratio

793900

793995

96

0.08

90

0.07

794037

794126

90

0.07

794302

90

0.05

794209

794302

94

0.05

1307391

1307480

90

0.05

1307211
1307391

1307300
1307522

90
132

0.06
0.05

0.04
0.11

1716584
1718450
1964637
2864945

1716876
1718539
1964713
2865144

293
90
77
200

0.07
0.04
0.11
0.04

1716584
1718450
1964637
2864942

1716855
1718550
1964768
2865144

272
101
132
203

0.07
0.06
0.09
0.03

0.04

2192605

2192696

92

2.27

2.21

2211476

2211528

53

2.37

Start

End

Length

0.07

794037

794126

90

0.05

794213

1307480

90

0.05

1718450
1964637

1718539
1964713

90
77

2865048

2865144

97

IR1

IR2

2211486

2211589

104

Ratio

Start

End

F4GL000753,
hypothetical protein
[Streptococcus
agalactiae]
F4GL000753
F4GL000754hypothetical
protein [Streptococcus
agalactiae] cadmium
efux system accessory
protein [Staphylococcus
aureus subsp. aureus
CIGC341D]
F4GL000755
hypothetical protein
[Staphylococcus aureus]

F4GL001229
transcriptional
regulator, GntR family
[Staphylococcus
epidermidis VCU041]

F4GL001615

F4GL002699 conserved
hypothetical protein
[Staphylococcus aureus
A8819]
F4GL002699 conserved
hypothetical protein
[Staphylococcus aureus
A8819]
F4GL002038
hypothetical protein
[Staphylococcus aureus]
F4GL002044 scaffold
protein Nfu/NifU
N-terminal domain
protein [Staphylococcus
aureus]

J. Guo et al. / Microbiological Research 170 (2015) 6168

DR1

Gene ID

LCT-SAS

J. Guo et al. / Microbiological Research 170 (2015) 6168

4. Discussion
MRSA is the leading pathogen causing nosocomial infection
due to its high level of antibiotic resistance, and it occurs worldwide (Lee et al., 2011). In the special environment of spaceight,
numerous changes in conditions may inuence the pathogens in
the containment vessel (Mermel, 2013). Moreover, it has been
observed that reversible increases in antibiotic resistance have
occurred during short-term spaceight (Taylor and Sommer, 2005).
In this study, for the rst time, we compared the whole-genome
sequences of 3 parallelly cultured MRSA strains in spaceight, in
simulated microgravity and on the ground to identify the molecular changes occurring after short-term spaceight. Limited by the
culturing time and spaceight environment, the strains studied
were not exposed to cosmic radiation and were not subcultured
during the experiment. Our research did not nd that the mutation rates in spaceight or in microgravity were higher than that
of the strain maintained in its natural cultured environment on the
ground. However, we found the space strain exhibited a different
location pattern of mutation sites compared to the strain cultured
on the ground. More importantly, our results show that the number
of intergenic mutations is much higher than the number of intragenic mutations. One of the possible reasons for this difference
is that S. aureus has a robust repair system responsible for recognizing and repairing DNA replication mismatch sites, targeting
more on mutations in genes that have essential functions associated with survival than in mutations occurring in intergenic regions
(Goodman, 2002). All of these results suggest a different strain evolution pattern after exposure to the space environment. Owing to
the high cost and scarce opportunities for spaceight, simulated
microgravity is widely used for space life studies (Herranz et al.,
2013). Simulated microgravity is a pre-ight study environment
and is commonly used for training astronauts to adapt before prolonged space travel. Compared to the ground-based in vitro strain,
the mutation sites observed in the strain cultured in microgravity
were more consistent with the strain cultured in spaceight. The
genomic variation was higher in the strain cultured in microgravity
than in the strain cultured on the ground. Thus, microgravity can be
used as a model environment to study the potential for microbial
evolution and for the possible spread of pathogens in space.
In the analysis of microbial source tracking and subtle
nucleotide variation in strains with highly similar genomes,
sequencing accuracy is of crucial importance and will be one of the
major factors impacting research results. In this study, the mutation
rates observed may be overestimated because of sequencing uncertainty. Errors could arise in two ways. One way involves sequence
assembly. In our studies, we could speculate that the 21 SNPs that
were consistent in the three derivative strains but different from
the original strain LCT-SAO were caused by assembly error. The
distribution pattern of these 21 SVs along the intergenic and gene
regions was abnormal compared to other sites with high-density
SVs in the intergenic region. Assembly errors may contribute to this
difference in performance, leading to an increased number of SVs
observed in genes. To validate these shared SVs, we mapped all of
the reads from LCT-SAO to the draft genome after sequence assembly. Among 21 SVs, 18 (85.7%) had site variations. The other source
of error may involve sequencing. This error could be removed by
high coverage (100) of reads in our mathematical calculations.
Five randomly selected loci containing candidate SNPs were PCRvalidated to be the result of one of these two types of errors.
Although high-throughput DNA sequencing inevitably introduces
high rates of errors in base calls due to the limitations of the technology, after removing these sites from the analysis, the patterns
remain the same.
A few virulence genes and antibiotic resistance-associated genes
are located in genomic islands, which are supposed to be recently

67

transferred from other pathogens (Boucher et al., 2003; Philippe

and Douady, 2003; Juhas et al., 2009). Theoretically, these genomic
islands are considered to be the fragile regions in the genome and
should have higher mutation probabilities. However, we did not
nd any mutations or InDels in these genomic islands. This lack was
likely due to the short time scale of the exposure to microgravity
in the extreme environment of space. In the future, research on
long-term space ight would be designed to verify this hypothesis.
Given the increase in space exploration, the effects and impact
of long-term space travel on bacteria has become an increasing concern. To our knowledge, this study represents the rst analysis to
explore the nucleotide structure variation of S. aureus strains cultured in space, under simulated microgravity and on the ground.
Although due to the short duration of spaceight, no signicant
variations affected the virulence and antibiotic resistance characteristics of MRSA, it is worth mentioning that unique SVs were
identied in the space strain LCT-SAS. Whether these SVs will affect
the phenotypic or biological characteristics of the bacteria remains
to be elucidated. Our ndings also have the potential to provide
valuable insight for understanding the mutation strategies of bacteria using space technology, which increases our knowledge about
the stability of the bacterial genome and could be benecial to the
eld of space microbiology.
Funding sources
This study was supported by the National Basic Research Program of China (973 program, no. 2014CB744400). It was partially
supported by the National Natural Science Foundation of China (no.
81290345) and the Program of Manned Spaceight (no. 040203).
The funding agencies had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing interests
The authors have declared that no competing interests exist.
Author contributions
Conceived and designed the experiments: CL, JG, XZ, NH, YZ.
Performed the experiments: JG, XZ, LS, CL, JL. Analyzed the data:
JG, NH, YZ, HW, CC. Wrote the paper: JG, NH, YZ, CC.
Acknowledgments
We thank Pengcheng Du and Zhen Wang from the National Institute for Communicable Disease Control and Prevention, Chinese
Center for Disease Control and Prevention for their generous technical support. The authors would like to thank International Science
Editing for critically revising the manuscript.
Appendix A. Supplementary data
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.micres.2014.09.001.
References
Arias CA, Murray BE. Antibiotic-resistant bugs in the 21st century a clinical superchallenge. N Engl J Med 2009;360(5):43943.
Aubry-Damon H, Soussy CJ, Courvalin P. Characterization of mutations in the rpoB
gene that confer rifampin resistance in Staphylococcus aureus. Antimicrob Agents
Chemother 1998;42(10):25904.
Benson G. Tandem repeats nder: a program to analyze DNA sequences. Nucleic
Acids Res 1999;27(2):57380.

68

J. Guo et al. / Microbiological Research 170 (2015) 6168

Boucher Y, Douady CJ, Papke RT, Walsh DA, Boudreau ME, Nesbo CL, Case RJ, Doolittle
WF. Lateral gene transfer and the origins of prokaryotic groups. Annu Rev Genet
2003;37:283328.
Carroll RK, Burda WN, Roberts JC, Peak KK, Cannons AC, Shaw LN. Draft genome
sequence of strain CBD-635, a methicillin-resistant Staphylococcus aureus
USA100 isolate. Genome Announc 2013;1(4), e00491-13.
CLSI. Performance standards for antimicrobial susceptibility testing: twenty-third
informational supplement M100-S23. Wayne, PA, USA: Clinical and Laboratory
Standards Institute, CLSI; 2013.
Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. Improved microbial gene identication with GLIMMER. Nucleic Acids Res 1999;27(23):463641.
Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus: infection,
genetics and evolution. J Mol Epidemiol Evol Genet Infect Dis 2008;8(6):74763.
Du P, Yang Y, Wang H, Liu D, Gao GF, Chen C. A large scale comparative genomic
analysis reveals insertion sites for newly acquired genomic islands in bacterial
genomes. BMC Microbiol 2011;11:135.
Edsall SC, Franz-Odendaal TA. An assessment of the long-term effects of simulated
microgravity on cranial neural crest cells in zebrash embryos with a focus on
the adult skeleton. PLOS ONE 2014;9(2):e89296.
Goodman MF. Error-prone repair DNA polymerases in prokaryotes and eukaryotes.
Annu Rev Biochem 2002;71:1750.
Grumann D, Nubel U, Broker BM. Staphylococcus aureus toxins their functions and genetics. Infect Genet Evol: J Mol Epidemiol Evol Genet Infect Dis
2014;21:58392.
Harro JM, Daugherty S, Bruno VM, Jabra-Rizk MA, Rasko DA, Shirtliff ME. Draft
genome sequence of the methicillin-resistant Staphylococcus aureus isolate
MRSA-M2. Genome Announc 2013;1(1), e00037-12.
Herranz R, Anken R, Boonstra J, Braun M, Christianen PC, de Geest M, Hauslage J,
Hilbig R, Hill RJ, Lebert M, Medina FJ, Vagt N, Ullrich O, van Loon JJ, Hemmersbach
R. Ground-based facilities for simulation of microgravity: organism-specic
recommendations for their use, and recommended terminology. Astrobiology
2013;13(1):117.
Hiramatsu K, Katayama Y, Yuzawa H, Ito T. Molecular genetics of methicillinresistant Staphylococcus aureus. Int J Med Microbiol 2002;292(2):6774.
Ilyin VK. Microbiological status of cosmonauts during orbital spaceights on Salyut
and Mir orbital stations. Acta Astronaut 2005;56(912):83950.
Ito T, Okuma K, Ma XX, Yuzawa H, Hiramatsu K. Insights on antibiotic resistance of
Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist
Updates: Rev Comment Antimicrob Anticancer Chemother 2003;6(1):4152.
Juhas M, van der Meer JR, Gaillard M, Harding RM, Hood DW, Crook DW. Genomic
islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol
Rev 2009;33(2):37693.
Klaus DM, Howard HN. Antibiotic efcacy and microbial virulence during space
ight. Trends Biotechnol 2006;24(3):1316.
Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, Harrison LH, Lyneld
R, Dumyati G, Townes JM, Craig AS, Zell ER, Fosheim GE, McDougal LK, Carey RB,
Fridkin SK. Active bacterial core surveillance MI, invasive methicillin-resistant
Staphylococcus aureus infections in the United States. JAMA: J Am Med Assoc
2007;298(15):176371.
Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus:
epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev
1997;10(3):50520.
Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki
K, Nagai Y, Lian J, Ito T, Kanamori M, Matsumaru H, Maruyama A, Murakami H,
Hosoyama A, Mizutani-Ui Y, Takahashi NK, Sawano T, Inoue R, Kaito C, Sekimizu
K, Hirakawa H, Kuhara S, Goto S, Yabuzaki J, Kanehisa M, Yamashita A, Oshima

K, Furuya K, Yoshino C, Shiba T, Hattori M, Ogasawara N, Hayashi H, Hiramatsu

K. Whole genome sequencing of methicillin-resistant Staphylococcus aureus.
Lancet 2001;357(9264):122540.
Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer:
consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res
2007;35(9):31008.
Lee AS, Huttner B, Harbarth S. Control of methicillin-resistant Staphylococcus aureus.
Infect Dis Clin N Am 2011;25(1):15579.
Leys NM, Hendrickx L, De Boever P, Baatout S, Mergeay M. Space ight effects on
bacterial physiology. J Biol Regul Homeost Agents 2004;18(2):1939.
Li R, Li Y, Fang X, Yang H, Wang J, Kristiansen K, Wang J. SNP detection for massively
parallel whole-genome resequencing. Genome Res 2009;19(6):112432.
Marasa BS, Revollo J, Iram S, Sung K, Xu J, Khan S. Draft genome sequence of a
methicillin-resistant Staphylococcus aureus ST1413 strain for studying genetic
mechanisms of antibiotic resistance. Genome Announc 2014;2(2), e00162-14.
Mermel LA. Infection prevention and control during prolonged human space travel.
Clin Infect Dis: Off Publ Infect Dis Soc Am 2013;56(1):12330.
Philippe H, Douady CJ. Horizontal gene transfer and phylogenetics. Curr Opin Microbiol 2003;6(5):498505.
Rosenzweig JA, Abogunde O, Thomas K, Lawal A, Nguyen YU, Sodipe A, Jejelowo
O. Spaceight and modeled microgravity effects on microbial growth and virulence. Appl Microbiol Biotechnol 2010;85(4):88591.
Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers
for the detection of tRNAs and snoRNAs. Nucleic Acids Res 2005;33(Web Server
issue):W6869.
Schiwon K, Arends K, Rogowski KM, Furch S, Prescha K, Sakinc T, Van Houdt R,
Werner G, Grohmann E. Comparison of antibiotic resistance, biolm formation and conjugative transfer of Staphylococcus and Enterococcus isolates from
International Space Station and Antarctic Research Station Concordia. Microb
Ecol 2013;65(3):63851.
Taylor GR. Recovery of medically important microorganisms from Apollo astronauts.
Aerosp Med 1974;45(8):8248.
Taylor PW, Sommer AP. Towards rational treatment of bacterial infections during
extended space travel. Int J Antimicrob Agents 2005;26(3):1837.
Vali L, Davies SE, Lai LL, Dave J, Amyes SG. Frequency of biocide resistance
genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates. J Antimicrob Chemother
2008;61(3):52432.
Warsa UC, Nonoyama M, Ida T, Okamoto R, Okubo T, Shimauchi C, Kuga A, Inoue M.
Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the
polymerase chain reaction. J Antibiot 1996;49(11):112732.
Wielgoss S, Barrick JE, Tenaillon O, Cruveiller S, Chane-Woon-Ming B, Medigue C,
Lenski RE, Schneider D. Mutation rate inferred from synonymous substitutions
in a long-term evolution experiment with Escherichia coli. Genes Genomes Genet
2011;1(3):1836.
Wilson JW, Ott CM, Honer zu Bentrup K, Ramamurthy R, Quick L, Porwollik S, Cheng
P, McClelland M, Tsaprailis G, Radabaugh T, Hunt A, Fernandez D, Richter E,
Shah M, Kilcoyne M, Joshi L, Nelman-Gonzalez M, Hing S, Parra M, Dumars P,
Norwood K, Bober R, Devich J, Ruggles A, Goulart C, Rupert M, Stodieck L, Stafford
P, Catella L, Schurr MJ, Buchanan K, Morici L, McCracken J, Allen P, Baker-Coleman
C, Hammond T, Vogel J, Nelson R, Pierson DL, Stefanyshyn-Piper HM, Nickerson
CA. Space ight alters bacterial gene expression and virulence and reveals a role
for global regulator Hfq. Proc Natl Acad Sci USA 2007;104(41):16299304.
Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S, Zhou X, Lam
TW, Li Y, Xu X, Wong GK, Wang J. SOAPdenovo-Trans: de novo transcriptome
assembly with short RNA-Seq reads. Bioinformatics 2014;30(12):16606.