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1.  Influence of wet distillers grains diets on beef cattle fecal bacterial community structure 
BMC Microbiology  2012;12:25.
The high demand for ethanol in the U.S. has generated large stocks of wet distillers grains (DG), a byproduct from the manufacture of ethanol from corn and sorghum grains. Little is known, however, about the potential influence of dietary DG on fecal microbial community structure. A better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. Five diets consisting of a traditional diet fed to finishing beef cattle in the Southern High Plains of Texas-CON (steam-flaked corn control with 0% DG), and four concentrations of DG in the dietary dry matter; 10 C (10% corn-based DG), 5S (5% sorghum-based DG), 10S (10% sorghum DG), and 15S (15% sorghum DG) were fed to steers at the Texas Tech University Burnett Animal Center. Diets were essentially isonitrogenous with a formulated crude protein value of 13.5%.
Fecal grab samples were obtained from 20 steers (n = 4 per diet) and the barcoded DNA pyrosequencing method was used to generate 127,530 16S operational taxonomic units (OTUs). A total of 24 phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla (core set) were observed in all animals regardless of dietary treatment. The average abundance and range of abundance, respectively of the core phyla were as follows: Firmicutes (61%, 19 to 83%), Bacteroidetes (28%, 11 to 63%), Proteobacteria (3%, 0.34 to 17.5%), Tenericutes (0.15%, 0.0 to 0.35%), Nitrospirae (0.11%, 0.03 to 0.22%), and Fusobacteria (0.086%, 0.017 to 0.38%). Feeding DG-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional CON. Four low abundance phyla significantly responded to dietary treatments: Synergistetes (p = 0.01), WS3 (p = 0.054), Actinobacteria (p = 0.06), and Spirochaetes (p = 0.06).
This is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet DG. Comparison of our results with other cattle DNA sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. These three phyla, in order of relative abundance are; Firmicutes, Bacteroidetes, and Proteobacteria. The presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others.
PMCID: PMC3305651  PMID: 22364310
2.  Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing 
BMC Microbiology  2011;11:6.
Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.
Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus.
This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.
PMCID: PMC3025832  PMID: 21211038
3.  Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches 
BMC Microbiology  2009;9:226.
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.
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, Staphylococcus, Corynebacterium, and Serratia. 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.
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.
PMCID: PMC2773781  PMID: 19860898
4.  The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing 
BMC Microbiology  2009;9:210.
Recent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing.
Pyrosequencing revealed a previously unrecognized species richness in the canine small intestine. Ten bacterial phyla were identified. Microbial populations were phylogenetically more similar during tylosin treatment. However, a remarkable inter-individual response was observed for specific taxa. Fusobacteria, Bacteroidales, and Moraxella tended to decrease. The proportions of Enterococcus-like organisms, Pasteurella spp., and Dietzia spp. increased significantly during tylosin administration (p < 0.05). The proportion of Escherichia coli-like organisms increased by day 28 (p = 0.04). These changes were not accompanied by any obvious clinical effects. On day 28, the phylogenetic composition of the microbiota was similar to day 0 in only 2 of 5 dogs. Bacterial diversity resembled the pre-treatment state in 3 of 5 dogs. Several bacterial taxa such as Spirochaetes, Streptomycetaceae, and Prevotellaceae failed to recover at day 28 (p < 0.05). Several bacterial groups considered to be sensitive to tylosin increased in their proportions.
Tylosin may lead to prolonged effects on the composition and diversity of jejunal microbiota. However, these changes were not associated with any short-term clinical signs of gastrointestinal disease in healthy dogs. Our results illustrate the complexity of the intestinal microbiota and the challenges associated with evaluating the effect of antibiotic administration on the various bacterial groups and their potential interactions.
PMCID: PMC2759960  PMID: 19799792
5.  Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) 
BMC Microbiology  2008;8:125.
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.
Ubiquitous 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).
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.
PMCID: PMC2515157  PMID: 18652685
6.  Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing 
BMC Microbiology  2008;8:43.
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.
PMCID: PMC2289825  PMID: 18325110
7.  Microarray based comparison of two Escherichia coli O157:H7 lineages 
BMC Microbiology  2006;6:30.
Previous research has identified the potential for the existence of two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to cluster primarily within one of these two lineages. To determine if there are virulence related genes differentially expressed between the two lineages we chose to utilize microarray technology to perform an initial screening.
Using a 610 gene microarray, designed against the E. coli O157 EDL 933 transcriptome, targeting primarily virulence systems, we chose 3 representative Lineage I isolates (LI groups mostly clinical isolates) and 3 representative Lineage II isolates (LII groups mostly bovine isolates). Using standard dye swap experimental designs, statistically different expression (P < 0.05) of 73 genes between the two lineages was revealed. Result highlights indicate that under in vitro anaerobic growth conditions, there is up-regulation of stx2b, ureD, curli (csgAFEG), and stress related genes (hslJ, cspG, ibpB, ibpA) in Lineage I, which may contribute to enhanced virulence or transmission potential. Lineage II exhibits significant up-regulation of type III secretion apparatus, LPS, and flagella related transcripts.
These results give insight into comparative regulation of virulence genes as well as providing directions for future research. Ultimately, evaluating the expression of key virulence factors among different E. coli O157 isolates has inherent value and the interpretation of such expression data will continue to evolve as our understanding of virulence, pathogenesis and transmission improves.
PMCID: PMC1431545  PMID: 16539702

Results 1-7 (7)