The influence of microbial colonization on inflammatory versus pro-healing trajectories in combat extremity wounds.

A combination of improved body armor, medical transportation, and treatment has led to the increased survival of warfighters from combat extremity injuries predominantly caused by blasts in modern conflicts. Despite advances, a high rate of complications such as wound infections, wound failure, amputations, and a decreased quality of life exist. To study the molecular underpinnings of wound failure, wound tissue biopsies from combat extremity injuries had RNA extracted and sequenced. Wounds were classified by colonization (colonized vs. non-colonized) and outcome (healed vs. failed) status. Differences in gene expression were investigated between timepoints at a gene level, and longitudinally by multi-gene networks, inferred proportions of immune cells, and expression of healing-related functions. Differences between wound outcomes in colonized wounds were more apparent than in non-colonized wounds. Colonized/healed wounds appeared able to mount an adaptive immune response to infection and progress beyond the inflammatory stage of healing, while colonized/failed wounds did not. Although, both colonized and non-colonized failed wounds showed increasing inferred immune and inflammatory programs, non-colonized/failed wounds progressed beyond the inflammatory stage, suggesting different mechanisms of failure dependent on colonization status. Overall, these data reveal gene expression profile differences in healing wounds that may be utilized to improve clinical treatment paradigms.

The military gear microbiome: risk factors surrounding the warfighter.

Combat extremity wounds are highly susceptible to contamination from surrounding environmental material. This bioburden could be partially transferred from materials in immediate proximity to the wound, including fragments of the uniform and gear. However, the assessment of the microbial bioburden present on military gear during operational conditions of deployment or training is relatively unexplored. Opportunistic pathogens that can survive on gear represent risk factors for infection following injury, especially following combat blasts, where fibers and other materials are embedded in wounded tissue. We utilized 16S rRNA sequencing to assess the microbiome composition of different military gear types (boot, trouser, coat, and canteen) from two operational environments (training in Hawai'i and deployed in Indonesia) across time (days 0 and 14). We found that microbiome diversity, stability, and composition were dependent on gear type, training location, and sampling timepoint. At day 14, species diversity was significantly higher in Hawai'i samples compared to Indonesia samples for boot, coat, and trouser swabs. In addition, we observed the presence of potential microbial risk factors, as opportunistic pathogenic species, such as Acinetobacter, Pseudomonas, and Staphylococcus, were found to be present in all sample types and in both study sites. These study outcomes will be used to guide the design of antimicrobial materials and uniforms and for infection control efforts following combat blasts and other injuries, thereby improving treatment guidance during military training and deployment.IMPORTANCECombat extremity wounds are vulnerable to contamination from environments of proximity to the warfighter, leading to potential detrimental outcomes such as infection and delayed wound healing. Therefore, microbial surveillance of such environments is necessary to aid the advancement of military safety and preparedness through clinical diagnostics, treatment protocols, and uniform material design.

Targeted metagenomic assessment reflects critical colonization in battlefield injuries.

IMPORTANCE: Microbial contamination in combat wounds can lead to opportunistic infections and adverse outcomes. However, current microbiological detection has a limited ability to capture microbial functional genes. This work describes the application of targeted metagenomic sequencing to profile wound bioburden and capture relevant wound-associated signatures for clinical utility. Ultimately, the ability to detect such signatures will help guide clinical decisions regarding wound care and management and aid in the prediction of wound outcomes.

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.

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach.

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing.

Sierra Nevada sweep: metagenomic measurements of bioaerosols vertically distributed across the troposphere.

To explore how airborne microbial patterns change with height above the Earth's surface, we flew NASA's C-20A aircraft on two consecutive days in June 2018 along identical flight paths over the US Sierra Nevada mountain range at four different altitudes ranging from 10,000 ft to 40,000 ft. Bioaerosols were analyzed by metagenomic DNA sequencing and traditional culturing methods to characterize the composition and diversity of atmospheric samples compared to experimental controls. The relative abundance of taxa changed significantly at each altitude sampled, and the diversity profile shifted across the two sampling days, revealing a regional atmospheric microbiome that is dynamically changing. The most proportionally abundant microbial genera were Mycobacterium and Achromobacter at 10,000 ft; Stenotrophomonas and Achromobacter at 20,000 ft; Delftia and Pseudoperonospora at 30,000 ft; and Alcaligenes and Penicillium at 40,000 ft. Culture-based detections also identified viable Bacillus zhangzhouensis, Bacillus pumilus, and Bacillus spp. in the upper troposphere. To estimate bioaerosol dispersal, we developed a human exposure likelihood model (7-day forecast) using general aerosol characteristics and measured meteorological conditions. By coupling metagenomics to a predictive atmospheric model, we aim to set the stage for field campaigns that monitor global bioaerosol emissions and impacts.

Crewmember microbiome may influence microbial composition of ISS habitable surfaces.

The International Space Station (ISS) is a complex built environment physically isolated from Earth. Assessing the interplay between the microbial community of the ISS and its crew is important for preventing biomedical and structural complications for long term human spaceflight missions. In this study, we describe one crewmember's microbial profile from body swabs of mouth, nose, ear, skin and saliva that were collected at eight different time points pre-, during and post-flight. Additionally, environmental surface samples from eight different habitable locations in the ISS were collected from two flights. Environmental samples from one flight were collected by the crewmember and samples from the next flight were collected after the crewmember departed. The microbial composition in both environment and crewmember samples was measured using shotgun metagenomic sequencing and processed using the Livermore Metagenomics Analysis Toolkit. Ordination of sample to sample distances showed that of the eight crew body sites analyzed, skin, nostril, and ear samples are more similar in microbial composition to the ISS surfaces than mouth and saliva samples; and that the microbial composition of the crewmember's skin samples are more closely related to the ISS surface samples collected by the crewmember on the same flight than ISS surface samples collected by other crewmembers on different flights. In these collections, species alpha diversity in saliva samples appears to decrease during flight and rebound after returning to Earth. This is the first study to compare the ISS microbiome to a crewmember's microbiome via shotgun metagenomic sequencing. We observed that the microbiome of the surfaces inside the ISS resemble those of the crew's skin. These data support future crew and ISS microbial surveillance efforts and the design of preventive measures to maintain crew habitat onboard spacecraft destined for long term space travel.

Putative parapoxvirus-associated foot disease in the endangered huemul deer (Hippocamelus bisulcus) in Bernardo O'Higgins National Park, Chile.

The huemul (Hippocamelus bisulcus) is an endangered cervid endemic to southern Argentina and Chile. Here we report foot lesions in 24 huemul from Bernardo O'Higgins National Park, Chile, between 2005 and 2010. Affected deer displayed variably severe clinical signs, including lameness and soft tissue swelling of the limbs proximal to the hoof or in the interdigital space, ulceration of the swollen tissues, and some developed severe proliferative tissue changes that caused various types of abnormal wear, entrapment, and/or displacement of the hooves and/or dewclaws. Animals showed signs of intense pain and reduced mobility followed by loss of body condition and recumbency, which often preceded death. The disease affected both genders and all age categories. Morbidity and mortality reached 80% and 40%, respectively. Diagnostics were restricted to a limited number of cases from which samples were available. Histology revealed severe papillomatous epidermal hyperplasia and superficial dermatitis. Electron microscopy identified viral particles consistent with viruses in the Chordopoxvirinae subfamily. The presence of parapoxvirus DNA was confirmed by a pan-poxvirus PCR assay, showing high identity (98%) with bovine papular stomatitis virus and pseudocowpoxvirus. This is the first report of foot disease in huemul deer in Chile, putatively attributed to poxvirus. Given the high morbidity and mortality observed, this virus might pose a considerable conservation threat to huemul deer in Chilean Patagonia. Moreover, this report highlights a need for improved monitoring of huemul populations and synergistic, rapid response efforts to adequately address disease events that threaten the species.

Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis.

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to determine its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

The biodistribution and pharmacokinetics of the oxime acetylcholinesterase reactivator RS194B in guinea pigs.

Organophosphorus-based (OP) nerve agents represent some of the most toxic substances known to mankind. The current standard of care for exposure has changed very little in the past decades, and relies on a combination of atropine to block receptor activity and oxime-type acetylcholinesterase (AChE) reactivators to reverse the OP binding to AChE. Although these oximes can block the effects of nerve agents, their overall efficacy is reduced by their limited capacity to cross the blood-brain barrier (BBB). RS194B, a new oxime developed by Radic et al. (J. Biol. Chem., 2012) has shown promise for enhanced ability to cross the BBB. To fully assess the potential of this compound as an effective treatment for nerve agent poisoning, a comprehensive evaluation of its pharmacokinetic (PK) and biodistribution profiles was performed using both intravenous and intramuscular exposure routes. The ultra-sensitive technique of accelerator mass spectrometry was used to quantify the compound's PK profile, tissue distribution, and brain/plasma ratio at four dose concentrations in guinea pigs. PK analysis revealed a rapid distribution of the oxime with a plasma t1/2 of ∼1 h. Kidney and liver had the highest concentrations per gram of tissue followed by lung, spleen, heart and brain for all dose concentrations tested. The Cmax in the brain ranged between 0.03 and 0.18% of the administered dose, and the brain-to-plasma ratio ranged from 0.04 at the 10 mg/kg dose to 0.18 at the 200 mg/kg dose demonstrating dose dependent differences in brain and plasma concentrations. In vitro studies show that both passive diffusion and active transport contribute little to RS194B traversal of the BBB. These results indicate that biodistribution is widespread, but very low quantities accumulate in the guinea pig brain, indicating this compound may not be suitable as a centrally active reactivator.

Whole metagenome profiles of particulates collected from the International Space Station.

BACKGROUND: The built environment of the International Space Station (ISS) is a highly specialized space in terms of both physical characteristics and habitation requirements. It is unique with respect to conditions of microgravity, exposure to space radiation, and increased carbon dioxide concentrations. Additionally, astronauts inhabit a large proportion of this environment. The microbial composition of ISS particulates has been reported; however, its functional genomics, which are pertinent due to potential impact of its constituents on human health and operational mission success, are not yet characterized. METHODS: This study examined the whole metagenome of ISS microbes at both species- and gene-level resolution. Air filter and dust samples from the ISS were analyzed and compared to samples collected in a terrestrial cleanroom environment. Furthermore, metagenome mining was carried out to characterize dominant, virulent, and novel microorganisms. The whole genome sequences of select cultivable strains isolated from these samples were extracted from the metagenome and compared. RESULTS: Species-level composition in the ISS was found to be largely dominated by Corynebacterium ihumii GD7, with overall microbial diversity being lower in the ISS relative to the cleanroom samples. When examining detection of microbial genes relevant to human health such as antimicrobial resistance and virulence genes, it was found that a larger number of relevant gene categories were observed in the ISS relative to the cleanroom. Strain-level cross-sample comparisons were made for Corynebacterium, Bacillus, and Aspergillus showing possible distinctions in the dominant strain between samples. CONCLUSION: Species-level analyses demonstrated distinct differences between the ISS and cleanroom samples, indicating that the cleanroom population is not necessarily reflective of space habitation environments. The overall population of viable microorganisms and the functional diversity inherent to this unique closed environment are of critical interest with respect to future space habitation. Observations and studies such as these will be important to evaluating the conditions required for long-term health of human occupants in such environments.

Predicting a Drug's Membrane Permeability: A Computational Model Validated With in Vitro Permeability Assay Data.

Membrane permeability is a key property to consider during the drug design process, and particularly vital when dealing with small molecules that have intracellular targets as their efficacy highly depends on their ability to cross the membrane. In this work, we describe the use of umbrella sampling molecular dynamics (MD) computational modeling to comprehensively assess the passive permeability profile of a range of compounds through a lipid bilayer. The model was initially calibrated through in vitro validation studies employing a parallel artificial membrane permeability assay (PAMPA). The model was subsequently evaluated for its quantitative prediction of permeability profiles for a series of custom synthesized and closely related compounds. The results exhibited substantially improved agreement with the PAMPA data, relative to alternative existing methods. Our work introduces a computational model that underwent progressive molding and fine-tuning as a result of its synergistic collaboration with numerous in vitro PAMPA permeability assays. The presented computational model introduces itself as a useful, predictive tool for permeability prediction.

Characterization of Genetic Variability of Venezuelan Equine Encephalitis Viruses.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broad panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Finally, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.

Metagenomic analysis of the airborne environment in urban spaces.

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers.

BACKGROUND: The ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma. METHODS: Wound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS. RESULTS: This approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy. CONCLUSION: These results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds.

Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

Vaccination with recombinant Mycobacterium tuberculosis PknD attenuates bacterial dissemination to the brain in guinea pigs.

BACKGROUND: We have previously identified Mycobacterium tuberculosis PknD to be an important virulence factor required for the pathogenesis of central nervous system (CNS) tuberculosis (TB). Specifically, PknD mediates bacillary invasion of the blood-brain barrier, which can be neutralized by specific antisera, suggesting its potential role as a therapeutic target against TB meningitis. METHODOLOGY/PRINCIPAL FINDINGS: We utilized an aerosol challenge guinea pig model of CNS TB and compared the protective efficacy of recombinant M. tuberculosis PknD subunit protein with that of M. bovis BCG against bacillary dissemination to the brain. BCG vaccination limited the pulmonary bacillary burden after aerosol challenge with virulent M. tuberculosis in guinea pigs and also reduced bacillary dissemination to the brain (P = 0.01). PknD vaccination also offered significant protection against bacterial dissemination to the brain, which was no different from BCG (P>0.24), even though PknD vaccinated animals had almost 100-fold higher pulmonary bacterial burdens. Higher levels of PknD-specific IgG were noted in animals immunized with PknD, but not in BCG-vaccinated or control animals. Furthermore, pre-incubation of M. tuberculosis with sera from PknD-vaccinated animals, but not with sera from BCG-vaccinated or control animals, significantly reduced bacterial invasion in a human blood-brain barrier model (P<0.01). CONCLUSION: Current recommendations for administering BCG at birth are based on protection gained against severe disease, such as TB meningitis, during infancy. We demonstrate that vaccination with recombinant M. tuberculosis PknD subunit offers a novel strategy to protect against TB meningitis, which is equivalent to BCG in a guinea pig model. Moreover, since BCG lacks the PknD sensor, BCG could also be boosted to develop a more effective vaccine against TB meningitis, a devastating disease that disproportionately affects young children.

Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tuberculosis.

BACKGROUND: Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously. RESULTS: We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum. CONCLUSIONS: Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.