Substrate Matters: Ionic Silver Alters Lettuce Growth, Nutrient Uptake, and Root Microbiome in a Hydroponics System.

Ionic silver (Ag+) is being investigated as a residual biocide for use in NASA spacecraft potable water systems on future crewed missions. This water will be used to irrigate future spaceflight crop production systems. We have evaluated the impact of three concentrations (31 ppb, 125 ppb, and 500 ppb) of ionic silver biocide solutions on lettuce in an arcillite (calcinated clay particle substrate) and hydroponic (substrate-less) growth setup after 28 days. Lettuce plant growth was reduced in the hydroponic samples treated with 31 ppb silver and severely stunted for samples treated at 125 ppb and 500 ppb silver. No growth defects were observed in arcillite-grown lettuce. Silver was detectable in the hydroponic-grown lettuce leaves at each concentration but was not detected in the arcillite-grown lettuce leaves. Specifically, when 125 ppb silver water was applied to a hydroponics tray, Ag+ was detected at an average amount of 7 µg/g (dry weight) in lettuce leaves. The increase in Ag+ corresponded with a decrease in several essential elements in the lettuce tissue (Ca, K, P, S). In the arcillite growth setup, silver did not impact the plant root zone microbiome in terms of alpha diversity and relative abundance between treatments and control. However, with increasing silver concentration, the alpha diversity increased in lettuce root samples and in the water from the hydroponics tray samples. The genera in the hydroponic root and water samples were similar across the silver concentrations but displayed different relative abundances. This suggests that ionic silver was acting as a selective pressure for the microbes that colonize the hydroponic water. The surviving microbes likely utilized exudates from the stunted plant roots as a carbon source. Analysis of the root-associated microbiomes in response to silver showed enrichment of metagenomic pathways associated with alternate carbon source utilization, fatty-acid synthesis, and the ppGpp (guanosine 3'-diphosphate 5'-diphosphate) stringent response global regulatory system that operates under conditions of environmental stress. Nutrient solutions containing Ag+ in concentrations greater than 31 ppb in hydroponic systems lacking cation-exchange capacity can severely impact crop production due to stunting of plant growth.

A hyperspectral plant health monitoring system for space crop production.

Compact and automated sensing systems are needed to monitor plant health for NASA's controlled-environment space crop production. A new hyperspectral system was designed for early detection of plant stresses using both reflectance and fluorescence imaging in visible and near-infrared (VNIR) wavelength range (400-1000 nm). The prototype system mainly includes two LED line lights providing VNIR broadband and UV-A (365 nm) light for reflectance and fluorescence measurement, respectively, a line-scan hyperspectral camera, and a linear motorized stage with a travel range of 80 cm. In an overhead sensor-to-sample arrangement, the stage translates the lights and camera over the plants to acquire reflectance and fluorescence images in sequence during one cycle of line-scan imaging. System software was developed using LabVIEW to realize hardware parameterization, data transfer, and automated imaging functions. The imaging unit was installed in a plant growth chamber at NASA Kennedy Space Center for health monitoring studies for pick-and-eat salad crops. A preliminary experiment was conducted to detect plant drought stress for twelve Dragoon lettuce samples, of which half were well-watered and half were under-watered while growing. A machine learning method using an optimized discriminant classifier based on VNIR reflectance spectra generated classification accuracies over 90% for the first four days of the stress treatment, showing great potential for early detection of the drought stress on lettuce leaves before any visible symptoms and size differences were evident. The system is promising to provide useful information for optimization of growth environment and early mitigation of stresses in space crop production.

Microbial isolation and characterization from two flex lines from the urine processor assembly onboard the international space station.

Urine, humidity condensate, and other sources of non-potable water are processed onboard the International Space Station (ISS) by the Water Recovery System (WRS) yielding potable water. While some means of microbial control are in place, including a phosphoric acid/hexavalent chromium urine pretreatment solution, many areas within the WRS are not available for routine microbial monitoring. Due to refurbishment needs, two flex lines from the Urine Processor Assembly (UPA) within the WRS were removed and returned to Earth. The water from within these lines, as well as flush water, was microbially evaluated. Culture and culture-independent analysis revealed the presence of Burkholderia, Paraburkholderia, and Leifsonia. Fungal culture also identified Fusarium and Lecythophora. Hybrid de novo genome analysis of the five distinct Burkholderia isolates identified them as B. contaminans, while the two Paraburkholderia isolates were identified as P. fungorum. Chromate-resistance gene clusters were identified through pangenomic analysis that differentiated these genomes from previously studied isolates recovered from the point-of-use potable water dispenser and/or current NCBI references, indicating that unique populations exist within distinct niches in the WRS. Beyond genomic analysis, fixed samples directly from the lines were imaged by environmental scanning electron microscopy, which detailed networks of fungal-bacterial biofilms. This is the first evidence of biofilm formation within flex lines from the UPA onboard the ISS. For all bacteria isolated, biofilm potential was further characterized, with the B. contaminans isolates demonstrating the most considerable biofilm formation. Moreover, the genomes of the B. contaminans revealed secondary metabolite gene clusters associated with quorum sensing, biofilm formation, antifungal compounds, and hemolysins. The potential production of these gene cluster metabolites was phenotypically evaluated through biofilm, bacterial-fungal interaction, and hemolytic assays. Collectively, these data identify the UPA flex lines as a unique ecological niche and novel area of biofilm growth within the WRS. Further investigation of these organisms and their resistance profiles will enable engineering controls directed toward biofilm prevention in future space station water systems.

Biofilm formation is correlated with low nutrient and simulated microgravity conditions in a Burkholderia isolate from the ISS water processor assembly.

The International Space Station (ISS) Water Processor Assembly (WPA) experiences intermittent dormancy in the WPA wastewater tank during water recycling events which promotes biofilm formation within the system. In this work we aimed to gain a deeper understanding of the impact of nutrient limitation on bacterial growth and biofilm formation under microgravity in support of biofilm mitigation efforts in exploration water recovery systems. A representative species of bacteria that is commonly cultured from the ISS WPA was cultured in an WPA influent water ersatz formulation tailored for microbiological studies. An isolate of Burkholderia contaminans was cultured under a simulated microgravity (SµG) treatment in a vertically rotating high-aspect rotating vessel (HARV) to create the low shear modeled microgravity (LSMMG) environment on a rotating wall vessel (RWV), with a rotating control (R) in the horizontal plane at the predetermined optimal rotation per minute (rpm) speed of 20. Over the course of the growth curve, the bacterial culture in ersatz media was harvested for bacterial counts, and transcriptomic and nutrient content analyses. The cultures under SµG treatment showed a transcriptomic signature indicative of nutrient stress and biofilm formation as compared to the R control treatment. Further analysis of the WPA ersatz over the course of the growth curve suggests that the essential nutrients of the media were consumed faster in the early stages of growth for the SµG treatment and thus approached a nutrient limited growth condition earlier than in the R control culture. The observed limited nutrient response may serve as one element to explain a moderate enhancement of adherent biofilm formation in the SµG treatment after 24 h. While nutrients levels can be modulated, one implication of this investigation is that biofilm mitigation in the ISS environment could benefit from methods such as mixing or the maintenance of minimum flow within a dormant water system in order to force convection and offset the response of microbes to the secondary effects of microgravity.

Predicting antimicrobial mechanism-of-action from transcriptomes: A generalizable explainable artificial intelligence approach.

To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action [MOA], represent a major research priority in biomedical science. However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.

Reference-guided metagenomics reveals genome-level evidence of potential microbial transmission from the ISS environment to an astronaut's microbiome.

Monitoring microbial communities aboard the International Space Station (ISS) is essential to maintaining astronaut health and the integrity of life-support systems. Using assembled genomes of ISS-derived microbial isolates as references, recruiting metagenomic reads from an astronaut's nasal microbiome revealed no recruitment to a Staphylococcus aureus isolate from samples before launch, yet systematic recruitment across the genome when sampled after 3 months aboard the ISS, with a median percent identity of 100%. This suggests that either a highly similar S. aureus population colonized the astronaut's nasal microbiome while the astronaut was aboard the ISS or that it may have been below detection before spaceflight, instead supporting a shift in community composition. This work highlights the value in generating genomic libraries of microbes from built-environments such as the ISS and demonstrates one way such data can be integrated with metagenomics to facilitate the tracking and monitoring of astronaut microbiomes and health.

Predictive Signatures of 19 Antibiotic-Induced Escherichia coli Proteomes.

Identifying the mode of action (MOA) of antibacterial compounds is the fundamental basis for the development of new antibiotics, and the challenge increases with the emerging secondary and indirect effect from antibiotic stress. Although various omics-based system biology approaches are currently available, enhanced throughput, accuracy, and comprehensiveness are still desirable to better define antibiotic MOA. Using label-free quantitative proteomics, we present here a comprehensive reference map of proteomic signatures of Escherichia coli under challenge of 19 individual antibiotics. Applying several machine learning techniques, we derived a panel of 14 proteins that can be used to classify the antibiotics into different MOAs with nearly 100% accuracy. These proteins tend to mediate diverse bacterial cellular and metabolic processes. Transcriptomic level profiling correlates well with protein expression changes in discriminating different antibiotics. The reported expression signatures will aid future studies in identifying MOA of unknown compounds and facilitate the discovery of novel antibiotics.

Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station.

The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.

Mechanism-of-Action Classification of Antibiotics by Global Transcriptome Profiling.

Antimicrobial resistance (AMR) is an ever-growing public health problem worldwide. The low rate of antibiotic discovery coupled with the rapid spread of drug-resistant bacterial pathogens is causing a global health crisis. To facilitate the drug discovery processes, we present a large-scale study of reference antibiotic challenge bacterial transcriptome profiles, which included 37 antibiotics across 6 mechanisms of actions (MOAs) and provide an economical approach to aid in antimicrobial dereplication in the discovery process. We demonstrate that classical MOAs can be sorted based upon the magnitude of gene expression profiles despite some overlap in the secondary effects of antibiotic exposures across MOAs. Additionally, using gene subsets, we were able to subdivide broad MOA classes into subMOAs. Furthermore, we provide a biomarker gene set that can be used to classify most antimicrobial challenges according to their canonical MOA. We also demonstrate the ability of this rapid MOA diagnostic tool to predict and classify the expression profiles of pure compounds and crude extracts to their expression profile-associated MOA class.

Following the Astrobiology Roadmap: Origins, Habitability and Future Exploration.

Astrobiology asks three fundamental questions as outlined by the NASA Astrobiology Roadmap: 1. How did Life begin and evolve?; Is there Life elsewhere in the Universe?; and, What is the future of Life on Earth? As we gain perspective on how Life on Earth arose and adapted to its many niches, we too gain insight into how a planet achieves habitability. Here on Earth, microbial Life has evolved to exist in a wide range of habitats from aquatic systems to deserts, the human body, and the International Space Station (ISS). Landers, rovers, and orbiter missions support the search for signatures of Life beyond Earth, by generating data on surface and subsurface conditions of other worlds. These have provided evidence for water activity, supporting the potential for extinct or extant Life. To investigate the putative ecologies of these systems, we study extreme environments on Earth. Several locations on our planet provide analog settings to those we have detected or expect to find on neighboring and distant worlds. Whereas, the field of space biology uses the ISS and low gravity analogs to gain insight on how transplanted Earth-evolved organisms will respond to extraterrestrial environments. Modern genomics allows us to chronicle the genetic makeup of such organisms and provides an understanding of how Life adapts to various extreme environments.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 µg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.

Alkaloids from the Sponge Stylissa carteri Present Prospective Scaffolds for the Inhibition of Human Immunodeficiency Virus 1 (HIV-1).

The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%-40% inhibition of HIV-1 at 3.1 µM and 13 µM, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 µM with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 µM. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery.