Sequencing the Astronaut Microbiome. Hernán Lorenzi PhD. July 17 th, 2014 The J. Craig Venter Institute

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1 Sequencing the Astronaut Microbiome Hernán Lorenzi PhD. July 17 th, 2014 The J. Craig Venter Institute

2 The human microbiome, an organ within the human body Number of cells Number of genes

3 A healthy human microbiome varies between habitats, people and time. E. Costello et al. Science Vol. 326 no pp

4 Taxonomic and functional variations in the human gut microbiome Bacterial Phylum COG category Diversity + - PJ Turnbaugh et al. Nature, 1-5 (2008 Chang et al. J. of Inf Diseases (2008)

5 The human microbiome in health and disease Microbial dysbiosis Reduced microbial diversity Makes environment unfavorable to colonization by Synthesis of pathogens essential Compounds and detoxification Endocrine regulation Health Disease Transient diseases (Atopic dermatitis, diarrhea, etc.) human microbiome Maturation and stimulation of the human immune system Processing and absorption of complex Lipids and polysaccharides Autism disorders, anxiety, depression, multiple sclerosis Chronic diseases (obesity, diabetes Cancer allergy, asthma, IBD, etc.) (colon, liver, etc.)

6 Interaction between immune system and gut microbiota Gut epithelial function Systemic immunity Balance between T-cell subsets Immune system human microbiome Bacterial stratification (lectin RecIIIγ and IgA) Bacterial compartmentalization Microbial composition (alpha-defensins)

7 Factors that modulate the human microbiota Radiation Maternal effect Stress Diet human microbiome Immune response Stochastic factors Host genetics Factors associated with space travel

8 So, what is the risk to astronauts health due to alterations in the interaction between humans and their microbiome during a space mission?

9 Space travel and the human microbiome: knowledge gaps AEH7: Is there any changes in the microorganisms during space travel that might affect human health? AEH8: What changes are occurring to host susceptibility during space travel that could affect crew health? AEH9: Is there any changes in host-microorganism interactions during human space exploration that could put in risk human health? AEH10: What changes are occurring to the efficiency of current countermeasures?

10 Space travel and the astronauts microbiome: what do we currently know?

11 Evidence of transference of microorganisms from person to person from Apollo 7-11 missions (1970).(risk of pathogen transmission) The composition of culturable intestinal, oral and nasal flora have been shown to change during spaceflight of Apollo and Skylab missions. Spaceflights lasting 30 or more dates cause a reduction in lactic acid-producing activity of commensal Bifidobacteria in the gut(1979). (risk of infection by gram neg. pathogenic bacteria) In the ISS Pseudomonas aeruginosa, a commensal bacteria from skin, shows altered biofilm structure and increased virulence in culture. (risk of infection) Alteration of commensal bacterial growth kinetic depends on culture conditions (nutrients, oxygen availability, etc). C. albicans shows ABC transporter overexpression (CDR4), random budding, cell aggregation, and oxidative stress resistance. (risk of increased pathogenesis and antifungal drug resistance)

12 Alteration in host-microorganism interactions D. melanogaster larval plasmatocytes show reduced E. coli phagocytic activity after spaceflight. Clearance of E. coli in adult flies remained robust (Marcu et al. Plos ONE. 2011). In-flight infection of C. elegans with S. aureus, Listeria monocystogenes, Enterococcus faecalis and Candida albicans showed reduced virulence compared to ground controls. (Hammond et al. Astrobiology. 2013). Other ongoing host-bacteria interaction in-flight experiments: (i) Infection of C. elegans with S. Typhimurium (Micro-5 experiment) ; (ii) Infection of human cells with S. Typhimurium.

13 Efficiency of current countermeasures Antibiotic resistance Evidence shows both increased sensitivity and resistance to antibiotics, depending on the culture conditions and antibiotic tested. Prebiotics and probiotics The ongoing PROBIOTICS experiment looks at changes in the intestinal flora after space flight. Astronauts will take a probiotic (containing Lactobacillus casei) for 14 consecutive days in the ISS.

14 Study of the gut microbiome of participants of the MARS-500 experiment Temporal taxonomic variation of the gut microbiome. Temporal changes in KEGG functional categories Carbohydrate metabolism Cell motility Firmicutes Bacteroidetes

15 Study of the impact of long-term space travel on the astronauts microbiome. Hypothesis: Long-term exposure to stressors associated with spaceflight affects the composition and relative abundance of the astronauts microbiome. Aim 1: To characterize changes occurring in the microbiome of astronauts from key body sites during a space mission Aim 2: To assess astronauts immune function and stress levels and their effect on the crew microbiome. Aim 3: To investigate whether the environmental microbiome of the ISS and other factors such as diet may promote changes in the astronaut's microbiome.

16 Experimental design Longitudinal study; N=9 astronauts that stay at least 6 months in the ISS pre-flight ISS post-flight L-240 L-150 L-90 L-60 FD7 FD90 R-14 R-1 R+0-3 R+30 R+60 R+180 body fecal saliva blood water ISS surface EHH survey time Data types astronauts microbiome -16S v1-v3 -Metagenomics (B+V) stress -cortisol -VZV/EBV virus reactivation immune system -cytokines environmental microbiome -16S v1-v3 EHH survey -Metadata: diet health status Temp / Humid.

17 Summary of sample collection

18 Preliminary results: taxonomic profiles of pre-flight samples Distribution of the most abundant genera across the five body-sites surveyed. PCA analysis of sample taxonomic profiles Forearm Forehead Nares Stool Tongue Intra vs. inter-site beta diversity

19 Preliminary results: temporal fluctuation in alpha and beta diversity before the mission p = p = Intra-sample diversity Inter-sample diversity

20 Other ongoing microbiome-related experiments The MULTI-OMICS experiment (JAXA) will look at changes in the microbial profile (feces and saliva), metabolite biomarkers (feces and saliva), immune response (blood and tissue) and transcriptomics (tissue) before, during and after a space mission.

21 Conclusions: 1. The experimental evidence presented in the evidence report Risk of adverse health effects due to alterations in host-microbial interactions, such as increased bacterial virulence, antibiotic resistance and altered biofilm formation, strongly suggest that the infection of crew members by obligate/opportunistic pathogens (some frequently found in the spaceship), pose a real threat to the health of astronauts during long-term space missions. 2. More evidence is needed to evaluate the risk of infection by (reactivated) viruses and fungi. 3. Studies on culturable commensal bacteria suggest that space travel might alter the normal composition of the human microbiome. Because microbiome changes are relevant to knowledge gaps EHA7 and EHA9, that information should be incorporated in future evidence reports.

22 Conclusions (cont.) 4. It is expected that the ongoing MICROBIOME study will determine the extent of the impact of space travel on the astronauts microbiome and the potential risk to crew health.

23 Other additional gaps in knowledge or areas of fundamental research that should be considered. 1. Does the interaction between environmental factors associated with space travel and current countermeasures pose a risk to astronauts health during long-term space exploration? Antibiotic resistance bacterial virulence Lifestyle Physical barriers Hygiene kits Clean foods High-quality air filters Environmental Stressors Microgravity Radiation Stress Antibiotic consumption Prebiotics Probiotics Adapted from Saei and Barzegari, Fut. Microbiol. 2012? Intestine Alterations in and washing out of microflora Impaired immune system Metabolic disease Facilitate colonization by opportunistic pathogens

24 Other additional gaps in knowledge or areas of fundamental research that should be considered. 2. Omics studies looking at the effect of space travel and changes in the transcriptome, proteome and metabolome of commensal microbiota. 3. Studies looking at the interaction between animals with an altered immune response and bacteria with enhanced virulence.

25 Acknowledgments Manny Torralba Karen Nelson Mark Ott Duane Pierson Satish Mehta ISSMP experiment team NASA s Human Research Program