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2015 Posters

Note to authors: Those wishing to present a poster at the conference should register your poster here. A $50 and $25 Amazon gift certificate will be awarded to the top poster and runner up. Tape will be provided to attach your poster to the wall. You may bring any size poster, typical sizes range from 3' x 3' to 4' x 4'. Please contact Derek Wood should you have questions. Thank you for your support of the NW ASM!

Microbial Presence and Organic Acid Content of Commercial Kombucha.
Katherine Leonard ¹, Lev Elson-Schwab², Kaleb Lund², and Rebecca Rashid Achterman².
¹School of Naturopathic Medicine, Bastyr University.
²School of Natural Health Arts and Sciences, Bastyr University.

Although the fermented tea beverage kombucha has recently increased in popularity, the probiotic content and benefits of commercial kombucha have not been rigorously examined. This project aims to identify presence and abundance of predominant probiotic microorganisms and organic acids in four different commercial brands of kombucha. We plated serial dilutions of the kombucha on selective media types to identify concentration of microorganisms including fungi (YEPD), Lactobacilli (MRS), and non-selective medium to promote bacterial growth (LB). Predominant colonies were examined by Gram stain and microscopy to determine (1) whether they were yeast or bacteria, and (2) bacterial cell wall type. HPLC analysis was used to identify the presence and abundance of organic acids, including acetic acid, lactic acid, propionic acid, glucuronic acid, gluconic acid. Prevalent colony types will be examined by sequence analysis to identify the organism at the species level.
Total microbial content varied between brands (175,248,000 CFU/16oz - 4,027,000,000 CFU/16oz) and within the same brand. Microscopy results indicate all brands predominantly contain yeast and one brand contains yeast and Gram-negative and Gram-positive bacteria. Interestingly, the predominant colony type was yeast, even on a medium we believed to be selective for Lactobacilli. HPLC analysis confirmed the presence of organic acids in kombucha, with Townshend’s brand having the highest average total organic acid content and the Revive brand having the lowest. There was high variability in organic acid levels between bottles of the Townshend’s brand. GT’s Enlightened Original brand contained acetic and lactic acid levels higher than claimed on the label whereas Revive brand contained less gluconic acid than claimed on the label. In conclusion, there was high variability of microbial content and organic acid levels in all brands of kombucha tested.
The Brucella Type IV effector BspB targets the Golgi apparatus to inhibit host secretory trafficking. Cheryl Miller, Erin Smith, Jennifer Cundiff, and Jean Celli.
Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University.

The bacterial pathogen Brucella abortus exploits functions of the host secretory pathway to grow within mammalian cells by generating a replicative Brucella Containing Vacuole (rBCV). One bacterial virulence factor necessary for Brucella to establish infection and grow intracellularly is the Type IV secretion system (T4SS), which delivers effector proteins into cells. The effectors are thought to modulate host functions to promote rBCV biogenesis, but their targets and mechanisms of action are unknown. We identified a novel Brucella T4SS effector, BspB, which inhibits host protein secretion, and is required for intracellular growth. We show that BspB interacts with the host Conserved Oligomeric Golgi (COG) complex, a membrane protein apparatus of the secretory pathway that orchestrates the tethering and fusion of intra-Golgi transport vesicles. We hypothesize that BspB exploits the functions of the COG complex to establish the rBCV by altering host secretory pathway functions. Consistently, BspB localizes to the Golgi apparatus when ectopically expressed in HeLa cells. The N-terminal transmembrane domain of BspB is necessary for Golgi localization, interaction with the COG complex, and for inhibiting the secretory pathway. When ectopically expressed, BspB disrupts the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) and induces dispersion of the Trans-Golgi Network (TGN), suggesting that BspB alters several steps in the secretory pathway. During infection, Brucella also disrupts the ERGIC as seen by immunofluorescence microscopy, however the TGN is not affected. These findings characterize the interaction between the Brucella effector protein BspB and its host target the COG complex, which is required for Brucella pathogenesis.
Genome Assembly and Investigation of Agrobacterium F5R19.
Loa Griesbach¹, Katherine Houmiel¹ and Derek Wood^1,2.
¹Department of Biology, Seattle Pacific University.
²Department of Microbiology, University of Washington.

Agrobacterium is a plant pathogen and biotechnology agent with the ability to transfer DNA across Kingdoms. In 2003, Martinez et al. published a study on plant-growth promoting symbiotic bacteria in banana (Musa spp.) in which they suggested that one of these bacteria, F5R19, had the ability to fix nitrogen. Biochemical analysis and next generation 454 sequencing by our group revealed that F5R19 was closely related to Agrobacterium species. Dr. Brad Goodner and Joe Moeller at Hiram College used Agrobacterium tumefaciens C58 as a scaffold to order and assemble the 454 contigs into larger pieces and identified genes that appear to be unique to F5R19. Combining the 326 454 contigs with 186 SMRT unitigs acquired in a recent project, a hybrid assembly of the genome has been created consisting of 14 segments. Currently these pieces are being compared tothe complete genome of Agrobacterium tumefaciens C58 to predict the relationship of these 14 segments. Finishing and gap closure using PCR and Sanger sequencing are in progress. Ultimately, the project seeks to identify the genetic source behind the novel nitrogen fixing phenotype. This search will begin by looking for classic nitrogen fixing genes amongst the novel genes identified by Goodner's lab.
Transmissible resistance to Salmonella infection in Naip5-/- mice.
Chunchi Lu, Xiaodan Zhao, Marvin A. Lai, Américo López-Yglesias, Ellen K. Quarles, Chia-Ta Lo, Kelly D. Smith.
Department of Pathology, University of Washington

Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised and elderly. Salmonella’s flagellin induces an innate immune response that is dependent on an inflammasome composed of Casp1, Nlrc4, and Naip5. We hypothesized that the Naip5 inflammasome was critical for detection of flagellin and protection against mucosal Salmonella infection. We demonstrate that during systemic infection, Salmonella efficiently evades flagellin detection by Naip5. Paradoxically during mucosal infections, Naip5-/- mice were protected from Salmonella infection, which was independent of flagellin expression by Salmonella. This protection against Salmonella infection was not inherited as a traditional Mendelian trait, but transmitted to all progeny regardless of the Naip5 genotype. Gavage of WT C57BL/6 mice with cecal contents from Naip5-/- mice was sufficient to transfer protection. The Naip5-/- colony was noted to harbor Trichomonas, a possible mediator of this phenotype. Treatment of Naip5-/- mice with metronidazole eliminated Trichomonas, but paradoxically augmented protection. The enhanced protection was associated with outgrowth of a commensal Escherichia coli strain. Gavage WT C57BL/6 mice with this strain of E. coli potently protected mice against Salmonella infection. These studies indicate that the protective phenotype noted in Naip5-/- mice can be attributed to a commensal E. coli strain that is capable of transmitting colonization resistance. Further characterization of this phenotype demonstrates that this strain of E. coli is capable of transmitting resistance to TLR5-/-, MyD88-/-, Rag1-/- and germ-free C57BL6 mice. Our studies illuminate the complexity of mucosal infections, where the multitude of interactions between microbiota, pathogens and the innate immune system shape host defenses.