Lipid vesicles chaperone an encapsulated RNA aptamer.

The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively 'chaperones' the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life

Improved single-swab sample preparation for recovering bacterial and phage DNA from human skin and wound microbiomes.

BackgroundCharacterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic.ResultsWe find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method.ConclusionUse of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples

Microbial predictors of healing and short-term effect of debridement on the microbiome of chronic wounds.

Chronic wounds represent a large and growing disease burden. Infection and biofilm formation are two of the leading impediments of wound healing, suggesting an important role for the microbiome of these wounds. Debridement is a common and effective treatment for chronic wounds. We analyzed the bacterial content of the wound surface from 20 outpatients with chronic wounds before and immediately after debridement, as well as healthy skin. Given the large variation observed among different wounds, we introduce a Bayesian statistical method that models patient-to-patient variability and identify several genera that were significantly enriched in wounds vs. healthy skin. We found no difference between the microbiome of the original wound surface and that exposed by a single episode of sharp debridement, suggesting that this debridement did not directly alter the wound microbiome. However, we found that aerobes and especially facultative anaerobes were significantly associated with wounds that did not heal within 6 months. The facultative anaerobic genus Enterobacter was significantly associated with lack of healing. The results suggest that an abundance of facultative anaerobes is a negative prognostic factor in the chronic wound microbiome, possibly due to the increased robustness of such communities to different metabolic environments

Characterization of the Bacterial and Viral Fractions of the Chronic Wound Microbiome and Their Associations to Clinical Outcomes

Chronic wounds represent a large and growing disease burden worldwide. Infection, biofilm formation, and associated pathological inflammation are some of the leading impediments to healing, suggesting an important role for the microbiome of these wounds. Studies of the bacterial fraction suggest that community composition, and its temporal variance, may be associated with healing outcomes, yet the forces that drive these dynamics are not well understood. The viral fraction of the microbiome, called the virome, may be a major contributing factor; other human viromes are dominated by bacteriophages, which not only infect and lyse bacteria, but can have profound impacts on host functionality. Despite its potential, the wound virome has not yet been described, largely due to the challenges associated with preparing and analyzing low-biomass clinical samples, like those obtained from skin and wounds. To facilitate the study of skin and wound viromes, we developed an improved sample processing method for obtaining both viral-enriched and bacterial DNA from a single swab sample, resulting in higher yields and viral purity when compared to traditional methods. The improved swab processing protocol was then employed in a small-cohort metagenomic survey of the skin and chronic wound microbiomes of 20 volunteers at an outpatient wound care clinic. We report taxonomic composition and diversity of the bacterial and viral fractions, and their associations to clinical features. Bacterial genera and viral species, as well as their respective oxygen requirements and auxiliary genes, were associated with both positive and negative healing outcomes. Additionally, we assessed how the microbiome is immediately impacted by sharp debridement, a standard of care procedure that physically removes necrotic tissue. There were no significant differences between microbiomes before and after a single debridement, suggesting that changes in microbial community structure are longitudinal and derived from repeated treatments. This work establishes novel methodology for studying the wound microbiome and virome, confirms previous findings in the field, presents the first chronic wound virome findings, and identifies correlations and associations to healing outcomes that causation studies may investigate in the future

Characterization of the Bacterial and Viral Fractions of the Chronic Wound Microbiome and Their Associations to Clinical Outcomes

Chronic wounds represent a large and growing disease burden worldwide. Infection, biofilm formation, and associated pathological inflammation are some of the leading impediments to healing, suggesting an important role for the microbiome of these wounds. Studies of the bacterial fraction suggest that community composition, and its temporal variance, may be associated with healing outcomes, yet the forces that drive these dynamics are not well understood. The viral fraction of the microbiome, called the virome, may be a major contributing factor; other human viromes are dominated by bacteriophages, which not only infect and lyse bacteria, but can have profound impacts on host functionality. Despite its potential, the wound virome has not yet been described, largely due to the challenges associated with preparing and analyzing low-biomass clinical samples, like those obtained from skin and wounds. To facilitate the study of skin and wound viromes, we developed an improved sample processing method for obtaining both viral-enriched and bacterial DNA from a single swab sample, resulting in higher yields and viral purity when compared to traditional methods. The improved swab processing protocol was then employed in a small-cohort metagenomic survey of the skin and chronic wound microbiomes of 20 volunteers at an outpatient wound care clinic. We report taxonomic composition and diversity of the bacterial and viral fractions, and their associations to clinical features. Bacterial genera and viral species, as well as their respective oxygen requirements and auxiliary genes, were associated with both positive and negative healing outcomes. Additionally, we assessed how the microbiome is immediately impacted by sharp debridement, a standard of care procedure that physically removes necrotic tissue. There were no significant differences between microbiomes before and after a single debridement, suggesting that changes in microbial community structure are longitudinal and derived from repeated treatments. This work establishes novel methodology for studying the wound microbiome and virome, confirms previous findings in the field, presents the first chronic wound virome findings, and identifies correlations and associations to healing outcomes that causation studies may investigate in the future

Lipid vesicles chaperone an encapsulated RNA aptamer.

The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively 'chaperones' the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life

The Chronic Wound Phageome: Phage Diversity and Associations with Wounds and Healing Outcomes.

Two leading impediments to chronic wound healing are polymicrobial infection and biofilm formation. Recent studies have characterized the bacterial fraction of these microbiomes and have begun to elucidate compositional correlations to healing outcomes. However, the factors that drive compositional shifts are still being uncovered. The virome may play an important role in shaping bacterial community structure and function. Previous work on the skin virome determined that it was dominated by bacteriophages, viruses that infect bacteria. To characterize the virome, we enrolled 20 chronic wound patients presenting at an outpatient wound care clinic in a microbiome survey, collecting swab samples from healthy skin and chronic wounds (diabetic, venous, arterial, or pressure) before and after a single, sharp debridement procedure. We investigated the virome using a virus-like particle enrichment procedure, shotgun metagenomic sequencing, and a k-mer-based, reference-dependent taxonomic classification method. Taxonomic composition, diversity, and associations with covariates are presented. We find that the wound virome is highly diverse, with many phages targeting known pathogens, and may influence bacterial community composition and functionality in ways that impact healing outcomes. IMPORTANCE Chronic wounds are an increasing medical burden. These wounds are known to be rich in microbial content, including both bacteria and bacterial viruses (phages). The viruses may play an important role in shaping bacterial community structure and function. We analyzed the virome and bacterial composition of 20 patients with chronic wounds. The viruses found in wounds are highly diverse compared to normal skin, unlike the bacterial composition, where diversity is decreased. These data represent an initial look at this relatively understudied component of the chronic wound microbiome and may help inform future phage-based interventions