Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds.

Past results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion, Bacillus subtilis cells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth-normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system. Only 9 compounds were identified by PM screening as exhibiting significant differences (P < 0.05, Student's t test) in FL versus GC samples: 6-mercaptopurine, cesium chloride, enoxacin, lomefloxacin, manganese(II) chloride, nalidixic acid, penimepicycline, rolitetracycline, and trifluoperazine. Testing of the same compounds by standard broth dilution assay did not reveal statistically significant differences in the 50% inhibitory concentrations (IC50s) between FL and GC samples. The results indicate that the susceptibility of B. subtilis cells to a wide range of antibiotics and growth inhibitors is not dramatically altered by space flight.IMPORTANCE This study addresses a major concern of mission planners for human space flight, that bacteria accompanying astronauts on long-duration missions might develop a higher level of resistance to antibiotics due to exposure to the space flight environment. The results of this study do not support that notion.

Microbial Tracking-2, a metagenomics analysis of bacteria and fungi onboard the International Space Station.

BACKGROUND: The International Space Station (ISS) is a unique and complex built environment with the ISS surface microbiome originating from crew and cargo or from life support recirculation in an almost entirely closed system. The Microbial Tracking 1 (MT-1) project was the first ISS environmental surface study to report on the metagenome profiles without using whole-genome amplification. The study surveyed the microbial communities from eight surfaces over a 14-month period. The Microbial Tracking 2 (MT-2) project aimed to continue the work of MT-1, sampling an additional four flights from the same locations, over another 14 months. METHODS: Eight surfaces across the ISS were sampled with sterile wipes and processed upon return to Earth. DNA extracted from the processed samples (and controls) were treated with propidium monoazide (PMA) to detect intact/viable cells or left untreated and to detect the total DNA population (free DNA/compromised cells/intact cells/viable cells). DNA extracted from PMA-treated and untreated samples were analyzed using shotgun metagenomics. Samples were cultured for bacteria and fungi to supplement the above results. RESULTS: Staphylococcus sp. and Malassezia sp. were the most represented bacterial and fungal species, respectively, on the ISS. Overall, the ISS surface microbiome was dominated by organisms associated with the human skin. Multi-dimensional scaling and differential abundance analysis showed significant temporal changes in the microbial population but no spatial differences. The ISS antimicrobial resistance gene profiles were however more stable over time, with no differences over the 5-year span of the MT-1 and MT-2 studies. Twenty-nine antimicrobial resistance genes were detected across all samples, with macrolide/lincosamide/streptogramin resistance being the most widespread. Metagenomic assembled genomes were reconstructed from the dataset, resulting in 82 MAGs. Functional assessment of the collective MAGs showed a propensity for amino acid utilization over carbohydrate metabolism. Co-occurrence analyses showed strong associations between bacterial and fungal genera. Culture analysis showed the microbial load to be on average 3.0 × 105 cfu/m2 CONCLUSIONS: Utilizing various metagenomics analyses and culture methods, we provided a comprehensive analysis of the ISS surface microbiome, showing microbial burden, bacterial and fungal species prevalence, changes in the microbiome, and resistome over time and space, as well as the functional capabilities and microbial interactions of this unique built microbiome. Data from this study may help to inform policies for future space missions to ensure an ISS surface microbiome that promotes astronaut health and spacecraft integrity. Video Abstract.

Evaluation of co-circulating pathogens and microbiome from COVID-19 infections.

Co-infections or secondary infections with SARS-CoV-2 have the potential to affect disease severity and morbidity. Additionally, the potential influence of the nasal microbiome on COVID-19 illness is not well understood. In this study, we analyzed 203 residual samples, originally submitted for SARS-CoV-2 testing, for the presence of viral, bacterial, and fungal pathogens and non-pathogens using a comprehensive microarray technology, the Lawrence Livermore Microbial Detection Array (LLMDA). Eighty-seven percent of the samples were nasopharyngeal samples, and 23% of the samples were oral, nasal and oral pharyngeal swabs. We conducted bioinformatics analyses to examine differences in microbial populations of these samples, as a proxy for the nasal and oral microbiome, from SARS-CoV-2 positive and negative specimens. We found 91% concordance with the LLMDA relative to a diagnostic RT-qPCR assay for detection of SARS-CoV-2. Sixteen percent of all the samples (32/203) revealed the presence of an opportunistic bacterial or frank viral pathogen with the potential to cause co-infections. The two most detected bacteria, Streptococcus pyogenes and Streptococcus pneumoniae, were present in both SARS-CoV-2 positive and negative samples. Human metapneumovirus was the most prevalent viral pathogen in the SARS-CoV-2 negative samples. Sequence analysis of 16S rRNA was also conducted to evaluate bacterial diversity and confirm LLMDA results.

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds.

Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

Investigation of Spaceflight Induced Changes to Astronaut Microbiomes.

The International Space Station (ISS) is a uniquely enclosed environment that has been continuously occupied for the last two decades. Throughout its operation, protecting the health of the astronauts on-board has been a high priority. The human microbiome plays a significant role in maintaining human health, and disruptions in the microbiome have been linked to various diseases. To evaluate the effects of spaceflight on the human microbiome, body swabs and saliva samples were collected from four ISS astronauts on consecutive expeditions. Astronaut samples were analyzed using shotgun metagenomic sequencing and microarrays to characterize the microbial biodiversity before, during, and after the astronauts' time onboard the ISS. Samples were evaluated at an individual and population level to identify changes in microbial diversity and abundance. No significant changes in the number or relative abundance of taxa were observed between collection time points when samples from all four astronauts were analyzed together. When the astronauts' saliva samples were analyzed individually, the saliva samples of some astronauts showed significant changes in the relative abundance of taxa during and after spaceflight. The relative abundance of Prevotella in saliva samples increased during two astronauts' time onboard the ISS while the relative abundance of other commensal taxa such as Neisseria, Rothia, and Haemophilus decreased. The abundance of some antimicrobial resistance genes within the saliva samples also showed significant changes. Most notably, elfamycin resistance gene significantly increased in all four astronauts post-flight and a CfxA6 beta-lactam marker significantly increased during spaceflight but returned to normal levels post-flight. The combination of both shotgun metagenomic sequencing and microarrays showed the benefit of both technologies in monitoring microbes on board the ISS. There were some changes in each astronaut's microbiome during spaceflight, but these changes were not universal for all four astronauts. Two antimicrobial resistance gene markers did show a significant change in abundance in the saliva samples of all four astronauts across their collection times. These results provide insight for future ISS microbial monitoring studies and targets for antimicrobial resistance screenings.

Comparisons of Transcriptome Profiles from Bacillus subtilis Cells Grown in Space versus High Aspect Ratio Vessel (HARV) Clinostats Reveal a Low Degree of Concordance.

Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data.

Comparison of Bacillus subtilis transcriptome profiles from two separate missions to the International Space Station.

The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium Bacillus subtilis was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. A total of 91 genes were significantly differentially expressed in both experiments; 55 exhibiting higher transcript levels in flight samples and 36 showing higher transcript levels in ground control samples. Genes upregulated in flight samples notably included those involved in biofilm formation, biotin and arginine biosynthesis, siderophores, manganese transport, toxin production and resistance, and sporulation inhibition. Genes preferentially upregulated in ground control samples notably included those responding to oxygen limitation, e.g., fermentation, anaerobic respiration, subtilosin biosynthesis, and anaerobic regulatory genes. The results indicated differences in oxygen availability between flight and ground control samples, likely due to differences in cell sedimentation and the toroidal shape assumed by the liquid cultures in microgravity.

Meta-analysis of data from spaceflight transcriptome experiments does not support the idea of a common bacterial "spaceflight response".

Several studies have been undertaken with the goal of understanding how bacterial transcriptomes respond to the human spaceflight environment. However, these experiments have been conducted using a variety of organisms, media, culture conditions, and spaceflight hardware, and to date no cross-experiment analyses have been performed to uncover possible commonalities in their responses. In this study, eight bacterial transcriptome datasets deposited in NASA's GeneLab Data System were standardized through a common bioinformatics pipeline then subjected to meta-analysis to identify among the datasets (i) individual genes which might be significantly differentially expressed, or (ii) gene sets which might be significantly enriched. Neither analysis resulted in identification of responses shared among all datasets. Principal Component Analysis of the data revealed that most of the variation in the datasets derived from differences in the experiments themselves.

Transcriptomic responses of Serratia liquefaciens cells grown under simulated Martian conditions of low temperature, low pressure, and CO2-enriched anoxic atmosphere.

Results from previous experiments indicated that the Gram-negative α-proteobacterium Serratia liquefaciens strain ATCC 27592 was capable of growth under low temperature (0 °C), low pressure (0.7 kPa), and anoxic, CO2-dominated atmosphere-conditions intended to simulate the near-subsurface environment of Mars. To probe the response of its transcriptome to this extreme environment, S. liquefaciens ATCC 27592 was cultivated under 4 different environmental simulations: 0 °C, 0.7 kPa, CO2 atmosphere (Condition A); 0 °C, ~101.3 kPa, CO2 atmosphere (Condition B); 0 °C, ~101.3 kPa, ambient N2/O2 atmosphere (Condition C); and 30 °C, ~101.3 kPa, N2/O2 atmosphere (Condition D; ambient laboratory conditions). RNA-seq was performed on ribosomal RNA-depleted total RNA isolated from triplicate cultures grown under Conditions A-D and the datasets generated were subjected to transcriptome analyses. The data from Conditions A, B, or C were compared to laboratory Condition D. Significantly differentially expressed transcripts were identified belonging to a number of KEGG pathway categories. Up-regulated genes under all Conditions A, B, and C included those encoding transporters (ABC and PTS transporters); genes involved in translation (ribosomes and their biogenesis, biosynthesis of both tRNAs and aminoacyl-tRNAs); DNA repair and recombination; and non-coding RNAs. Genes down-regulated under all Conditions A, B, and C included: transporters (mostly ABC transporters); flagellar and motility proteins; genes involved in phenylalanine metabolism; transcription factors; and two-component systems. The results are discussed in the context of Mars astrobiology and planetary protection.

Expeditionary Resuscitation Surgical Team: The US Army's Initiative to Provide Damage Control Resuscitation and Surgery to Forces in Austere Settings.

Improvements in surgical care on the battlefield have contributed to reduced morbidity and mortality in wounded Servicemembers. 1 Point-of-injury care and early surgical intervention, along with improved personal protective equipment, have produced the lowest casualty statistics in modern warfare, resulting in improved force strength, morale, and social acceptance of conflict. It is undeniable that point-of-care injury, followed by early resuscitation and damage control surgery, saves lives on the battlefield. The US Army's Expeditionary Resuscitation Surgical Team (ERST) is a highly mobile, interprofessional medical team that can perform damage control resuscitation and surgery in austere locations. Its configuration and capabilities vary; however, in general, a typical surgical element can perform one major surgery and one minor surgery without resupply. The critical care element can provide prolonged holding in garrison, but this diminishes in the austere setting with complex and acutely injured patients.