Bacteriology Commons^™

3d Printing Of Human Microbiome Constituents To Understand Spatial Relationships And Shape Parameters In Bacteriology, Jacques Izard, Teklu Kuru Gerbaba, Shara R. P. Yumul

Department of Food Science and Technology: Faculty Publications

Effective laboratory and classroom demonstration of microbiome size and shape, diversity, and ecological relationships is hampered by a lack of high-resolution, easy-to-use, readily accessible physical or digital models for use in teaching. Three-dimensional (3D) representations are, overall, more effective in communicating visuospatial information, allowing for a better understanding of concepts not directly observable with the unaided eye. Published morphology descriptions and microscopy images were used as the basis for designing 3D digital models, scaled at 20,000×, using computer-aided design software (CAD) and generating printed models of bacteria on mass-market 3D printers. Sixteen models are presented, including rod-shaped, spiral, flask-like, vibroid, …

Comparisons Of Oral, Intestinal, And Pancreatic Bacterial Microbiomes In Patients With Pancreatic Cancer And Other Gastrointestinal Diseases, Mei Chung, Naisi Zhao, Richard Meier, Devin C. Koestler, Guojun Wu, Erika De Castillo, Bruce J. Paster, Kevin Charpentier, Jacques Izard, Karl T. Kelsey, Dominique S. Michaud

Department of Food Science and Technology: Faculty Publications

Background: Oral microbiota is believed to play important roles in systemic diseases, including cancer. Methods: We collected oral samples (tongue, buccal, supragingival, and saliva) and pancreatic tissue or intestinal samples from 52 subjects, and characterized 16S rRNA genes using high-throughput DNA sequencing.

Results: Bray–Curtis plot showed clear separations between bacterial communities in the oral cavity and those in intestinal and pancreatic tissue samples. PERMANOVA tests indicated that bacterial communities from buccal samples were similar to supragingival and saliva samples, and pancreatic duct samples were similar to pancreatic tumor samples, but all other samples were significantly different from each …

Mucosa-Associated Microbiota In Barrett’S Esophagus, Dysplasia, And Esophageal Adenocarcinoma Differ Similarly Compared With Healthy Controls, Shajan Peter, Amanda Pendergraft,, William Vanderpol, Mel Wilcox, Kondal R. Kyanam Kabir Baig, Casey Morrow, Jacques Izard, Peter J. Mannon

Department of Food Science and Technology: Faculty Publications

Introduction: Alterations in the composition of the human gut microbiome and its metabolites have been linked to gut epithelial neoplasia. We hypothesized that differences in mucosa-adherent Barrett’s microbiota could link to risk factors, providing risk of progression to neoplasia.

Methods: Paired biopsies from both diseased and nonaffected esophagus (as well as gastric cardia and gastric juice for comparison) from patients with intestinal metaplasia (n = 10), low grade dysplasia (n = 10), high grade dysplasia (n = 10), esophageal adenocarcinoma (n = 12), and controls (n = 10) were processed for mucosa-associated bacteria …

Metagenomics For Bacteriology, Erika Del Castillo, Jacques Izard

Department of Food Science and Technology: Faculty Publications

The study of bacteria, or bacteriology, has gone through transformative waves since its inception in the 1600s. It all started by the visualization of bacteria using light microscopy by Antonie van Leeuwenhoek, when he first described “animalcules.” Direct cellular observation then evolved into utilizing different wavelengths on novel platforms such as electron, fluorescence, and even near-infrared microscopy. Understanding the link between microbes and disease (pathogenicity) began with the ability to isolate and cultivate organisms through aseptic methodologies starting in the 1700s. These techniques became more prevalent in the following centuries with the work of famous scientists such as Louis Pasteur …

A Prospective Study Of Periodontal Disease And Risk Of Gastric And Duodenal Ulcer In Male Health Professionals, Matthew R. Boylan, Hamed Khalili, Edward S. Huang, Dominique S. Michaud, Jacques Izard, Kaumudi J. Joshipura, Andrew T. Chan

Department of Food Science and Technology: Faculty Publications

Periodontal disease is characterized by microbial shifts within the oral cavity and chronic inflammation of the gingival tissue, and can lead to tooth loss. Several studies support an association between periodontal disease and risk of chronic systemic conditions, including vascular disease, chronic obstructive pulmonary disease, diabetes mellitus, and cancer, including gastric adenocarcinoma. These associations may be mediated by systemic inflammation, the immune response to periodontal infection, or direct invasion by pathogens. Previous studies of periodontal disease and risk of peptic ulcer have been limited by cross-sectional design, diagnoses that were not confirmed by medical record review, and a lack of …

Neisseria Oralis Sp. Nov., Isolated From Healthy Gingival Plaque And Clinical Samples, William J. Wolfgang, Teresa V. Passaretti, Reashma Jose, Jocelyn Cole, An Coorevits, Andrea N. Carpenter, Sherly Jose, Anita Van Landschoot, Jacques Izard, Donna J. Kohlerschmidt, Peter Vandamme, Floyd E. Dewhirst, Mark A. Fisher, Kimberlee A. Musser

Department of Food Science and Technology: Faculty Publications

A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida, and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7–100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA–DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbor, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the …

Composition Of The Adult Digestive Tract Bacterial Microbiome Based On Seven Mouth Surfaces, Tonsils, Throat And Stool Samples, Nicola Segata, Susan Kinder Haake, Peter Mannon, Katherine P. Lemon, Levi Waldron, Dirk Gevers, Curtis Huttenhower, Jacques Izard

Department of Food Science and Technology: Faculty Publications

Background: To understand the relationship between our bacterial microbiome and health, it is essential to define the microbiome in the absence of disease. The digestive tract includes diverse habitats and hosts the human body’s greatest bacterial density. We describe the bacterial community composition of ten digestive tract sites from more than 200 normal adults enrolled in the Human Microbiome Project, and metagenomically determined metabolic potentials of four representative sites.

Results: The microbiota of these diverse habitats formed four groups based on similar community compositions: buccal mucosa, keratinized gingiva, hard palate; saliva, tongue, tonsils, throat; sub- and supra-gingival plaques; …

Efficient And Robust Rna-Seq Process For Cultured Bacteria And Complex Community Transcriptomes, Georgia Giannoukos, Dawn M. Ciulla, Katherine Huang, Brian J. Haas, Jacques Izard, Joshua Z. Levin, Jonathan Livny, Ashlee M. Earl, Dirk Gevers, Doyle V. Ward, Chad Nusbaum, Bruce W. Birren, Andreas Gnirke

Department of Food Science and Technology: Faculty Publications

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.

The Human Oral Microbiome, Floyd E. Dewhirst, Tuste Chen, Jacques Izard, Bruce J. Paster, Anne C. R. Tanner, Wen-Han Yu, Abirami Lakshmanan, William G. Wade

Department of Food Science and Technology: Faculty Publications

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome comprises over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene …

Killing Of Treponema Denticola By Mouse Peritoneal Macrophages, P. Gaibani, C. Vocale, S. Ambretti, F. Cavrini, Jacques Izard, L. Miragliotta, M. T. Pellegrino, V. Sambri

Department of Food Science and Technology: Faculty Publications

Treponema denticola has been identified as an important cause of periodontal disease and hypothesized to be involved in extra-oral infections. The objective of this study was to investigate the role of T. denticola cell length and motility during mouse peritoneal macrophages in vitro uptake. Macrophages, incubated under aerobic and anaerobic conditions, produced a similar amount of TNF-α when stimulated with Escherichia coli LPS. The uptake of FlgE- and CfpA-deficient mutants of T. denticola was significantly increased compared with the wild-type strain, due to cell size or lack of motility. Opsonization with specific antibodies considerably improved the treponemes’ uptake. …

Native Cellular Architecture Of Treponema Denticola Revealed By Cryo-Electron Tomography, Jacques Izard, Chyong-Ere Hsieh, Ronald J. Limberger, Carmen A. Mannella, Michael Marko

Department of Food Science and Technology: Faculty Publications

Using cryo-electron tomography, we are developing a refined description of native cellular structures in the pathogenic spirochete Treponema denticola. Tightly organized bundles of periplasmic flagella were readily observed in intact plunge-frozen cells. The periplasmic space was measured in both wild-type and aflagellate strains, and found to widen by less than the diameter of flagella when the latter are present. This suggests that a structural change occurs in the peptidoglycan layer to accommodate the presence of the flagella. In dividing cells, the flagellar filaments were found to bridge the cytoplasmic cylinder constriction site. Cytoplasmic filaments, adjacent to the inner membrane, …

Pyramidobacter Piscolens Gen. Nov., Sp. Nov., A Member Of The Phylum ‘Synergistetes’ Isolated From The Human Oral Cavity, Julia Downes, Sonia R. Vartoukian, Floyd E. Dewhirst, Jacques Izard, Tsute Chen, Wen-Han Yu, Iain C. Sutcliffe, William G. Wade

Department of Food Science and Technology: Faculty Publications

Four strains of anaerobic, gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA–DNA reassociation data revealed that the strains constituted a novel group within the phylum ‘Synergistetes’ and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455T and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus …

Cryo-Electron Tomography Elucidates The Molecular Architecture Of Treponema Pallidum, The Syphilis Spirochete, Jacques Izard, Christian Renken, Chyong-Ere Hsieh, Daniel C. Desrosiers, Star Dunham-Ems, Carson La Vake, Linda L. Gebhardt, Ronald J. Limberger, David L. Cox, Michael Marko, Justin D. Radolf

Department of Food Science and Technology: Faculty Publications

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure’s fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly …

Periplasm Organization In Treponema Denticola As Studied By Cryo-Electron Tomography, Jacques Izard, C.-E. Hsieh, C. A. Mannella, Ronald J. Limberger, M. Marko

Department of Food Science and Technology: Faculty Publications

As a spirochete, the genus Treponema is one of the few major bacterial groups whose natural phylogenic relationships are evident at the level of gross phenotypic characteristics such as their morphology. Treponema spp. are highly invasive due to their unique motility in dense media, and their ability to penetrate cell layers. This feature is associated with the helical cell body and the presence of flagellar filaments in the periplasm. Treponema denticola is an oral pathogen involved in endodontic infections and periodontal diseases. The presence and quantity of T. denticola in the subgingival biofilm is correlated with the severity of periodontal …

Cytoplasmic Filament-Deficient Mutant Of Treponema Denticola Has Pleiotropic Defects, Jacques Izard, William A. Samsonoff, Ronald J. Limberger

Department of Food Science and Technology: Faculty Publications

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further …

Genetic And Structural Analyses Of Cytoplasmic Filaments Of Wild-Type Treponema Phagedenis And A Flagellar Filament-Deficient Mutant, Jacques Izard, William A. Samsonoff, Mary Beth Kinoshita, Ronald J. Limberger

Department of Food Science and Technology: Faculty Publications

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments, cfpA, was identified in various treponemal species. Treponema phagedenis cfpA was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from T. denticola, T. vincentii, and T. pallidum subsp. pertenue. Wild-type T. phagedenis and a …

Insertional Inactivation Of Treponema Denticola Tap1 Results In A Nonmotile Mutant With Elongated Flagellar Hooks, Ronald J. Limberger, Linda L. Slivienski, Jacques Izard, William A. Samsonoff

Department of Food Science and Technology: Faculty Publications

The treponemal fla operon is comprised of numerous motility-related genes; however, the initial gene of this operon, tap1, has no known function. A recently developed system to generate specific mutants in Treponema denticola was utilized to determine if Tap1 was essential for motility. T. denticola tap1 and flanking DNA were identified, cloned, and sequenced, and a suicide plasmid that contained tap1 interrupted with an erythromycin resistance cassette (ermF and ermAM) was constructed. Because of potential polar effects from this cassette, a second plasmid that contained tap1 interrupted with a modified erythromycin resistance cassette that lacked the putative ermF transcription …

Membrane Topology Of The Colicin A Pore-Forming Domain Analyzed By Disulfide Bond Engineering, Denis Duche, Jacques Izard, Juan M. Gonzalez-Manas, Michael W. Parker, Marcel Crest, Daniel Baty

Department of Food Science and Technology: Faculty Publications

Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected a-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the poreforming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that …