The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Evolution of Protein Molecules

Background: Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control Methods and Findings: The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2 The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N=20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P=003) Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C coccoides-E rectale group correlated positively and significantly with plasma glucose concentration (P=004) but not with BMIs Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P=002) and positively correlated with plasma glucose (P=004) Conclusions: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota

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Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults

The skin is the human body's largest organ, colonized by a diverse milieu of microorganisms, most of which are harmless or even beneficial to their host. Colonization is driven by the ecology of the skin surface, which is highly variable depending on topographical location, endogenous host factors and exogenous environmental factors. The cutaneous innate and adaptive immune responses can modulate the skin microbiota, but the microbiota also functions in educating the immune system. The development of molecular methods to identify microorganisms has led to an emerging view of the resident skin bacteria as highly diverse and variable. An enhanced understanding of the skin microbiome is necessary to gain insight into microbial involvement in human skin disorders and to enable novel promicrobial and antimicrobial therapeutic approaches for their treatment.

The skin microbiome

The cellular and molecular mechanisms underpinning tissue repair and its failure to heal are still poorly understood, and current therapies are limited. Poor wound healing after trauma, surgery, acute illness, or chronic disease conditions affects millions of people worldwide each year and is the consequence of poorly regulated elements of the healthy tissue repair response, including inflammation, angiogenesis, matrix deposition, and cell recruitment. Failure of one or several of these cellular processes is generally linked to an underlying clinical condition, such as vascular disease, diabetes, or aging, which are all frequently associated with healing pathologies. The search for clinical strategies that might improve the body’s natural repair mechanisms will need to be based on a thorough understanding of the basic biology of repair and regeneration. In this review, we highlight emerging concepts in tissue regeneration and repair, and provide some perspectives on how to translate current knowledge into viable clinical approaches for treating patients with wound-healing pathologies.

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Wound repair and regeneration: Mechanisms, signaling, and translation

Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P). There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community compositions of chronic wound pathogenic biofilms and other medical biofilm infections.

/pdf/survey-of-bacterial-diversity-in-chronic-wounds-using-4r1b1c70h8.pdf

Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

Background: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. Methods and Findings: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. Conclusions: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to highresolution molecular diagnostic methods such as bTEFAP.

/pdf/polymicrobial-nature-of-chronic-diabetic-foot-ulcer-biofilm-3xej4ptxqy.pdf

Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

Objective: This phase 1 trial set out to examine the safety of a bacteriophage-based preparation for difficult-to-treat wounds. Method: The intention-to-treat sample comprised 42 patients with chronic venous leg ulcers (VLUs); 39 patients completed the trial. The ulcers were treated for 12 weeks with either a saline control or bacteriophages targeted against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Follow-up continued until week 24. Results: No adverse events were attributed to the study product. No signifi cant difference (p>0.05) was determined between the test and control groups for frequency of adverse events, rate of healing, or frequency of healing. Conclusion: This study found no safety concerns with the bacteriophage treatment. Efficacy of the preparation will need to be evaluated in a phase II efficacy study. Declaration of interest: One of the authors (AS) holds an equity interest in Intralytix. The other authors do not have any interest in commercial activities.

Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial.

In contrast to the commonly accepted hypothesis of host-centred pathology, it is possible that surface bacteria, not host dysfunction, cause the chronicity and perpetual inflammation associated with chronic non-healing wounds.

Biofilms and chronic wound inflammation.

There is a growing recognition that biofilms are the principal cause of wound chronicity. The development of treatments for wound biofilms raises the prospect that chronic wounds can be treated, potentially saving many patients' lives.

Daniel D. Rhoads

Papers

Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial.

Biofilms and chronic wound inflammation.

Chronic wounds and the medical biofilm paradigm.