Ott, C. Mark

NASA Technical Reports Server

Microbiology and the International 
Space Station
C. Mark Ott, PhD
Microbiology Laboratory
NASA Johnson Space Center, Houston, TX
https://ntrs.nasa.gov/search.jsp?R=20180004288 2019-08-31T15:18:44+00:00Z

Microbiological Monitoring on the ISS
AirSurfaces Water
Quantified in-flight and returned to JSC for identification
3
ISS Air and Surface Monitoring
FungaI Isolates
Pierson, et. al. Environmental Monitoring: A Comprehensive Handbook 2012
4
ISS Air and Surface Monitoring
Bacterial Isolates
Pierson, et. al. Environmental Monitoring: A Comprehensive Handbook 2012
5
“…(fungi) feeding behind control panels, slowly digesting the ship’s 
air conditioner, communications unit, and myriad other surfaces.”
Gareth Cook, Boston Globe Staff (10-1-00)
Adverse Effects of Microorganisms
• Biodegradation
• Systems failure
• Food spoilage
• Release of volatiles
6
Microbial Mutation and Evolution
• Craig Everroad, NASA Ames Research Center
– Experimental Evolution of Bacillus subtilis Populations 
in Space; Mutation, Selection and Population 
Dynamics
• Wayne Nicholson, University of Florida
– Global Transcriptome Profiling to Identify Cellular 
Stress Mechanisms Responsible for Spaceflight-
Induced Antibiotic Resistance
• Cheryl Nickerson, Arizona State University
– High Dimensional Biology to Understand the 
Functional Response of Salmonella to Long-Term 
Multigenerational Growth in the Chronic Stress of 
Microgravity
Biofilm Studies
• Robert McLean, Texas State
– Polymicrobial Biofilm Growth and Control 
during Spaceflight
• Luis Zea, University Colorado, Boulder
– Characterization of Biofilm Formation, 
Growth, and Gene Expression on Different 
Materials and Environmental Conditions in 
Microgravity
Human Health
• Cheryl Nickerson, Arizona State University
– Investigation of Host-Pathogen Interactions, Conserved Cellular 
Responses, and Countermeasure Efficacy During Spaceflight using 
the Human Surrogate Model Caenorhabditis elegans
• Clay Wang, University of Southern California
– Influence of Microgravity on the Production of Aspergillus
Secondary Metabolites (IMPAS) - a Novel Drug Discovery 
Approach with Potential Benefits to Astronauts' Health
• Sheila Nielsen, Montana State University
– Genotypic and phenotypic responses of Candida albicans to 
spaceflight
• Grace Douglas, NASA Johnson Space Center
– The Integrated Impact of Diet on Human Immune Response, the 
Gut Microbiota, and Nutritional Status During Adaptation to 
Spaceflight
Human and Environmental 
Microbiomes
• Hernan Lorenzi, J. Craig Venter Institute
– Study of the Impact of Long-Term Space Travel on the 
Astronauts' Microbiome 
• Fred Turek, Northwestern
– Effects of Spaceflight on Gastrointestinal Microbiota in Mice:  
Mechanisms and Impact on Multi-System Physiology
• Crystal Jiang, Lawrence Livermore National Laboratory
– International Space Station, Microbial Observatory of 
Pathogenic Virus, Bacteria, and Fungi (ISS-MOP) Project
• Kasturi Venkateswaran, NASA Jet Propulsion Laboratory
– ISS Microbial Observatory - a Genetic Approach
– Bacterial, Archaeal, & Fungal Diversity of the ISS--HEPA 
Filter System
The International Space Station: 
A Research Platform to Understand Environmental and 
Human Microbiomes
C. Mark Ott, PhD
Microbiology Laboratory
NASA Johnson Space Center, Houston, TX
Microbiology Laboratory
NASA Johnson Space Center
• Debbie Aldape
• Audry Almengor, Ph.D.
• Bekki Bruce
• Victoria Castro
• Christian Castro
• Brandon Dunbar
• Todd Elliott
• Tanner Hamilton
• Jane McCourt
• Cherie Oubre, Ph.D.
• Duane Pierson, Ph.D.
• Joan Robertson
• Melanie Smith
• Sarah Stahl
• Sarah Wallace, Ph.D.
Prevention
12
Vehicle Design Controls
• HEPA air filters
• In-line water filters
• Contamination resistant 
surfaces
• Water biocides
• Water pasteurization 
systems
• Minimize condensation
• Contain trash and 
human waste
13
Operational Controls 
Mission Illness (Crew)
Apollo 7 Upper respiratory infection (3) 
Apollo 8 Viral gastroenteritis (3)
Apollo 9 Upper respiratory infection (3) 
Apollo 10 Upper respiratory infection (2)
Apollo 11
Apollo 12 Skin infection (2)
Apollo 13 Rubella (1)
Apollo 14
Apollo 15
Apollo 16
Apollo 17 Skin infection (1)
Skylab-2
Skylab-3 Skin infection (2)
Skylab-4 Skin infection (2)
*Billica, Pool, Nicogossian, Space Physiology and Medicine, 1994
Health Stabilization Program
14
Preflight Microbiological Monitoring
• Crewmembers
• Food
• Potable water
• Vehicle surfaces
• Vehicle air
• Cargo
• Biosafety review 
of payloads
Acceptability Limits
Air
• Total bacteria 1,000 CFU/m3
• Total fungi 100 CFU/m3
Surfaces
• Total bacteria 10,000 CFU/100 cm2
• Total fungi 100 CFU/100 cm2
Water
• Heterotrophic plate count 50 CFU/ml
• Total coliform bacteria Not detected in 100 ml
Preflight Monitoring Synopsis
• Few reported clinical infections
– Dermatitis
– Urinary tract infection
– Upper respiratory infection
• Common environmental flora*
• Opportunistic pathogens*
– Burkholderia cepacia
– Pseudomonas aeruginosa
– Staphylococcus aureus
*Castro, et al, Microb Ecol 2004 17
Disqualified Food Samples
International Space Station (ISS)
Freeze dried shrimp Salmonella enterica serovar Typhimurium
Oatmeal with raisins Aspergillus flavus
Miso soup Staphylococcus aureus
Berry medley Total aerobic (TNTC) - Bacillus species
Chicken Pineapple salad Enterobacter cloacae
Freeze dried chopped pecans Aspergillus fumigatus, Penicillium species
Freeze dried corn Klebsiella pneumoniae, Enterobacter cloacae
San Francisco seasoning Total aerobic (TNTC) - Bacillus species
Onion medley seasoning Total aerobic (TNTC) - Bacillus species
Almond M&Ms Yeast species
Japanese sugar candy Yeast species
Trail mix Aspergillus niger, Aspergillus fumigatus
Chicken salad Enterobacter cloacae, Enterobacter
intermedius, Pantoea agglomerans 18
Contamination Potential
Spacecraft are 
complex (cluttered)
Preflight 
contamination
Astronaut 
activities, such as 
eating and hygiene 19
Microbial Monitoring during Spaceflight
• Safety concerns
• Minimal
– Power
– Weight
– Volume
– Crew Time
• No phase 
separation
20
Microbiological Monitoring of Water
21
U. S. Potable Water Dispenser
• Provides “hot” and 
“ambient” potable water
• Processing includes:
– Catalytic oxidizer
– Iodine disinfection
– In-line filter (0.2 micron)
• Common isolates
– Ralstonia pickettii
– Burkholderia multivorans
– Sphingomonas sanguinis
– Cupriavidas metallidurans
22
Staphylococcus aureus
• No Methicillin Resistant S. 
aureus (MRSA) have been 
recovered from ISS
• 48% of coagulase negative 
staphylococci were methicillin 
resistant
• Blue - isolated from the crew of 
ISS-5, the crew of ISS-4, and in-
flight environmental isolates
• Green - isolated from the crews 
of ISS-1, ISS-4, and ISS-5
• Red - isolated from the crew of 
ISS-1 and ISS-4 and from an in-
flight environmental surface
23
Wolf spent several hours working with Vinagradov to mop 
up a basketball-size drop of water…“I didn’t realize I 
bought myself anywhere from two to six hours per day 
doing this for the rest of the mission.”
From DRAGONFLY by Bryan Burrough
Free Floating Condensate
24
MIR Condensate Samples
NASA 6 “Slimy”
Fungi
– Acremonium species
– Candida guilliermondii
– Candida krusei
– Cladosporium species
– Fusarium species
– Penicillium species
– Rhodotorula rubra
Bacteria
– Alcaligenes eutrophus
– Alcaligenes latus
– Citrobacter freundii
– Corynebacterium aquaticum
– Corynebacterium jeikeium
– Enterobacter agglomerans
– Escherichia coli
– Hydrogenophaga flava
– Kingella denitrifican
– Methylobacterium species
– Pseudomonas vesicularis
– Serratia liquefaciens
– Stentrophomonas maltophilia
NASA 7 “Slimy”
Fungi
– Acremonium species
– Candida guilliermondii
– Candida krusei
– Cladosporium species
– Fusarium species
– Penicillium species
– Rhodotorula rubra
Bacteria
– Alcaligenes faecalis
– Bacillus species
– Bacillus circulan
– Bacillus coagulans
– Bacillus licheniformis
– Bacillus pumulis
– Citrobacter brackii
– Citrobacter freundii
– Comamonas acidovorans
– Corynebacterium species
– Flavobacterium meningosepticum
– Legionella species
– Pseudomonas fluorescens
– Ralstonia paucula
– Serratia liquefaciens
– Serratia marcesens
– Yersinia frederiksenii
– Yersinia intermedia
NASA 7 “Fresh”
Fungi
– Acremonium species
– Candida guilliermondii
– Candida krusei
– Cladosporium species
– Fusarium species
– Penicillium species
– Rhodotorula rubra
Bacteria
– Bacillus coagulans
– Bacillus licheniformis
– Bacillus pumilus
– Bacillus species
– Comamonas acidovorans
– Corynebacterium species
– Enterobacter cloacae
– Legionella species
– Pseudomonas species
– Rhodococcus species
– Serratia liquefaciens
– Serratia marcerans
– Sphingobacterium thalpophilum
– Yersinia frederiksenii
– Yersinia intermedia
Escherichia coli
Serratia marcescens
Legionella species
Ott, et al, Microb Ecol 2004
MIR Condensate Residents
Spirochetes
Dust Mites
Ciliated Protozoa
Ott, et al, Microb Ecol 2004
“Establish a “microbial observatory” program on the ISS”
– National Research Council
Microbiological Spaceflight Research
• Multiple experiments over the 
past 50 years indicate unique 
microbial responses when 
cultured during spaceflight
• The environmental 
stimulus/stimuli initiating the 
response mechanisms are 
unclear
• The vast majority of microbial 
ecology data is based on media-
based analysis
• The impact of radiation on 
microbial responses/mutational 
rates is not known
28
28
Ground-based Analogue
The Rotating Wall Vessel (RWV)
• Solid body rotation in the 
reactor simulates several 
aspects of culture in 
microgravity
• Enables relatively high 
throughput
• Provides good indicators 
for spaceflight experiments
• Capability to follow up 
spaceflight findings without 
the delays associated with 
true spaceflight 
experiments The low shear culture conditions has 
initiated the term Low Shear Modeled 
Microgravity (LSMMG) environment
29
Microgravity Analogue Model Control
Gravity
(A) Analogue (B) Control
Rotation
Rotation
30
Microgravity Analogue Model Results
Virulence  
Gene 
expression
Stress 
Resistance
(Macrophage, acid, thermal, 
osmotic, oxidative)
Rotating Wall Vessel 
bioreactor
Nickerson et. al. Infect Immun 2000;  Wilson et al., Proc Natl Acad Sci USA 2002;  
Wilson et al. Appl Environ Microbiol 2002;  Nickerson, et al. Microbiol Mol Biol Rev 2004.
Salmonella typhimurium
31
Unique Microbial Responses
• Investigations by Dr. Cheryl 
Nickerson at Arizona State 
University evaluating 
microbial gene expression, 
morphology, and virulence
• Experiments aboard space 
shuttle missions STS-115 
(2006) and STS-123 (2008)
32
MICROBE 
Shuttle Atlantis, STS-115, launch September 2006
Salmonella Typhimurium experiment design
Gene 
expression
Disease 
potential
Cellular 
morphology
In-flight
hardware
Wilson et al. Proc Natl Acad Sci USA 2007
* Synchronous ground controls maintained under identical conditions as those on-
board Shuttle - ground and in-flight hardware loaded with same sample.
33
Flight Sample Ground Control
Salmonella Typhimurium Response to 
Spaceflight Culture
• In-flight grown S. Typhimurium 
showed the presence of an 
extracellular material not seen in 
ground control
• In-flight grown S. Typhimurium 
grown in LB broth killed mice 
faster and killed mice at lower 
doses than identical bacterial 
cultures grown on the ground
• LD50 was decreased 2.7 fold0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9 11 13 15 17 19 21 23 25
Days post-infection
Flight
Ground
P
e
r
c
e
n
t 
S
u
r
v
iv
a
l
Wilson et al. Proc Natl Acad Sci USA 2007 34
0500000
1000000
1500000
20000002500000
3000000
3500000
4000000
4500000
Salmonella enterica
serovar Typhimurium
genome
4,857 kb
Global Proteomic Profiling 
(MudPIT) identified 73 proteins 
differentially regulated by 
spaceflight
Hfq - Master molecular 
regulator identified
Microarray Analysis identified 
167 genes differentially 
regulated by spaceflight 
• Protein secretion
• Outer membrane proteins
• Iron metabolism and storage
• Ion response pathways
• Plasmid transfer functions
• Energy and metabolism
• Ribosomal proteins
• Small regulatory RNAs
• Biofilm formation 
• Transcriptional regulators 
• Unknown function
Spaceflight Globally Alters
S. Typhimurium Gene Expression
Wilson et al. Proc Natl Acad Sci USA 2007 35
Media composition
Disease potential
In-flight
hardware
* Synchronous ground controls maintained under identical conditions as those on-
board Shuttle - ground and in-flight hardware loaded with same sample.
Independent validation of the 
STS-115 results
Wilson et al. PLOS One 2008
MDRV
Shuttle Endeavour, STS-123, launch March 2008
Salmonella Typhimurium experiment design
36
The Impact of Media on Spaceflight 
Changes in S. Typhimurium Virulence
• Using Lennox Broth, the LD50 in the second 
spaceflight experiment again decreased (6.9 fold)
• This trend did not occur when M9 media was used or 
when the Lennox Broth media was supplemented 
with the inorganic ions used in M9 media
Wilson et al. PLOS One 2008 37
Media 
Growth
Location LD50 (CFU)
Fold Increase 
Relative to LB 
Media - Flight
LB media Flight 5.81 x 104 1.0
LB-M9 salts media Flight 7.45 x 105 12.8
M9 media Flight 3.30 x 106 56.8
Media 
Growth
Location LD50 (CFU)
Fold Increase 
Relative to LB 
Media - Ground
LB Media Ground 4.02 x 105 1.0
LB-M9 salts media Ground 5.73 x 105 1.4
M9 media Ground 2.30 x 106 5.7
Is there a Spaceflight Contribution?
Wilson et al. PLOS One 2008 38
Increased phosphate 
ion concentration 
prevents altered S. 
typhimurium acid 
tolerance in 
analogue culture 
Which component of the media?
Spaceflight data 
supplemented 
with ground-
based model
Wilson et al. PLOS One 2008 39
Summary Salmonella Typhimurium
Virulence
Gene Expression
• 167 genes
• Virulence genes down
•Hfq - evolutionarily conserved low 
fluid shear response regulator  found 
in other microorganisms
Stress Resistance 
and Biofilm 
formation
RWV Bioreactor
LSMMG
Control
Modulate ion levels 
Phosphate controls low fluid 
shear-associated pathogenesis 
response 
Spaceflight
Salmonella Typhimurium
Nickerson et. al. Infect Immun 2000;  Wilson et al., Proc Natl Acad Sci USA 2002; Wilson et al. Appl Environ Microbiol 2002;  
Nickerson, et al. Microbiol Mol Biol Rev 2004; Wilson et al. Proc Natl Acad Sci USA 2007; Wilson et al. PLOS One 2008
Gene Expression
Stress Resistance
Pseudomonas aeruginosa
Hfq - evolutionarily conserved 
low fluid shear response 
regulator in other pathogens
Crabbé et. al. Environ Microbiol 2008; Crabbé et al. Environ Microbiol 2010; Crabbé et al. Appl Eviron Microbiol 2011
Biofilm 
production
RWV Bioreactor
LSMMG
Control
Spaceflight
Summary Pseudomonas aeruginosa
Production 
virulence 
factors  
Gene Expression
Stress Resistance
Hfq - evolutionarily conserved low 
fluid shear response regulator in 
other pathogens
Castro et al. Appl Eviron Microbiol 2011
EPS
production
RWV Bioreactor
LSMMG
Control
Summary Staphylococcus aureus
Staphylococcus aureus
Carotenoid Production 
(Virulence Factor)
LSMMG
LSMMG
Control
Control
42
Current Studies in Microbiology
• Astronaut Microbiome
– Hernan Lorenzi, J. Craig 
Venter Institute
– Designed to gather 
information on changes in 
the crew microbiome during 
a spaceflight missions
– Investigation will include 
preflight, in-flight, and post-
flight samples from 9 
astronauts
– Tightly monitored conditions 
(e.g., temperature, humidity, 
diet)
43
Current Studies in Microbiology
• Latent viral reactivation in 
crewmembers
– Dr. Duane Pierson, NASA
– A series of experiments 
investigating the reactivation of 
Epstein Barr Virus (EBV), 
Cytomegalovirus, and Varicella 
Zoster Virus (VZV) in 
crewmembers during a mission
– Increased concentrations of 
EBV and VZV in astronaut 
saliva during a mission
– VZV can reactivate subclinically
in healthy individuals after 
acute stress.
44
Current Studies in Microbiology
• Picture Microbial Ecology
The ISS as a Microbial Observatory
• The ISS is a semi-closed, well controlled 
research platform advancing our ability to 
mitigate microbiological risk to the crew and 
their vehicle enabling space exploration
• The unique research enabled by access to 
space provides novel insight into our 
scientific understanding of life on Earth



The Risk of Astronaut Infection
Positives
• Preflight medical exams
• Preflight crew quarantine
• Stringent microbiological 
monitoring
• Limited exposure to many 
public health pathogens
• Healthy, well-conditioned 
crew
• Medical consult throughout 
a mission
Negatives
• Small enclosed environment
• Recycled air/water
• Stressful conditions
• Dysfunctional aspects of the  
immune system
• Altered microbial 
characteristics, including 
virulence
• Limited diagnostics and 
treatment on board
• Limited remediation 
capabilities
50
Infectious Disease during Spaceflight
• Upper respiratory infections
• Ear infections
• Various fungal infections
• Herpes Zoster
• Gastroenteritis
• Stye
• Allergic reactions
• Rashes & skin disorders
51


Microbiology and the International Space Station

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