Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches

<p>Abstract</p> <p>Background</p> <p>Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections.</p> <p>Results</p> <p>Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, <it>Staphylococcus</it>, <it>Corynebacterium</it>, and <it>Serratia</it>. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds.</p> <p>Conclusion</p> <p>The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.</p

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

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Results</p> <p>There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including <it>Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia</it>, and <it>Serratia </it>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 <it>Abiotrophia para-adiacens </it>and <it>Rhodopseudomonas </it>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.</p> <p>Conclusion</p> <p>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.</p

A Comparison of Bacterial Composition in Diabetic Ulcers and Contralateral Intact Skin

An extensive portion of the healthcare budget is allocated to chronic human infection. Chronic wounds in particular are a major contributor to this financial burden. Little is known about the types of bacteria which may contribute to the chronicity, biofilm and overall bioburden of the wound itself. In this study we compare the bacteriology of wounds and associated intact skin. Wound and paired intact skin swabs (from a contralateral location) were collected. The bacterial diversity was determined using bacterial Tag-encoded FLX amplicon pyrosequencing (bTEFAP). Diversity analysis showed intact skin to be significantly more diverse than wounds on both the species and genus levels (3% and 5% divergence). Furthermore, wounds show heightened levels of anaerobic bacteria, like Peptoniphilus, Finegoldia, and Anaerococcus, and other detrimental genera such as Corynebacterium and Staphylococcus. Although some of these and other bacterial genera were found to be common between intact skin and wounds, notable opportunistic wound pathogens were found at lower levels in intact skin. Principal Component Analysis demonstrated a clear separability of the two groups. The findings of the study not only greatly support the hypothesis of differing bacterial composition of intact skin and wounds, but also contribute additional insight into the ecology of skin and wound microflora. The increased diversity and lowered levels of opportunistic pathogens found in skin make the system highly distinguishable from wounds

Black Box Chimera Check (B2C2): a Windows-Based Software for Batch Depletion of Chimeras from Bacterial 16S rRNA Gene Datasets

The existing chimera detection programs are not specifically designed for "next generation" sequence data. Technologies like Roche 454 FLX and Titanium have been adapted over the past years especially with the introduction of bacterial tag-encoded FLX/Titanium amplicon pyrosequencing methodologies to produce over one million 250-600 bp 16S rRNA gene reads that need to be depleted of chimeras prior to downstream analysis. Meeting the needs of basic scientists who are venturing into high-throughput microbial diversity studies such as those based upon pyrosequencing and specifically providing a solution for Windows users, the B2C2 software is designed to be able to accept files containing large multi-FASTA formatted sequences and screen for possible chimeras in a high throughput fashion. The graphical user interface (GUI) is also able to batch process multiple files. When compared to popular chimera screening software the B2C2 performed as well or better while dramatically decreasing the amount of time required generating and screening results. Even average computer users are able to interact with the Windows .Net GUI-based application and define the stringency to which the analysis should be done. B2C2 may be downloaded from http://www.researchandtesting.com/B2C2

Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)

<p>Abstract</p> <p>Background</p> <p>The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows.</p> <p>Results</p> <p>Ubiquitous bacteria detected from the cattle feces included <it>Clostridium</it>, <it>Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus</it>, and <it>Acidaminococcus </it>spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for <it>Salmonella </it>spp (tentative <it>enterica</it>) and 6 cows were positive for <it>Campylobacter </it>spp. (tentative <it>lanienae</it>).</p> <p>Conclusion</p> <p>Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.</p

Comparison of Culture and Molecular Identification of Bacteria in Chronic Wounds

Clinical diagnostics of chronic polymicrobial infections, such as those found in chronic wounds, represent a diagnostic challenge for both culture and molecular methods. In the current retrospective study, the results of aerobic bacterial cultures and culture-free bacterial identification using DNA analyses were compared. A total of 168 chronic wounds were studied. The majority of bacteria identified with culture testing were also identified with molecular testing, but the majority of bacteria identified with the molecular testing were not identified with culture testing. Seventeen (17) different bacterial taxa were identified with culture, and 338 different bacterial taxa were identified with molecular testing. This study demonstrates the increased sensitivity that molecular microbial identification can have over culture methodologies, and previous studies suggest that molecular bacterial identification can improve the clinical outcomes of patients with chronic wounds

Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds

Background: Despite a growing consensus that biofilms contribute to a delay in the healing of chronic wounds, conflicting evidence pertaining to their identification and management can lead to uncertainty regarding treatment. This, in part, has been driven by reliance on in vitro data or animal models, which may not directly correlate to clinical evidence on the importance of biofilms. Limited data presented in human studies have further contributed to the uncertainty. Guidelines for care of chronic wounds with a focus on biofilms are needed to help aid the identification and management of biofilms, providing a clinical focus to support clinicians in improving patient care through evidence-based medicine. Methods: A Global Wound Biofilm Expert Panel, comprising 10 clinicians and researchers with expertise in laboratory and clinical aspects of biofilms, was identified and convened. A modified Delphi process, based on published scientific data and expert opinion, was used to develop consensus statements that could help identify and treat biofilms as part of the management of chronic nonhealing wounds. Using an electronic survey, panel members rated their agreement with statements about biofilm identification and treatment, and the management of chronic nonhealing wounds. Final consensus statements were agreed on in a face-to-face meeting. Results: Participants reached consensus on 61 statements in the following topic areas: understanding biofilms and the problems they cause clinicians; current diagnostic options; clinical indicators of biofilms; future options for diagnostic tests; treatment strategies; mechanical debridement; topical antiseptics; screening antibiofilm agents; and levels of evidence when choosing antibiofilm treatments. Conclusion: This consensus document attempts to clarify misunderstandings about the role of biofilms in clinical practice, and provides a basis for clinicians to recognize biofilms in chronic nonhealing wounds and manage patients optimally. A new paradigm for wound care, based on a stepped-down treatment approach, was derived from the consensus statements

Evaluation of the bacterial diversity of Pressure ulcers using bTEFAP pyrosequencing

<p>Abstract</p> <p>Background</p> <p>Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories.</p> <p>Methods</p> <p>Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities.</p> <p>Results</p> <p>Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics.</p> <p>Conclusions</p> <p>Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.</p

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

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. spp. 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 high-resolution molecular diagnostic methods such as bTEFAP