Ott, C. Mark

English

NASA Technical Reports Server

Spaceflight Microbiology
From Small Things, Big Things One Day Come
C. Mark Ott, PhD
Microbiology Laboratory
NASA Johnson Space Center, Houston, TX
University of  Notre Dame 2020
https://ntrs.nasa.gov/search.jsp?R=20200001426 2020-03-28T19:17:42+00:00Z


4
Microbiological Areas of  Concern
• Astronaut health
• Vehicle integrity
• Life support and other systems failure
− Biofilm formation/biofouling
− Biocorrosion and biodegradation
− Trash and human waste containment
− Astronaut hygiene areas
• Spaceflight foods
− In addition to packaged food, NASA is developing  
“pick and eat” vegetable growth systems
• Planetary protection
− How do we track the microorganisms we are 
bringing versus what we are finding in our search 
for life on other planets?
5
Astronaut Health
• Preventative measures
− Preflight medical exams and medical consult throughout a mission
− Preflight crew quarantine
− Stringent microbiological monitoring of  spacecraft and its cargo
• Examples of  diseases during spaceflight missions 
− Upper respiratory infections
− Urinary tract infections
− Ear infections
− Herpes Zoster
− Rashes & skin disorders
− Gastroenteritis
6
Astronaut Health
• Generally healthy crew
− Survey of  Space Shuttle missions (STS-1 through STS-89) indicated 
infectious disease accounted for 1.4% of  all medical events (not 
including skin and subcutaneous tissue) (Risin 2009)
− Infectious disease and allergic symptom rate on the International Space 
Station (ISS) has been estimated at 3.4 events/flight year (Crucian et al. 
2016)
• Immune system evidence for long duration spaceflight
− Reduced function and distribution of  some immune cells (e.g., natural 
killer (NK) cells, T cells, monocytes, and neutrophils)
− Reactivation of  latent viruses 
− Altered cytokine production patterns
7
Infectious Disease during Spaceflight
• Diagnosis is often based on symptomology
− Headache
− Rash
− Dry hacking cough
− Diarrhea
• Attributed to infectious disease
− Upper respiratory infections
− Ear infections
− Various fungal infections
− Herpes Zoster
− Gastroenteritis
− Stye
− Allergic reactions
− Rashes & skin disorders
Prevention - Spacecraft Design
• Vehicle surfaces
− Use of  materials that are not conducive to 
microbial growth
− Remediation: Disinfectant wipes
• Vehicle air
− Air flow through HEPA filters
• Potable water recovery system
− Recovers humidity condensate and urine
− Final water recycling steps of  the NASA 
water system include catalytic oxidizer (267 
°F for 10 minutes); iodination; iodine 
removal; 0.22 micron filtration
− Remediation: High concentration iodine 
solution
9
Prevention - Operations 
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
10
Prevention - Environmental Monitoring
• Extensive microbial monitoring and preflight disinfection
• Preflight monitoring targets environmental areas that are 
probable routes of  infection, including vehicle air and surfaces, 
spaceflight food, potable water, cargo, and a biosafety review 
of  experimental payloads.
• Flight requirements
− Vehicle air - 100 CFU/m3 fungi; 1000 CFU/m3 bacteria
− Vehicle surfaces - 100 CFU/100 cm2 fungi; 10,000 CFU/100 cm2
bacteria
11
− Potable water - 50 CFU/ml heterotrophic 
plate count; no detectable coliforms in 100 
ml; no detectable fungi in 100 ml; treatment 
technique to prevent transmission of  
parasitic protozoa.
Contamination Potential
Spacecraft are 
complex
Preflight 
contamination
Astronaut activities, 
such as eating and 
hygiene
12
Constraints of  Spaceflight
• When considering spaceflight 
hardware (e.g., monitoring 
equipment) and cargo (e.g., 
disinfection wipes, clothing, 
food), the benefit of  the item is 
balanced against the need for:
− Safe operation
− Minimal power
− Minimal mass
− Minimal volume
− Minimal crew time
13
No phase separation
Microbiological Monitoring on the ISS
14
AirSurfaces Water
Quantified in-flight and returned to JSC for identification
Microbiological Monitoring of  Water
15
ISS Air and Surface Monitoring
FungaI Isolates
16Pierson, et al. 2012 
ISS Air and Surface Monitoring
Bacterial Isolates
Pierson, et. al. Environmental Monitoring: A Comprehensive Handbook 2012 17Pierson, et al. 2012 
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
Pierson, et al. 2012 
Current Studies in Microbiology
• Picture Microbial Ecology
Environmental Health
• Microbial monitoring by JSC 
and others indicate ISS 
environmental flora reflect 
human-associated 
microorganisms similar to a 
home.
20
• Media-based monitoring occasionally identifies opportunistic 
bacterial pathogens, such as Staphylococcus aureus and 
Bacillus cereus. No methicillin resistant S. aureus has been 
identified. Enterobacter and Enterococcus species are 
occasionally identified throughout the ISS. 
Pierson, et al. 2012; Lang, et al. 2017; Blaustein, et al. 2019
Environmental Health
• The most prevalent fungal genera are 
Aspergillus and  Penicillium. A. flavus, A. 
niger, and A. fumigatus have been 
identified from spaceflight samples. 
Stachybotrys chartarum has been 
isolated preflight.
• Potable water reflects common 
environmental bacterial species. The 
0.22 micron filter upstream of  the 
water ports have limited increases in 
diversity.
• The ISS is only semi-closed and “new” 
bacterial and fungal isolates are 
regularly identified.
21
Environmental Anomalies
• Mir Space Station
− Power failures and problems with 
temperature control eventually 
caused large amounts of  free-
floating condensate
− Samples of  condensate revealed 
high microbial diversity, including 
Escherichia coli, Serratia
marcescens, Legionella species, 
and protozoa (Ott et al., 2005)
22
• ISS anomalies
− Surface contamination is often associated with uncontrolled water
− Air contamination and unpleasant odors do occur; however, the sources 
are often unclear. These events have been transient and/or handled by 
the HEPA filtration system
ISS Environmental Anomalies
23
Advances in Environmental Monitoring
• DNA sequencing in space 
− First performed in 2016 using Oxford 
Nanopore’s MinION using lambda 
bacteriophage, E. coli, and mouse DNA
• In-flight identification of  
environmental samples
− Three microbial colonies from a media 
plate used for ISS surface sampling were 
processed and sequenced on ISS.
− The MinION accurately identified the 
three isolates that were selected.
• Major push to convert our current 
monitoring hardware to this 
technology, especially for deep space 
missions. 
24Castro-Wallace et al. 2017; Burton et al. 2020
Spaceflight Food
25
Early 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 26
Spaceflight Food
http://www.newhealthguide.org/Fruit-and-Vegetable-Only-Diet.html
The Future of  Food Requirements
• The VEGGIE spaceflight 
experiment grew lettuce on ISS
− Can the crew eat it?
− What about other crops besides 
lettuce?
• We are planning to prepare and 
cook more food on spaceflight
− How do we control contamination 
during processing?
• Probiotics are being evaluated as 
an immune booster
− What organisms do we monitor 
for safety?
28
Microbial Research in Space
• Multiple experiments over the past 50 to 60 
years have demonstrated unexpected 
microbial responses when microorganisms are 
cultured during spaceflight, including:
− Antibiotic resistance
− Growth kinetics
− Virulence
− Gene expression
• Spaceflight research has been burdened by 
sensationalized or unsubstantiated conclusions. 
− Highly visible field with limited access to rerun 
experiments for confirmation
− Misconceptions from these reports can last for years 
and actually be damaging to many of  the real 
scientific breakthroughs
29
Ground Based Spaceflight Analogues
• Rotating Wall Vessel (RWV) bioreactor
− Solid body rotation in the reactor 
simulates several aspects of  culture in 
microgravity
− Provides predictive trends for spaceflight 
experiments
− Enables follow up to spaceflight findings 
without the delays associated with true 
spaceflight experiments
• Earth based habitats
− Controlled habitats that mimic certain 
aspects of  spaceflight conditions
− Examples of  study topics include human 
behavior, life support system 
development, immune system function, 
environmental microbiome 
30
Concordia Station (Antarctica)
The low shear culture conditions has 
prompted the term Low Shear Modeled 
Microgravity (LSMMG) environment
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.
31
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 fold
0
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
er
ce
n
t 
S
u
rv
iv
al
Wilson et al. Proc Natl Acad Sci USA 2007 32
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 33
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
34
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
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
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 37
Gene Expression
Stress Resistance
Pseudomonas aeruginosa
Hfq - evolutionarily 
conserved low fluid 
shear response 
regulator in other 
pathogens
Crabbé et. al. 2008; Crabbé et al. 2010; Crabbé et al. 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. 2011
EPS
production
RWV Bioreactor
LSMMG
Control
Summary Staphylococcus aureus
Staphylococcus aureus
Carotenoid Production 
(Virulence Factor)
LSMMG
LSMMG
Control
Control
39
Studies by our team and others have implicated Hfq in the response of  at 
least four other microbes to spaceflight and/or spaceflight-analogue culture
Biofilms and Microbial Evolutional
41
• PI: Cheryl Nickerson, Arizona State University
• As most spaceflight studies deal with short duration, 
batch cultures of  microorganisms, no information is 
available on the long term evolution of  microbial 
pathogens cultured in the spaceflight environment.
• This study will focus on the mutation rate and 
phenotypic characteristics of  S. Typhimurium actively 
cultured over 100 generations aboard ISS.
• PI: Bob McLean, Texas State University
• Investigation into polymicrobial biofilm development during 
spaceflight by P. aeruginosa and E. coli.
• The study focuses on biofilm architecture, disinfection, and 
corrosion potential during spaceflight.
• Biofilm studies have been ongoing since the first study was 
reported by McLean in 2001.
Crew and Vehicle Microbiology
42
• PI: Hernan Lorenzi, J. Craig Venter Institute
• Baseline study of  skin sites, nostrils, fecal samples from 9 
astronauts collected preflight, in-flight, and post-flight 
• The composition of  the intestinal microbiota became more 
similar across astronauts in space
• Alterations in the skin microbiome were observed that 
might contribute to the high frequency of  skin 
rashes/hypersensitivity episodes.
• PI: Cherie Oubre, NASA
• Expansion of  our nominal crew health environmental 
monitoring to include the VEGGIE plant growth system to 
determine if  the bacterial and fungal composition near the 
plants is different than the general habitat area.
• Information will be used to support future requirements for 
“pick and eat” foods

Microbiology Laboratory
NASA Johnson Space Center
44
Debbie Aldape
Audry Almengor, Ph.D.
Bekki Bruce
Victoria Castro
Christian Castro
Audrie Colorado, Ph.D.
Brandon Dunbar
Todd Elliott
Tanner Hamilton
Jacob Harris
Jane McCourt
Christian Mena
Hang Ngoc Nguyen, Ph.D.
Duane Pierson, Ph.D. (Emeritus)
Joan Robertson
Melanie Smith
Sarah Stahl
Sarah Wallace, Ph.D.



Spaceflight Microbiology From Small Things, Big Things One Day Come

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