Browsing by Subject "bacteria"
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Item ELUCIDATING THE DIVERSITY OF MICROBIAL RETTING COMMUNITIES ON HIBISCUS CANNABINUS USING NEXT-GENERATION SEQUENCING(2014-03) Visi, David K.; Allen, Michael S.Purpose (a): Global environmental concerns have led to a growing interest in renewable resources such as plant-based fibers. Beyond textiles and cordage, plant fibers have the potential for incorporation into renewable, bio-based composite materials for the building and manufacturing sectors. Successful commercialization of fiber production requires optimization of fiber extraction. Retting is the traditional method of fiber extraction, whereby endogenous microorganisms break down heteropolysaccharides to release fiber bundles. Previous studies have analyzed the retting solution for its bacterial constituents, but none have followed changes in the microbial community through the retting process. This research aims to track the bacterial components of the retting community through time, and determine the effects of bacterial augmentation with isolated pectinolytic bacteria using next generation sequencing of 16S rRNA gene amplicons. Methods (b): Batch C1 included plant material and an inoculum of pond water represented a “traditional” retting environment, while C2 contained autoclaved pond water to represent the endogenous microorganisms associated with the plant material. Experimental batch E1 included the addition of three pectinolytic bacterial isolates: Bacillus DP1, Paenibacillus DP2, and Bacillus K1. Total DNA was extracted from the surface-adhering biofilm bacteria and used for PCR with full-length 16S primers 27F and 1492R. Amplification products were used as templates for a nested PCR with primers 786F and 939R targeting variable region 5 of the 16S molecule. The nested 16S rRNA amplicons were then sequenced on the next-generation Ion Torrent PGM platform. Results (c): The E1 environment showed a marked increase in phylum Firmicutes (55 to 94%), while phylum Proteobacteria showed a progressive decrease from Day 1 to 4 (36% to 5%). This was correlated with easy separation of fibers by mechanical movement. At a finer taxonomic level, E1 showed a rapid loss of inoculated Bacillus species DP1 and K1, and a more gradual loss of the family Paenibacillaceae 1 (P. DP2) as the time course progressed. In contrast, C1 was co-dominated by phyla Firmicutes and Proteobacteria, while C2 was composed in large part by the phylum Bacteroidetes. Additionally, comparison of the microbial communities under the different conditions revealed differences in diversity and composition at day 4 time points between the three conditions. Conclusions (d): Introduction of pectinolytic bacteria into the batch reactions increased production rate and increased fiber quality. Introduction of Paenibacillus DP2 is likely the driving force behind the community shifts detected in E1, which warrants further study to determine the mechanism of action. The findings confirmed previous studies that suggest a gradual replacement of aerobic organisms to an environment that is dominated by strict anaerobes. Moreover, the efforts shed light on conditions and mechanisms for the manipulation of microbiomes. These approaches may have relevance to the treatment of dysbiosis in the gut flora in humans and the treatment of related diseases.Item EXTRACELLULAR SUPEROXIDE DISMUTASE MODULATES THE ADAPTIVE IMMUNE RESPONSE DURING SECONDARY INTRACELLULAR BACTERIAL INFECTION(2013-04-12) Witter, AlexandraPurpose: Listeria monocytogenes (LM) is an intracellular foodborne pathogen that causes severe disease in immunocompromised individuals, spontaneous abortion in pregnant women, and results in ~25% mortality rate in infected individuals, making it one of the leading causes of death from foodborne infection. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. While it has been shown that ecSOD decreases innate immune responses during LM infection, its role in a secondary infection model has not been explored. Methods: Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background, were infected with LM/OVA ΔActA and after 40 days challenged with LM/OVA. Colony forming units (CFUs) were counted to determine bacterial burden in the spleen and liver. Percentages of different cell types were determined by flow cytometry. IFN-𝛾 production and concentrations were determined by flow cytometry and ELISA respectively. Results: Our results indicate that ecSOD is protective in a secondary infection since ecSOD HI mice are better able to control bacterial burden than ecSOD KO mice with the ecSOD WT mice showing intermediate CFUs. The ecSOD KO mice exhibit a significantly lower percentage of dendritic cells and corresponding decreases in percentages of both overall CD8 T cells as well as memory CD8 T cells. There was also a decrease in percentages of overall CD4 T cells and memory CD4 T cells, primarily in the spleen. Additionally, there was a significant decrease in CD8 T cell specific IFN-𝛾 production in the spleen after overnight culture with both specific and non-specific stimulation. Conclusions: Our data indicate that ecSOD plays an important role in modulating cell-mediated adaptive immune responses during secondary intracellular bacterial infection. The combination of decreased dendritic cell and CD8 T cell populations, as well as decreased ability of CD8 T cells to produce IFN-𝛾, in ecSOD KO mice suggest that ecSOD may play a role in facilitating the activation of CD8 T cells and their ability to effectively respond during secondary LM infection.Item Genetic profiling of skin microbiomes for forensic human identification(2017-12-01) Schmedes, Sarah E.; Budowle, Bruce; Ge, Jianye; He, Johnny J.The field of microbial forensics has expanded from a focus in biodefense and biocrime attribution to include various metagenomics and microbiome applications made possible by advancements in sequencing and bioinformatics technologies. Recent developments in metagenomics and microbiome research with application to the forensic sciences, include post-mortem interval, body fluid identification, recent geolocation, and human identification. The primary goal of the dissertation described herein was to assess the feasibility of human identification from skin microbiomes using both shotgun metagenomic sequencing and targeted enrichment strategies. The main studies of this dissertation were conducted under the hypothesis that genes from stable, universal microbial species from the core skin microbiome can differentiate skin microbiomes of individuals and be applied towards forensic human identification purposes. The initial study presented describes the development of a tool, AutoCurE, used to identify errors in bacterial genome metadata from public databases and curate the data for subsequent use in comparative genomic studies. This study highlights the types of inconsistencies and errors which may be present in public genome databases and describes the development of a curated local bacterial database for use in subsequent studies. This doctoral research herein presents the development of a novel approach for human identification using stable, universal clade-specific markers from skin microbiomes. Initially, publically available shotgun metagenomic datasets generated from skin microbiome samples collected from 17 body sites from 12 individuals, sampled over three time points over the course of ~3-year period, were mined to identify stable, universal microbial markers. Supervised learning, specifically regularized multinomial logistic regression and 1-nearest-neighbor classification, were performed using the nucleotide diversities of clade-specific markers to predict the correct classification of skin microbiomes to their respective host individuals. Reduced subsets of markers were developed into a novel targeted metagenomics sequencing panel, the hidSkinPlex, to generate individual-specific skin microbiome profiles to use for human identification. Finally, the hidSkinPlex was evaluated on skin microbiome samples collected from eight individuals and three body sites, in triplicate, to demonstrate a proof-of-concept to differentiate individuals with high accuracy. The hidSkinPlex, comprised of 282 bacterial and 4 phage markers from 22 family-, genus-, species-, and subspecies-level clades, was used to correctly identify skin microbiomes from their respective donors with up to 92%, 96%, and 100% accuracy using samples from the foot, manubrium, and hand, respectively. Additionally, skin microbiomes were classified with up to 97% accuracy when the body site was unknown, and body site origin could be predicted with up to 86% accuracy. The hidSkinPlex is the first targeted metagenomics sequencing panel and method designed specifically for skin microbiomes with the intent of forensic human identification applicationsItem Improving Human Identification Using the Human Skin Microbiome(2021-12) Sherier, Allison J.; Budowle, Bruce; Leudtke, Robert; Phillips, Nicole R.There are times when biological evidence has too low of quality or quantity of human DNA to provide enough information for human identification (HID). However, nucleic acids from the human skin microbiome are sources of genetic material that may be useful for HID. The studies in this dissertation test the hypothesis that specific single nucleotide polymorphisms (SNPs) of selected human skin microorganisms can be used to attribute an unknown microbiome sample to an individual. The first study investigated how Wright's fixation index (FST) can be used to select potentially informative SNPs for HID. SNPs with high estimated FST were ascertained in three different ways to examine three distinct hypotheses. The hypotheses focused on testing whether a high FST, increased taxonomic abundance, and/or using a predetermined panel would be the most effective for HID. Classification accuracies ranged from 88 – 95%, and the method using the most taxa possible performed the best. Results from the study support that using genetic distance to select informative markers from the human skin microbiome for HID was viable. The predetermined panel only achieved an 88% accuracy, although it would be the most applicable of the tested method for forensic case work. The second study focused on using FST estimations to select SNPs abundant in 51 individuals sampled at three body sites in triplicate for HID. The most common SNPs (present in ≥ 75% of the samples) which had FST estimates ≥ 0.1 were used with least absolute shrinkage and selection operator (LASSO) to select a list of informative SNPs for HID. The final list (i.e., hidSkinPlex+) contains 365 SNPs and achieved a 95% classification accuracy on 459 samples. The hidSkinPlex+ lays the foundation for a targeted sequencing panel that can be used to further study the stability and specificity of human skin microorganism SNPs for HID applications.