The bodies of most animals are populated by highly complex and genetically diverse communities of microorganisms. The majority of these microbes reside within the intestines in largely stable but dynamically interactive climax communities that positively interact with their host. Studies from this laboratory have shown that stressor exposure impacts the stability of the microbiota and leads to bacterial translocation. The biological importance of these alterations, however, is not well understood. To determine whether the microbiome contributes to stressor-induced immunoenhancement, mice were exposed to a social stressor called social disruption (SDR), that increases circulating cytokines and primes the innate immune system for enhanced reactivity. Bacterial populations in the cecum were characterized using bacterial tag-encoded FLX amplicon pyrosequencing. Stressor exposure significantly changed the community structure of the microbiota, particularly when the microbiota were assessed immediately after stressor exposure. Most notably, stressor exposure decreased the relative abundance of bacteria in the genus Bacteroides, while increasing the relative abundance of bacteria in the genus Clostridium. The stressor also increased circulating levels of IL-6 and MCP-1, which were significantly correlated with stressor-induced changes to three bacterial genera (i.e., Coprococcus, Pseudobutyrivibrio, and Dorea). In follow up experiments, mice were treated with an antibiotic cocktail to determine whether reducing the microbiota would abrogate the stressor-induced increases in circulating cytokines. Exposure to SDR failed to increase IL-6 and MCP-1 in the antibiotic treated mice. These data show that exposure to SDR significantly affects bacterial populations in the intestines, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. and remarkably also suggest that the microbiota are necessary for stressor-induced increases in circulating cytokines. NIH Public Access

BackgroundThe 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.ResultsUbiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae).ConclusionUsing 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.

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Background: 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).

BackgroundThe advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications.ResultsWe herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample.ConclusionsLaboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-016-0103-7) contains supplementary material, which is available to authorized users.

BackgroundDiabetic 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 FindingsUsing 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.ConclusionsIn 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 high-resolution molecular diagnostic methods such as bTEFAP.

Within days after birth, rapid surface colonization of infant skin coincides with significant functional changes. Gradual maturation of skin function, structure, and composition continues throughout the first years of life. Recent reports have revealed topographical and temporal variations in the adult skin microbiome. Here we address the question of how the human skin microbiome develops early in life. We show that the composition of cutaneous microbial communities evolves over the first year of life, showing increasing diversity with age. Although early colonization is dominated by Staphylococci, their significant decline contributes to increased population evenness by the end of the first year. Similar to what has been shown in adults, the composition of infant skin microflora appears to be site specific. In contrast to adults, we find that Firmicutes predominate on infant skin. Timely and proper establishment of healthy skin microbiome during this early period might have a pivotal role in denying access to potentially infectious microbes and could affect microbiome composition and stability extending into adulthood. Bacterial communities contribute to the establishment of cutaneous homeostasis and modulate inflammatory responses. Early microbial colonization is therefore expected to critically affect the development of the skin immune function.

This study evaluated the fecal microbiota of 12 healthy pet dogs and 12 pet cats using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing. A total of 120,406 pyrosequencing reads for bacteria (mean 5017) and 5359 sequences (one pool each for dogs and cats) for fungi were analyzed. Additionally, group-specific 16S rRNA gene clone libraries for Bifidobacterium spp. and lactic acid-producing bacteria (LAB) were constructed. The most abundant bacterial phylum was Firmicutes, followed by Bacteroidetes in dogs and Actinobacteria in cats. The most prevalent bacterial class in dogs and cats was Clostridia, dominated by the genera Clostridium (clusters XIVa and XI) and Ruminococcus. At the genus level, 85 operational taxonomic units (OTUs) were identified in dogs and 113 OTUs in cats. Seventeen LAB and eight Bifidobacterium spp. were detected in canine feces. Ascomycota was the only fungal phylum detected in cats, while Ascomycota, Basidiomycota, Glomeromycota, and Zygomycota were identified in dogs. Nacaseomyces was the most abundant fungal genus in dogs; Saccharomyces and Aspergillus were predominant in cats. At the genus level, 33 different fungal OTUs were observed in dogs and 17 OTUs in cats. In conclusion, this study revealed a highly diverse bacterial and fungal microbiota in canine and feline feces.