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.

A Bayesian nonparametric analysis for zero-inflated multivariate count data with application to microbiome study.

High-throughput sequencing technology has enabled researchers to profile microbial communities from a variety of environments, but analysis of multivariate taxon count data remains challenging. We develop a Bayesian nonparametric (BNP) regression model with zero inflation to analyse multivariate count data from microbiome studies. A BNP approach flexibly models microbial associations with covariates, such as environmental factors and clinical characteristics. The model produces estimates for probability distributions which relate microbial diversity and differential abundance to covariates, and facilitates community comparisons beyond those provided by simple statistical tests. We compare the model to simpler models and popular alternatives in simulation studies, showing, in addition to these additional community-level insights, it yields superior parameter estimates and model fit in various settings. The model's utility is demonstrated by applying it to a chronic wound microbiome data set and a Human Microbiome Project data set, where it is used to compare microbial communities present in different environments.

EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides.

In vitro evolution is a well-established technique for the discovery of functional RNA and peptides. Increasingly, these experiments are analyzed by high-throughput sequencing (HTS) for both scientific and engineering objectives, but computational analysis of HTS data, particularly for peptide selections, can present a barrier to entry for experimentalists. We introduce EasyDIVER (Easy pre-processing and Dereplication of In Vitro Evolution Reads), a simple, user-friendly pipeline for processing high-throughput sequencing data from in vitro selections and directed evolution experiments. The pipeline takes as input raw, paired-end, demultiplexed Illumina read files. For each sample provided, EasyDIVER outputs a dereplicated list of unique nucleic acid and/or peptide sequences and their count reads.

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.

High throughput sequencing of in vitro selections of mRNA-displayed peptides: data analysis and applications.

In vitro selection using mRNA display is currently a widely used method to isolate functional peptides with desired properties. The analysis of high throughput sequencing (HTS) data from in vitro evolution experiments has proven to be a powerful technique but only recently has it been applied to mRNA display selections. In this Perspective, we introduce aspects of mRNA display and HTS that may be of interest to physical chemists. We highlight the potential of HTS to analyze in vitro selections of peptides and review recent advances in the application of HTS analysis to mRNA display experiments. We discuss some possible issues involved with HTS analysis and summarize some strategies to alleviate them. Finally, the potential for future impact of advancing HTS analysis on mRNA display experiments is discussed.

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

BACKGROUND: Characterization 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. RESULTS: We 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. CONCLUSION: Use of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

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.