Identification and Characterization of Pb-Resistant Bacterial Strains Isolated from Saint Claire River Sediments

Dominic Azzopardi, Adam Oest, Nichole Bowman, Renee Gismondi, Drashti Patel, Mitchell Fenner, and Sonia M. Tiquia-Arashiro, University of Michigan-Dearborn, Dearborn

Potential use of Dehydrogenase Activity as a Marker of Microbial Activity in Lake St. Clair and St. Clair River Sediments

Mitchell Fenner, Renee Gismondi, Drashti Patel Adam Oest, Nichole Bowman, Dom Azzopardi, and Sonia M. Tiquia-Arashiro, University of Michigan-Dearborn, Dearborn

Temporal and Spatial Analysis of Heterotrophic Diversity in the Sediments of Lake St. Claire and the St. Claire River

Adam Oest, Dom Azzopardi, Nichole Bowman and Sonia M. Tiquia-Arashiro, University of Michigan-Dearborn, Dearborn

The diversity of heterotrophic microorganisms in the sediments of the St. Clair River and Lake St. Clair were determined using Biolog Ecoplates, which test for oxidation of 31 carbon sources. Sediment suspensions (1:10 w/v sediment: saline water ratio) were applied to the Biolog and the color change was measured daily using a microplate reader set at 595 nm. Data was recorded daily until an average well color development (AWCD) closest to 0.75 was achieved. T-test was carried out to compare carbon utilization patterns between lake and river sediments, and ANOVA was used to differentiate utilization patterns among sites (river sites: SC1, SC2, SC3; lake sites: LC1, LC2, LC3) and seasons.  The utilization patterns of carbon substrates by the heterotrophic communities revealed differences between lake and river sediments in spring. Sediments from sites LC1, LC2, LC3, and SC2 utilized less of the carbon sources compared to sites SC1 and SC3, which used nearly all of them.  As time went on this began to change, in summer all samples used almost all of the carbon sources with small discrepancies between them, and this trend continued into the fall, with the only change being LC1 utilizing less carbon sources. The main reason for this change in growth is likely linked to the weather, as increased heat and precipitation likely increased the nutrients and microbial growth within the sediments. This research is important because it provides insight into the basic microbial diversity between the lake and river, and what conditions may influence that.

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Dissecting the Role of RidA in Carbamoyl Phosphate Metabolism

Jenna Olson, Kelsey Hodge-Hanson and Diana Downs, University of Michigan-Dearborn, Dearborn

Resistance to Antibiotic and Lead of Bacteria Isolated from Sediments of St. Clair River

Renee Gismondi, Drashti Patel, Mitchell Fenner and Sonia M. Tiquia-Arashiro, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn

Bacteria are generally the first organisms affected by the increased presence of toxic compounds in the aquatic environments including antibiotics and heavy metals.  The purpose of this study is to determine natural susceptibility levels of bacterial isolates to Pb and antibiotics. Two enrichment experiments (LB and R2A broths) containing 10 g of sediments and increasing concentrations of Pb(NO3)2 were carried out to isolate Pb-resistant strains. Cells that grew on enrichments containing the highest Pb concentrations (1500 mg/L for LB and 1250 mg/L for R2A) were isolated and characterized for their antibiotic resistance using eight different antibiotics.  The results showed that the majority of the LB strains were resistant to chloramphenicol (80% of the isolates), erythromycin (71%), novobiocin (100%), penicillin G (100%) and tetracycline (73%).  For the R2A strains, the majority were resistant to erythromycin (98% of isolates), neomycin (81%), novobiocin (100%), and penicillin G (100%).  However, LB strains showed sensitivity to streptomycin (98% of isolates) while the R2A strains showed sensitivity to both streptomycin (75%) and chloramphenicol (72%). In general, Pb-resistant strains isolated from Saint Clair River sediments showed natural resistance to a variety of antibiotics.  This result suggests that the combined expression of antibiotic resistance and metal tolerance is caused by selection resulting from the metals present in the environment.  Bioremediation requires organisms that are tolerant to an array of toxins.  As such, Pb-resistant strains isolated in this study could be successful candidates for bioremediation of Pb-contaminated sites in a more economical way in comparison to current chemical remediation methods.

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Characterization of the Role of Glycine in Germination of Clostridium difficile Spores

Travis J. Kochan and Philip C. Hanna, University of Michigan, Ann Arbor MI

Siderophore Vaccine Conjugates Protect Against Uropathogenic Escherichia coli Urinary Tract Infection

Laura A. Mike, Sara N. Smith, and Harry L.T. Mobley, University of Michigan Medical School, Ann Arbor, Michigan

Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). While most UTIs are isolated cases, 2.4% of women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. During infection, UPEC must acquire essential metal nutrients such as iron in order to successfully colonize the urinary tract. UPEC expresses iron acquisition systems that include multiple iron, heme, and siderophore receptors. E. coli isolates encode up to four iron-chelating siderophores of which salmochelin, aerobactin and yersiniabactin tend to be associated with pathogenic strainsas these “stealth siderophores” evade the host immune system. Previous work has determined that vaccination with yersiniabactin and aerobactin receptors, but not the salmochelin receptor, protect in a murine model of UTI. Siderophore receptors are integral outer membrane proteins with limited solubility in aqueous solutions. Their cognate siderophores, however, are highly water soluble. Since yersiniabactin and aerobactin receptors restrict UPEC pathogenesis, we hypothesized that the cognate siderophores would also protect against UTI. To test this hypothesis, mice were immunized intranasally with aerobactin and yersiniabactin conjugated to an immunogenic carrier protein. The siderophore-protein conjugates protect against UTI individually, and co-immunization further restricts UPEC pathogenesis. Protection is lost when mice are infected with yersiniabactin and aerobactin null strains. This is the first report of protective vaccination with siderophores. A vaccine specifically targeting stealth siderophores offers an exciting therapeutic strategy that may protect against a broad range of pathogenic bacteria without disrupting commensal bacteria.

These data were presented at the 2015 ASM General Meeting and the 2015 Midwest Microbial Pathogenesis Conference.

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Invasive populations of Medicago polymorpha rely less on co-evolved rhizobia

Katherine J. Wozniak and Maren L. Friesen, Michigan State University

The ecological and evolutionary concepts underlying pathogenesis are common throughout the tree of life and include the effects of mixed culture microbial communities. In the plant kingdom, initial infection occurs in colonization of both rhizobia and plant-specific bacterial pathogens. Colonization of non-co-evolved microbes could hinder overall growth when compared to co-evolved microbes, which is similar to pathogenesis. Medicago polymorpha and its rhizobial symbiont, Ensifer medicae originated in the Mediterranean coast but have evolved to cultivate all continents except for Antarctica. Specialized mutualisms that were heavily relied upon for legume growth are now less utilized in the invasive ranges, suggesting these locations favor more generalist plant genotypes. Evolving a generalist approach in the invasive ranges requires recruitment of bacterial diversity from the rhizosphere, instead of forming a symbiosis with only a few efficient rhizobia. Our project aimed to elucidate the rhizobial dependence of native and invasive Medicago polymorpha genotypes. To do this, we compared legume growth when given a strong N-fixing strain mixed with soils from either native or invasive regions. We were also interested in understanding whether the presence and density of foreign soil microbes affected the mutualistic relationship between the rhizobia and plants. Additionally, native plants that were inoculated formed significantly more rhizobia-containing nodules than inoculated invasive plants in general. Our results support the hypothesis that invasive mutualisms are less specialized compared to native mutualisms and pairing genotypes with non-co-evolved bacteria has a detrimental effect. Investigating plant-bacterial associations in invasive regions could help us understand how plant genotypes are able to colonize and flourish in the invaded range, and could have implications for prevention of plant invasions.

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Protein Modeling and Docking of Predicted AHS/ALS Proteins in Methanocaldococcus jannaschii

Laura K. Harris, Davenport University, Rutgers University

Several bioinformatics studies on Methanocaldococcus jannaschii’s open reading frame (ORF) numbers 0277 and 0063, originally thought to encode large sub-units of acetolactate synthase (ALS) based on BLAST analysis, demonstrate a difference in the sequence patterning between the genes. Experimental study has shown MJ0277 to have ALS function where MJ0663 does not.  Ergo, the aim of this work is to examine why MJ0663 is not functional using GOLD ligand docking software algorithms.  Structure models of MJ0277 and MJ0663 were developed by homology modeling algorithms in SYBYL suite and their affinity for docking known ALS ligands; TPP, FAD, and pyruvate measured.  These models were compared to the affinity that models established from x-ray crystallography structures of ALS proteins have for the same ligands.  Relatedness of proteins involved were confirmed via BLAST sequence identity and phylogenetic tree analyses.  These findings suggest that MJ0277 is phylogenetically related to ALS proteins and has a similar affinity for the same ligands based on Chemscore fitness scores.  While MJ0663 has a similar affinity for ALS ligands, it does not have a similar sequence identity percentage or phylogeny to other AHS/ALS proteins.  Further examination using different algorithms is needed to address why MJ0663 is not functional.

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Genomic and Epidemiological Determinants of Enterobacter cloacae Transmission

Shawn Whitefield, Washer, Laraine,L, Scipone, Christy,A, Dombecki, Carolyn, R, Newton, Duane, W, Snitkin, Evan, S. University of Michigan, Ann Arbor, MI

One in twenty-five patients seeking healthcare acquire an infection that they did not have prior to treatment.  These healthcare associated infections (HAI) are a source of excess healthcare costs and patient morbidity. Carbapenem resistant Enterobacteriaceae (CRE) are an emerging cause of HAI, whose increasing prevalence is concerning because carbapenems are a last line treatment for resistant Gram-negative infections.  Recent reports link healthcare associated CRE outbreaks to contaminated medical equipment.  In early 2015, the infection control team at our hospital identified a cluster of Enterobacter cloacae CRE infections among patients who had undergone endoscopy.  The type of endoscope associated with the affected patients has been linked to CRE outbreaks at other hospitals, raising concerns that contaminated scopes mediated transmission in our hospital.  To understand if endoscopes at our institution were a vehicle for CRE transmission, we undertook a combined epidemiological and whole genome sequencing investigation of all CRE infections identified in our hospital in the last 3 years.  Genomic analysis of isolates from patients undergoing endoscopy provided definitive evidence ruling out endoscope-mediated transmission.  However, we did identify putative transmission clusters among several patients without endoscope exposure, suggesting alternative routes of transmission.  These genetically homogeneous transmission clusters were in stark contrast with the diverse background population of E. cloacae circulating in our hospital.  This motivated us to ask if specific genetic determinants contribute to the preferential dissemination of abundant E. cloacae strains. To test this, we searched for virulence and antibiotic resistance genes common to transmission clusters. By overlaying the presence of these genes onto the phylogeny, we identified a diverse array of resistance mechanisms and virulence factors that are unevenly distributed throughout phylogenetic clusters.  Overall, these results provide a first glimpse into genetic and epidemiological determinants of E. cloacae prevalence in hospitals.

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Enhanced Ability to Survive Phagosomal Stress in Type III Group B Streptococcus Isolates

Michelle L. Korir1, Clare Laut1, Lisa M. Rogers2, David M. Aronoff2, and Shannon D. Manning1, 1Michigan State University, East Lansing, MI 48824; and 2Vanderbilt University, Nashville, TN 37235

Group B Streptococcus (GBS) is commonly found asymptomatically colonizing up to 30% of women and can persistently colonize even after antibiotic treatments. GBS is also a leading cause of neonatal sepsis and meningitis. Previous studies have shown that GBS survives inside macrophages while residing in the phagosome, but the mechanism by which it survives this harsh environment remains unknown. To better understand persistent colonization and intracellular survival, we compared two GBS strains, one representing the hypervirulent lineage sequence type (ST)-17 (serotype III) and the other ST-12 (serotype II), in their ability to persist during antibiotic exposure and survive inside PMA-differentiated THP-1 cells. Additionally, a phagosome-mimicking media was developed using several stressors found in the phagosome to more easily asses the ability of GBS to survive phagosomal stress. The ST-17 strain was more readily phagocytosed and intracellularly survived longer when compared to the ST-12 strain, but the ST-12 strain was tolerant to ampicillin exposure whereas the ST-17 strain was not. Interestingly, exposure to sub-inhibitory concentrations of ampicilin increased the level of phagocytosis of the ST-17 strain, but had no effect on the ST-12 strain. In addition, blocking acidification of the phagosome decreased the survival of the ST-17 strain, but had no affect on the survival of the ST-12 strain. Congruent with the macrophage experiments, the ST-17 strain had a higher survival rate in the phagosome-like media compared to the ST-12 strain. Examining the ability of a number of different GBS isolates in their ability to survive the phagosome-like media showed that serotype III isolates survive significantly better than other serotypes. Taken together, these results indicate that the ST-17 strain persistently colonizes by hiding inside macrophages using a pH dependent mechanism to withstand phagosomal stress and that serotype III GBS strains have an enhanced ability to survive the phagosome relative to other serotypes.

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Investigating Petrobactin Export by Bacillus anthracis

Ada K. Hagan, McColm, Sarah, Carlson Jr., Paul E., Dixon, Shandee, Nunsca, Tyler, and Philip C. Hanna, University of Michigan Medical School, Ann Arbor, MI 48109

Bacillus anthracis, a Gram-positive spore forming bacterium, is the causative agent of anthrax. Anthrax is a highly lethal disease accompanied by growth to high titers in the blood. Iron is an important nutrient required for multiple cellular processes. Accordingly, iron acquisition by bacterial pathogens in the iron-limited host environment is vital to pathogenesis. Siderophores, small molecules with high binding affinities for iron, are secreted into the environment under iron-limited conditions by many pathogens to retrieve iron for growth. B. anthracis has multiple methods to acquire iron but the siderophore petrobactin (PB) is the only system required in murine models of virulence. Much is known about PB biosynthesis and import. However, the protein(s) responsible for PB export are unknown. Putative PB exporters have been identified using a combination of bioinformatics and microarray data from B. anthracis Sterne cultures grown in iron deficient conditions including iron-depleted media (IDM), bovine blood, and macrophages. The putative PB exporters belong to four different transport families: ATP-binding cassette (ABC), major facilitator superfamily (MFS), multidrug and toxic compound extrusion (MATE) and resistance nodulation diffusion (RND). Marker-less deletion mutants lacking each of these genes have been created and screened for reduced growth rates in IDM and reduced PB export into the supernatant. The single deletion mutants screened thus far have not revealed a role for an individual transporter in PB export. Due to the amount of redundancy in the B. anthracis genome the generation of double deletion mutants is in progress. In order to narrow the scope of the study, we sought to determine if ATPases were required for PB export. To test this, two small molecules, gambogic acid and mecloclycine, identified previously in our lab for their ability to inhibit ATPases, were analyzed for their ability to reduce PB export at sub-lethal concentrations. A reduction in supernatant PB levels in the presence of these ATPase inhibitors support the importance of ABC transporters in PB export.

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Binding Affinity of Caldicellulosiruptor bescii Predicted Major Type IV Pili Protein To Model Plant Polysaccharides

Valerie Bell, Catherine Fick, and Sara Blumer-Schuette, Oakland University

Caldicellulosiruptor bescii is an extremely thermophilic bacterium that has the ability to deconstruct high lignin plant biomass by hydrolyzing crystalline cellulose and other plant polysaccharides. Past studies using another cellulolytic thermophile, Clostridium thermocellum, indicated that microbe-substrate proximity is important during cellulose hydrolysis (Lu et al., 2006). Therefore, we are interested in similar mechanisms, namely type IV pili, which are extracellular filaments that mediate adherence to surfaces. Analysis of the genome sequence from C. bescii determined that it encodes for a type IV pilus. Using protein functional domain annotations, we predicted that Athe_1880 from this locus is the major pilin gene. Proteomics analysis determined that Athe_1880 was the fourth highest produced protein during growth on cellulose (Blumer-Schuette et al., 2012), and transcripts were up regulated over 30-fold in a comparison to growth on glucose (Dam et al., 2010). Given this preliminary data, we propose a working hypothesis that type IV pili from C. bescii docontribute to plant biomass deconstruction by attaching to plant biomass. Our initial aim is to determine if Athe_1880 contributes to cell-substrate proximity. To investigate the binding ability of the major pilin, Athe_1880 was cloned and purified using affinity chromatography for qualitative substrate binding assays. We observed that Athe_1880 had an affinity for beech wood xylan, both Sigmacell and Whatman No. 1 filter paper, but not to glass beads or the microcentrifuge tube surface. Overall, more protein appears to adhere to filter paper than to either type of microcrystalline cellulose tested. Further quantitative measures of affinity for filter paper are being estimated using Langmuir isotherms. Additionally, four other predicted pilin genes (Athe_1872, Athe_1876, Athe_1877 and Athe_1881) have also been cloned for determination of their affinity to plant polysaccharides. Our results indicate that Athe_1880 is capable of binding to many plant polysaccharides, not just cellulose suggesting that Type IV pili are playing a role in cell-substrate attachment.

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Testing the Efficacy of Hydrogen Peroxide and Sodium Hypochlorite Based Disinfectants in Killing Clostridium difficile Spores

Tricia J. O’Brien, Leslie, Jhansi L., and Young, Vincent B., University of Michigan, Ann Arbor, MI 48109

Clostridium difficile is a Gram-positive, spore forming bacterium. C.difficile spores are incredibly durable, which is believed to help the transmission of the bacterium. Disinfectants are critically important in preventing the spread of spores in hospitals and laboratories, thus preventing costly C. difficile colitis. Therefore, potential differences in the amount of spores killed and the time needed to kill them are significant when considering which commercially available disinfect is best to use. Two of the three disinfectants, Perisept and Sanotracin, used in this experiment contain the active ingredient hydrogen peroxide, which is thought to alter spore germination. The third disinfectant, bleach, contains sodium hypochlorite, an oxidizing agent. Perisept and Sanotracin claim to take two to three minutes to kill C. difficile spores while bleach instructions advise wait 10 minutes. Therefore, if Sanotracin, bleach, and Perisept are tested for how well they have killed spores within a three-minute period, then Sanotracin and Perisept should work better than bleach. Bleach, Sanotracin, and Perisept were separately mixed with a high concentration of C. difficile strain R20291 spores in eppendorf tubes. After three minutes, these mixtures were diluted and then plated. The plates were incubated overnight and then were observed to see if colonies had grown. To determine if the disinfectants had been properly diluted out of the spores’ tube after the killing period, Whatman paper disks were soaked in the diluted disinfectants and placed on a lawn of C. difficile. There were no C. difficile colonies seen on the bleach, Perisept, or Sanotracin treated plates. No zones of clearing were seen on the Whatman paper plates, which suggest that the disinfectant had been properly diluted after the three-minute killing period. These results suggest that all of the disinfectants mentioned above killed C. difficile strain R20291 spores equally effectively.

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In Search of the Realized Niche for Clostridium difficile During Infection

Matthew L. Jenior, Leslie, Jhansi L., & Schloss, Patrick D., University of Michigan

The Role of Intraspecific Competition and Host Adaptive Immunity in Protection from C. difficile Infection

Jhansi L. Leslie, Jenior, Matthew L., and Young, Vincent B., University of Michigan, Ann Arbor MI 48109

It is clear that disturbances in the intestinal microbiota generally precede susceptibility to Clostridium difficile infection (CDI). A growing body of research has studied how the indigenous microbiota can influence the ability of C. difficile to establish colonization and persist within the gastrointestinal tract. However, disease is a manifestation of the production of the toxins TcdA and TcdB, which has led to the strategy of neutralizing these variables factors to abrogate disease. An alternative experimental approach for dealing with CDI is using non-toxigenic strains of C. difficile as a form of biocontrol. We describe here experiments in a murine model of CDI that examines the relative role of bacterial competition versus development of anti-toxin antibodies in meditating protection from CDI. We find that previous colonization with a less virulent but toxigenic strain of C. difficile provides protection from subsequent infection with a more virulent strain. These data suggest that the development of an adaptive immune response to C. difficile toxins and not bacterial competition between strains is important for mediating protection. While sufficient for protection, repeating these studies with antibody-deficient RAG-/- mice demonstrates that interspecific competition between C. difficile strains can also protect mice from lethal challenge. Thus, stimulation of anti-toxin antibody responses and fostering bacteria-bacteria competition can represent complementary methods for controlling this important nosocomial infection.

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Microbiota-Based Model Complements Fecal Immunochemical Test for Improved Detection of Colonic Lesions

Nielson T. Baxter1, Mack T. Ruffin IV2, Mary A.M. Rogers3, and Patrick D. Schloss1, 1Department of Microbiology and Immunology, 2Department of Family Medicine, 3Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109

Colorectal cancer is the second leading cause of death among cancers in the United States. Although individuals diagnosed early have a greater than 90% chance of survival, more than one-third of individuals do not adhere to screening recommendations partly because the standard diagnostics, colonoscopy and sigmoidoscopy, are expensive and invasive. Thus, there is a great need to improve the sensitivity of non-invasive tests to detect early stage cancers and adenomas. Numerous studies have identified shifts in the composition of the gut microbiota associated with the progression of colorectal cancer, suggesting that the gut microbiota may represent a reservoir of biomarkers that would complement existing non-invasive methods such as the widely used fecal immunochemical test (FIT). We sequenced the 16S rRNA genes from the stool samples of 490 patients and used the relative abundances of the bacterial populations within each sample to develop a cross-validated random forest classification model that detects colonic lesions using the relative abundance of gut microbiota and the concentration of hemoglobin in stool. The microbiota-based random forest model detected 95.0% of cancers and 57.1% of adenomas while FIT alone detected 75.0% and 15.7%, respectively. Of the colonic lesions missed by FIT, the model detected 80.0% of cancers and 49.1% of adenomas. Although we confirmed known assocaitions of Porphyromonas assaccharolytica, Peptostreptococcus stomatis, Parvimonas micra, and Fusobacterium nucleatum with CRC, we found that the loss of potentially beneficial organisms, like Lachnospiraceae, was more predictive for identifying patients with adenomas when used in combination with FIT. These findings demonstrate the potential for microbiota analysis to complement existing screening methods to improve detection of colonic lesions.

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The relationship between biofilm production, persistent colonization, and disease in Streptococcus agalactiae

Robert Parkera, Clare Lauta, Ruth N. Zadoksb,c, H. Dele Daviesd, and Shannon D. Manninga, aDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA; bInstitute for Biodiversity, Animal Health, and Comparative Medicine, University of Glasgow, Glasgow, UK and  cMoredun Research Institute, Penicuik, UK; and the dUniversity of Nebraska Medical School, Omaha, NE, USA.

Group B Streptococcus (GBS) is a leading cause of sepsis and meningitis and an important factor in premature and still births.  Biofilm production has been suggested to be important for GBS pathogenesis alongside many other elements, including phylogenetic lineage, defined by multilocus sequence type (ST), and virulence factors, such as pili and capsule type.  A complete understanding of the confluence of these components, however, is lacking.  To identify associations between biofilm phenotype, pilus profile and lineage, 293 isolates from asymptomatic carriers, invasive disease cases, and bovine mastitis cases, were assessed for biofilm production using an in-vitroassay.  Multilocus sequence type (ST) profile, pilus island profile, and source were all found to be associated with biofilm production, and unexpectedly, isolates from invasive disease cases, and/or belonging to the ST-17 lineage, were significantly more likely to form weak biofilms.  The effect of biofilm variation on host cell adherence was further investigated through an in-vitro assay with 32 strains representing 3 commonly-isolated STs encompassing differential biofilm phenotypes and clinical outcomes. Isolates belonging to more commensal-associated lineages displayed increased attachment to host cells compared to the hypervirulent ST-17 strains. Taken together, these data suggest an evolutionary trade-off between invasiveness and colonizing ability, while also identifying biofilm production as a driver of carriage and heavy colonization, two major determinants of clinical outcome in GBS.

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Individual Capsular Polysaccharides Provide an Advantage In Vivo to the Intestinal Symbiont Bacteroides thetaiotaomicron

Nathan T. Porter1, Peterson, Daniel A.2, and Martens, Eric C.1, 1University of Michigan, Ann Arbor, MI 48109, 2Johns Hopkins University, Baltimore, MD 21205

Loci for the synthesis of capsular polysaccharides (CPS) are replete within the highly abundant, gut-dwelling Bacteroides genus. Though much is known about the role of CPS in bacterial pathogenesis, little is known about their role in more symbiotic relationships. In order to determine the advantages that individual CPS provide in different host environments, we generated a set of 8 B. theta strains that each encode and express one of its CPS.
We first competed these strains in germ free mice of different host immune backgrounds. The cps5 locus-expressing strain (cps5 strain), as well as the cps4 and cps6 strains, outcompeted the other strains regardless of host immune proficiency. However, this occurred more quickly in mice with defects in innate immune signaling (MyD88-/-/Trif-/-), and less quickly in mice with defects in adaptive immunity (Rag1-/-). Interestingly, we recovered intestinal IgA against all of the CPS, even those that had been quickly outcompeted. To determine if dietary alterations would affect fitness of the different strains, we competed the strains in mice fed one of 5 different diets. Host diet did not affect the experimental outcome.
Next, we competed the wild type (WT) strain (which produces multiple CPS) against the single CPS-expressing strains. WT either equaled or outcompeted the single CPS-expressing strains when competed in germ free mice, with a greater advantage in mice proficient in adaptive immunity. Surprisingly, WT expressed all of its cps loci, including those encoded by the most rapidly outcompeted strains. To determine whether the cps5 strain would still compete equally with the WT in the presence of an established immune response, we depleted the strains with antibiotics and allowed them to regrow after cessation of treatment. Though not differentially resistant to the antibiotic, WT outcompeted all other strains after treatment. Our data indicate that specific CPS provide an advantage in vivo, especially in the presence of an adaptive immune response, and that multiple CPS provide an advantage following antibiotic perturbation.
This data was also presented at the Cayman Microbiome Symposium.

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Novel Insights Into the Evolutionary and Functional Implications of Hypervariable Loci Within the Skin Virome

Geoffrey D. Hannigan, Qi Zheng, Jacquelyn S. Meisel, Elizabeth A. Grice, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA,19104, USA

Targeted genomic variability is crucial for the ongoing conflicts between infectious microbes and their hosts. An understanding of the evolutionary and adaptive patterns associated with this variability will guide our basic understanding of virus community interactions and therapeutic design, particularly vaccine development and infection treatment. Because major shifts in these interactions can be influenced by a small number of nucleotide polymorphisms, nucleotide resolution analysis is essential for studying these systems. Here we go beyond previous approaches by employing high-resolution analytical methods on multiple, independently collected skin shotgun metagenomic libraries to investigate the hypervariable loci of skin viral communities at the nucleotide level. We find that skin virus communities contain diverse hypervariable loci that are associated with purifying selection and targeted substitutions. The degree of selective pressure and the potential impacts of sequence variants differ by virus more than gene type. A wide range of gene types are affected by hypervariable loci, including those involved in host tropism, immune evasion, and the hijacking gene expression associated with viruses. Identified contigs maintain a variety of phylogenetic states, suggesting a range of evolutionary lineages resulting in strain diversification. These findings are highly reproducible across multiple datasets that were collected by independent groups. This is the first community-wide description of skin viral hypervariable loci and the potential impacts of these elements on virome evolutionary selective pressure and functionality. Together our data suggest the skin virome harbors numerous hypervariable regions, and that the potential functional and evolutionary implications of these regions are diverse and depend on virus type. By studying nucleotide variance, we are able to outline the different roles of these regions among the different skin viruses, and evaluate their potential roles in pathogenicity.

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Enrichment and Isolation of Lead-Resistant Bacteria from Saint Claire River Sediments

Nichole Bowman, Mitchell Fenner, Renee Gismondi, Drashti Patel, Adam Oest, Dominic Azzopardi, and Sonia M. Tiquia-Arashiro, Department of Natural Sciences, University of Michigan-Dearborn

One of the most common heavy metals present in polluted waters is lead (Pb). Certain bacteria have the ability to resist and reduce lead’s toxic effects and play a role in bioremediation. Two enrichment experiments using LB and R2A broths containing 10g of sediments and increasing concentrations of Pb (NO3)2 were carried out to isolate Pb-resistant strains.  The enrichments were incubated at room temperature and shook at 250 rpm; aliquots were transferred to fresh media after 48 hours three times.  Cells that grew in LB (1500 mg/L) and R2A (1250 mg/L) were serially-diluted and plated.  Colonies on the plates were isolated, purified, and the resulting 92 pure cultures were transferred onto slants.  Optical density (550 nm) was used to measure cell viability and Pb-resistance.  The specific growth rates in LB and R2A enrichments were 0.604h-1 and 0.426h-1, respectively. Relative growth rates of cells in high Pb concentration enrichments did not grow as much as lower concentration enrichments, however they grew well compared to the control. Bio-flocculation assay was performed on each isolate using Kaolin solution and CaCl to assess their ability for extracellular Pb sequestration.  For R2A, seven out of 20 isolates produced optimal flocculent product, ranging from 23-40%.  For LB, eight out of 21 isolates produced optimal flocculent product, ranging from 25-57%. To continue the research, various lead resistant mechanisms such as efflux mechanism, extracellular sequestration, biosorption, precipitation, enhanced siderophore production and intracellular lead bioaccumulation must be tested on these isolates to determine the best strains for Pb bioremediation.

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Double Trouble: Coinfection with Proteus mirabilis and Providencia stuartii Enhances the Severity of Catheter-Associated Urinary Tract Infection

Chelsie E. Armbruster, Smith, Sara N., Eaton, Kathryn A., Mobley, Harry L.T., University of Michigan Medical School, Ann Arbor, MI 48109

Catheter-associated urinary tract infections (CAUTIs) account for >30% of hospital-acquired infections and are frequently polymicrobial, yet few studies have experimentally addressed the consequences of polymicrobial infection.  Proteus mirabilis and Providencia stuartii are two uropathogens reported to co-colonize catheterized individuals.  Both bacteria are urease-positive, and infection with either can result in formation of urinary stones (urolithiasis) and potentially bacteremia.  We previously observed an enhancement of urease activity during co-culture of a 50:50 mixture of P. mirabilis and P. stuartii in urine.  Using a murine model of ascending UTI, we also observed an increased incidence of bacteremia and urolithiasis during coinfection compared to infection with either species alone.  Development of both complications requires P. mirabilis urease as mice coinfected with P. stuartii and a P. mirabilis urease mutant (ureF) did not exhibit urolithiasis or bacteremia.  Our present work tests the hypothesis that enhanced urease activity occurs during coinfection with P. mirabilis and P. stuartii and potentiates tissue damage and entry to the bloodstream.  To measure the extent of urease activity in vivo, mice were transurethrally inoculated with P. mirabilis, a P. mirabilis urease mutant (ureF), P. stuartii, or a 50:50 mixture of P. mirabilis and P. stuartii.  Urine pH was measured at 6, 24, 48, 72, and 96 hours post infection.  Mice infected with the P. mirabilis urease mutant exhibited the lowest urine pH values and coinfected mice exhibited the highest values, indicating that enhanced urease activity occurs in vivo.  Coinfection resulted in the greatest tissue damage and inflammation based on scoring of bladder and kidney sections, but only when P. mirabilis produced active urease.  Similar findings were observed using a model of catheter-associated UTI compared to the model of ascending UTI.  This work was previously presented at the 2015 Federation of American Societies for Experimental Biology (FASEB) Meeting on Molecular Pathogenesis: Mechanisms of Infectious Diseases (Keystone, CO).

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Elucidating the Role of the EpsE Zinc Binding Domain in Type II Secretion

Chelsea S. Rule, Patrick, Marcella, Sandkvist, Maria, University of Michigan Medical School, Ann Arbor, MI 48109

The type II secretion (T2S) system mediates the extracellular transport of virulence factors, such as toxins and hydrolytic enzymes, in many Gram-negative pathogens. Vibrio cholerae uses the T2S system to secrete cholera toxin, which causes the severe diarrhea that characterizes cholera. The T2S apparatus in V. cholerae is composed of 12 Eps (extracellular protein secretion) proteins, including the cytoplasmic ATPase EpsE that provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique domain called the C-terminal metal binding domain (CM) that coordinates zinc through a tetracysteine motif (CXXCX29CXXC). The CM domain is conserved among T2S and Type IV pilus (T4P) assembly ATPases, while it is noticeably absent in T4P retraction ATPases and other homologous ATPases, suggesting that it may be required for a process unique to T2S and T4P assembly. We thus sought to characterize the role of the EpsE CM domain in T2S. We have constructed several EpsE variants in which the CM domain was removed or the cysteine residues were substituted with serines. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogate secretion in an EpsE-deficient strain and have a dominant negative effect on secretion in the presence of WT EpsE. Additionally, these variant proteins were not amenable to purification due to aggregation when overproduced in E. coli. Chemical depletion of zinc from purified EpsE hexamers results in conformational changes and a loss of in vitro ATPase activity. Replacement of the 29 residues comprising the CM loop between the two dicysteines with those from the homologous T2S ATPase XcpR does not affect EpsE function or activity. These results indicate that, although the individual residues in the metal binding domain may be interchangeable, zinc coordination is essential for the activity and function of EpsE and supports overall protein stability.

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Siderophores secreted by K. pneumoniae induce inflammation and bacterial dissemination

Victoria I Holden, Paul Breen, and Michael A Bachman, University of Michigan

Klebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores- low molecular-weight, high affinity iron chelators- for full virulence and is attenuated by Lipocalin 2 (Lcn2), an inflammatory host protein that sequesters the prototypic siderophore Enterobactin (Ent). Infection with a siderophore-secreting strain of K. pneumoniae induces increased lung bacterial load, increased spleen bacterial load, and increased lung inflammation. Increased bacterial dissemination and lung inflammation could be due to increased bacterial density in the lung, however, recent research has indicated an increased role for non-iron delivery functions of siderophores. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to Lcn2-dependent inflammation and bacterial dissemination during pneumonia. Because siderophore secretion affects bacterial load, we developed strains lacking TonB, a protein responsible for siderophore import. Mutants in tonB secrete siderophores but are unable to utilize them, uncoupling siderophore secretion and bacterial growth. Infection of wild-type mice with siderophore-producing tonB K. pneumoniae induced increased bacterial dissemination to the spleen, as well as increased secretion of the cytokines Interleukin (IL)-6, MIP2, and KC, compared to infection with a siderophore-null tonB mutant. Infection of Lcn2-deficient mice did not indicate Lcn2-dependent dissemination to the spleen or modulation of cytokine secretion. Infection of wild-type mice with isogenic siderophore-secreting mutants indicated that secretion of multiple siderophores is required for enhanced bacterial dissemination and lung inflammation. Further results indicate a role for the master transcription factor HIF-1α in the dissemination of bacteria to the spleen. These results indicate a novel role for siderophores in the induction of inflammation and bacterial dissemination during pneumonia.

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Genetically engineered tanapox virus expressing interleukin-2 suppresses human melanoma tumors in nude mice

Tiantian Zhang, Dennis H. Kordish, and Karim Essani, Western Michigan University, Kalamazoo, MI, USA, 49006

Interleukin 2 (IL-2) is a pleiotropic cytokine that plays a key role both in innate and adaptive immune systems. It has been shown to be involved in the differentiation of T cells and activation of NK cells. Oncolytic (tumor lysing) virotherapy has become an appealing addition to the current therapies of cancers due to its ability to specifically infect, replicate and lyse tumor cells, and activate the host immune systems to regress the tumor. Tanapox virus (TPV), which possesses large genome and only causes mild diseases in human, is potentially a perfect candidate for virotherapy. In this study, a recombinant TPV expressing mouse interleukin-2 (TPVΔ66R/mIL-2) was generated, where the 66R/thymidine kinase (TK) gene was replaced with mouse IL-2 transgene. In cell culture, the replication of TPVΔ66R/mIL-2 was less than that of both wild-type TPV (wtTPV) and TPVΔ66R at a low multiplicity of infection (MOI) in owl monkey kidney (OMK) cells. However, TPVΔ66R/mIL-2 replicates as efficiently as wtTPV and TPVΔ66R in human melanoma SK-MEL-3 cells. The antitumor potential of TPVΔ66R/mIL-2 was studied on xenografted nude mice carrying SK-MEL-3 tumors. Results showed that TPVΔ66R/mIL-2 significantly regress the induced tumor, which was even more effective than that of wtTPV and TPVΔ66R. Taken together, these results suggest that TPVΔ66R/mIL-2 has potential for immunotherapy and oncolytic therapy of melanoma.

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Elucidation of Phage-Mediated PLE Induction in Vibrio cholerae

Amelia C McKitterick, O’Hara, Brendan J, Seed, Kimberley D, University of Michigan, Ann Arbor, MI 48104

Vibrio cholerae is the causative agent of cholera and thrives in both aquatic environments and the human intestinal tract. In addition to threats such as host immune factors and antibiotics, V. cholerae is also susceptible to predation by bacteriophages, such as ICP1. Interestingly, V. cholerae has adapted a unique defense mechanism referred to as a PLE (Phage-inducible chromosomal island Like Element) that specifically responds to ICP1 infection. The PLE is an ~18-kb island that integrates into the small chromosome and has been identified in clinical isolates of V. cholerae. Five PLE variants have been identified and are characterized by the ability to respond to phage infection and inhibit progeny phage production through an unknown mechanism. We speculate that a PLE encoded repressor keeps the island quiescent during normal cellular growth, and upon infection, a phage protein interacts with the repressor, alleviating repression and stimulating PLE activity. Each PLE is induced by a different set of ICP1 phage isolates. We hypothesize that the observed variations in phage host range are due to evolution at the level of the inducer-repressor complex. We exploited the differences in host range in order to select for recombinants with altered induction phenotypes. ICP1 and ICP1_2011_A have opposite host ranges, where ICP1 induces PLE1 but not PLE2, and, conversely, ICP1_2011_A induces PLE2 but not PLE1. We constructed a randomly sheared ICP1_2011_A genomic library and introduced it into PLE- V. cholerae. The wild-type ICP1 was propagated on this host in the presence of the genomic library in a single round of infection at an MOI of 1. The resulting lysate was screened on V. cholerae PLE2 to identify recombinant phage lacking the inducing cue. Using this method, we isolated eight ICP1 recombinant phage that are no longer blocked by PLE2. We investigated the ability of these recombinants to induce other PLEs and propose whole-genome sequencing to identify recombinant regions and determine the induction cue.

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Comprehensive Validation of a Deep Sequencing Pipeline for Assessing Intrahost Viral Diversity

John T. McCrone, Adam S. Lauring, University of Michigan, Ann Arbor, MI 48109

Until recently, studies of intahost viral diversity have been limited by labor-intensive, low-throughput sequencing methods. With the advent of next generation sequencing, it is now feasible to efficiently sequence viral populations at a depth of coverage sufficient to detect rare single nucleotide variants (SNV). However, each sequencing platform has error prone tendencies, and sample collection, target amplification, and library preparation are additional processes whereby errors are produced and propagated. Many statistical models have been reported to accurately distinguish rare SNVs from sequencing errors. However, studies of viral diversity often require complex sample preparation, and the reliable discrimination of true and false positive SNVs under these conditions is a largely, unresolved issue. To meet this need, we have developed and robustly benchmarked a variant calling pipeline designed to identify rare SNVs in patient-derived influenza populations. Samples are enriched for influenza genomes through multiplex RT-PCR prior to sequencing on the Illumina platform. SNVs are then identified using custom scripts and the R package DeepSNV. This variant caller relies on a clonal plasmid-derived genome that serves as an internal control for context-dependent errors. We have applied this method to defined influenza populations and are able to detect variants at frequencies as low as 0.5% with 99.8% specificity. These thresholds are robust over the input titers found in patient samples. Additionally, we used our data set to benchmark the popular variant caller Lofreq. We found the accuracy of this statistical method was largely dependent on experimental design and input titer. We are currently applying our deep sequencing method to measure the diversity of epidemiologically linked, patient-derived influenza populations. Our work will allow us to confidently identify the impact of inter- and intrahost factors on the short-term evolution of influenza virus, and will promote more accurate studies of viral diversity.

This is an updated version of work presented at the 2015 American Society of Virology meeting.

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Development of a Surrogate to Assess the Efficacy of Inactivation of Protozoa in Ozone Based Water Treatment Systems

Nishita G. D'Souza, Rose Joan B., Shrivastava Sandhya*, Michigan State University, East Lansing, *Bhavan's College, Mumbai 400058, Maharashtra, India

Cryptosporidium parvum infections account for a majority of water associated parasitic disease worldwide. Protozoan oocysts have been detected in finished drinking water and persist through traditional halogen based water treatment processes. Operational practices to reduce the risk of these infections to acceptable levels are not well understood. Ozone has shown promise in inactivating chlorine resistant protozoa. Ozone based disinfection systems worldwide, particularly in developing countries are monitored only on the basis of chemical parameters and bacterial indicators as direct monitoring of protozoa is a cost and competency driven process. There is a need to be able to evaluate disinfection efficacy of water treatment systems with respect to protozoan disinfection in a practical and cost effective manner. Several reports recommend the use of bacterial spores as surrogates to assess inactivation efficacy of Cryptosporidium spp. and Giardia spp., however no protocols have been established by regulatory standards, the purpose of the study was to establish the same. 75 spore producing, resistant bacterial strains were screened and isolated from water and soil environments exposed to disinfectants. Of these, 36 were characterized, sporulated [EN13704:2002(E)] and subjected to seeding experiments in the presence of ozone. The dissolved ozone concentration was determined using standard Indigo method (SM 4500-B). The inactivation was assessed by calculation of CT values (concentration x time) required for 3 log reduction of the initial spike. Previous reports indicate CTs of 8-18 mg.min/L will inactivate 99.9% of Cryptosporidium oocysts. The most resistant isolates CCH-1, RG-2, RG-5, GR-1 showed CT values of 24, 24, 18 and 18 mg.min/L respectively (as compared to the suggested surrogate Bacillus subtilis 6633- 15 mg.min/L) indicating that the isolates from the present study could be explored as potential surrogates for protozoa and integrated into an evaluation programs base for water treatment based on ozone.

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Acidithiobacillus ferrooxidans, an iron-oxidizing, acidophilic bacterium, prefers hydrophobic surfaces for cell attachment and planktonic growth

James Iordanou, Popko, Sarah P. and Sara E. Blumer-Schuette, Oakland University, Rochester, MI, 48309

Acidophiles are microbes that thrive in harsh, low pH environments. In many instances, acidophiles are found in microenvironments directly around metal substrates. Acidithiobacillus ferrooxidans is one such bacterium that can oxidize ferrous iron to generate energy in highly acidic environments. As a consequence, A. ferrooxidans can thrive in highly acidic mine drainage that is rich in both iron and sulfur. Past studies have focused on optimal growth conditions and molecular mechanisms for metal oxidation by A. ferrooxidans. Here, we propose that the specific mechanisms behind substrate attachment and the development of biofilms are also important for metal oxidation to occur. In order to better understand how A. ferrooxidans attaches to surfaces, the rate of biofilm formation, cell density, and iron oxidation were monitored during growth on soluble iron (II). Here, we describe the effects of various surfaces with different charge states on cell-surface attachment and iron oxidation. We observed that a greater number of cells attach to polystyrene that mimics the hydrophobic state of metal sulfide ores. Subsequently, attachment assays have been conducted on pyrite to measure the influence iron sulfide ores have on growth phase properties and biofilm recruitment. A. ferrooxidans possesses three different pilus systems within its genome, the tight adherence pilus, chaperone-usher pilus, and the type IV pilus. We predict that one or more are involved in attachment to surfaces, and more importantly, to metal sulfide ores. Primer sets have been designed for qPCR to monitor expression of key genes in each of three distinct pilus gene clusters. These results are providing data on the overall conditions for microbial attachment by A. ferrooxidans, as well as the contribution the microenvironment has on overall iron oxidation in solution. This project will be expanded further by using prior community analysis studies to analyze the attachment capabilities of microbial enrichments from soil samples harvested around Michigan.

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Acinetobacter baumannii is Dependent on the Type II Secretion System and its Substrate LipA for Lipid Utilization and in vivo Fitness

Ursula Waack#, Johnson, Tanya L.2#; Smith, Sara1, Mobley, Harry1 and Sandkvist, Maria1, 1 University of Michigan, Ann Arbor, MI, 481019, 2 Eastern Michigan University, Ypsilanti, MI, 48197, # These authors contributed equally to this work

Acinetobacter baumannii is an increasingly common nosocomial pathogen that has developed multi-drug resistance. Infections by A. baumannii typically occur in immune-compromised patients such as those in the ICU, in individuals with invasive devices or extensive wounds, and in those undergoing or having recently undergone antibiotic regimens. A. baumannii causes pneumonia, bacteremia, meningitis, urinary tract infection, and wound infection. While there is a breadth of research on antibiotic resistance in A. baumannii, understanding of A. baumannii pathogenesis is lagging. Here, we focus on the Type II Secretion (T2S) system, a multiprotein complex that is responsible for extracellular secretion of toxins, proteases, lipases, and enzymes that break down complex carbohydrates. The T2S system in A. baumannii has a unique genetic organization as the 13 T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this difference, we show here that A. baumannii has a functional T2S system. T2S  mutants lacking either the gspD or gspE genes, which encode the outer membrane secretin and the cytoplasmic ATPase, respectively, are unable to secrete a lipase, LipA, to the extracellular environment. As a consequence, these mutants are, similarly to a lipA deletion mutant, unable to grow on lipid agar containing olive oil as sole carbon source, but show no growth defect on LB agar or other rich media. However, growth on lipid agar is restored by  plasmid-encoded genes in trans. To determine a putative role for the T2S system in pathogenesis, we separately subjected the gspD and lipA mutants to competition assays with the wild type A. baumannii strain in a neutropenic murine bacteremia model.  Both mutants were outcompeted by the WT strain suggesting that the ability of A. baumannii to utilize exogenous lipids contributes to its fitness in vivo.

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Prevalence of antibiotic resistant Shiga toxin- producing Escherichia coli (STEC) in Michigan

Sanjana Mukherjee1, Pallavi Singh1, Rebekah Mosci1, James T. Rudrik2, and Shannon D. Manning1, 1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan; 2 Michigan Department of Health and Human Services (MDHHS), Lansing, Michigan

The burden of STEC, an important food-borne pathogen, is enormous causing ~265,000 cases per year in the US. From a public health perspective, antibiotic resistant STEC is important due to the possibility of transfer of antibiotic resistance to other bacteria. Resistance to antibiotics, however, has not been widely researched among STEC. In this study, STEC O157 isolates, obtained from the MDHHS for the periods 2001-2006 (n=40) and 2010-2015 (n=91), were tested for antibiotic resistance by disk diffusion; a subset of non-O157 STEC (n=86) from 2010-2015 were also examined. Overall, 10 (7.6%) of the O157 isolates and 7 (8.13%) of the non-O157 isolates were resistant to at least one antimicrobial. The percentage of ampicillin resistant STEC O157 decreased from 15% in 2001-2006 to 3.29% in 2010-2015 while trimethoprim-sulfamethoxazole (SXT) resistance decreased from 5% (2001-2006) to 1.09% (2010-2015). Genotypic studies revealed that resistance to ampicillin for most isolates was not associated with chromosomal ampC while SNP genotyping of the O157 isolates demonstrated that the resistant isolates belonged to separate clades. Although there was a significant decrease in ampicillin resistance over time, which may be a result of decreased use of ampicillin, resistance to important antimicrobials was detected in both O157 and non-O157 STEC in Michigan. Future studies should focus on identifying genotypic factors associated with antibiotic resistance and link resistance with demographic, geographic and other risk factors to help in disease management. Continuous monitoring of antibiotic resistance frequencies in STEC and other enteric pathogens is important for assessing the efficacy of public health measures used to combat antibiotic resistance.

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Analysis of Vibrio Cholerae ToxR Translocation via Reporter Protein Alkaline Phosphatase

Morgan F. Rice, Jeremiah G. Johnson, and Victor J. DiRita, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Vibrio cholerae is a Gram-negative bacterium that colonizes the small intestine of humans, causing an extensive efflux of water into the lumen, resulting in watery diarrhea, vomiting and, if left untreated, death. Cholera is contracted via consumption of contaminated food and water, with the majority of cases occurring in countries that have inadequate sanitation systems and limited access to clean water. V. cholerae uses a complex regulatory network, the ToxR regulon, to control expression of two essential virulence factors, the cholera toxin (CT) and the toxin coregulated pilus (TCP). ToxR is a membrane protein that, when transported to the periplasm, interacts with ToxS and various transcriptional activators to activate expression of toxT, whose product directly activates genes encoding CT and TCP. As such, translocation of ToxR through the inner membrane is an important step in virulence gene regulation. We are interrogating a V. cholerae transposon library for mutants unable to translocate a ToxR-PhoA fusion through the inner membrane. Using 5-Bromo-4-choloro-3-indolyl phosphate (BCIP) indicator plates, mutants unable to cleave the substrate – indicating a lack of ToxR-PhoA fusion translocation - will be studied further. We successfully optimized indicator plates using various concentrations of BCIP and glucose. Currently, we are constructing a conjugatable reporter plasmid that will allow us to assess ToxR translocation in the mutant library.  This data will help us to better understand the steps involved in ToxR induction of the virulence cascade.

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Genetic and Virulence Characteristics of Serratia marcescens and Citrobacter freundii Bacteremia Isolates

Mark T. Anderson, Lindsay A. Bradford, and Harry L. T. Mobley, University of Michigan Medical School, Ann Arbor, MI 48109

Serratia marcescens and Citrobacter freundii are opportunistic pathogens that are capable of colonizing a wide range of host sites causing wound, urinary tract, and bloodstream infections. Both bacterial species are significant causes of hospital-acquired infections and are noted for frequently encoding antibiotic resistance determinants, making infections difficult to treat. To advance our understanding of the pathogenic mechanisms of these organisms, 18 total S. marcescens and C. freundii bacteremia isolates were collected from infected individuals through the University of Michigan Health System and characterized using a combination of comparative genomics and phenotypic analyses. Isolates were identified by MALDI-TOF mass spectrometry in conjunction with 16S rRNA sequencing. The minimum inhibitory concentration of selected clinically relevant antibiotics was determined for each isolate with multiple isolates demonstrating complete or partial resistance to beta-lactam, aminoglycoside, or fluoroquinolone antibiotics. Selected isolates were also assessed for virulence in a murine model of bacteremia. Following intravenous inoculation at an infectious dose of ≤ 5 x 107 colony forming units, both S. marcescens and C. freundii could be readily detected in the spleen, liver, and kidneys by 4 hours post-infection and robust colonization was maintained for at least 48 hours. Variations in overall bacterial burden and murine lethality were observed when different isolates within each species were compared, suggesting an enhanced pathogenic potential for certain strains in this model system. The complete genome sequence of each isolate was also determined to facilitate comparative genetic analyses and provide a basis for further investigation of the virulence mechanisms for these bacterial species.

This work was presented at the 2015 ASM General Meeting.

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Comparative Genomics of Clinical Isolates of Pseudomonas fluorescens and the Discovery of a Novel Disease-associated Subclade Associated with Chronic Lung Disease

Brittan Scales, Erb-Downward JR, Dickson RP, Falkowski NR, Pandit CR, LiPuma JJ, Huffnagle GB, University of Michigan, Ann Arbor

Background
Taxonomically, there are over 52 Pseudomonas species that group within the Pseudomonas fluorescens species-complex. Prior to beginning this project, we identified that the lung microbiome of COPD patients contained abundant levels of P. fluorescens. This finding was highly unexpected; P. fluorescens colonization was not previously reported in chronic respiratory disease and there was no taxonomic information on clinical P. fluorescens strains. This raised the question of whether there were differences between P. fluorescens strains isolated from clinical vs. environmental sources.
Methods
We sequenced a collection of Pseudomonas spp. isolates from individuals with chronic lung disease, including 22 clinical P. fluorescens strains. Through multi-locus sequence analysis (MLSA), we phylogenetically classified the Pseudomonas spp. strains, with a focus on those that grouped within the P. fluorescens species complex. Using a comparative genomics approach, we identified genomic attributes associated with growth in humans in the clinical P. fluorescens species complex isolates. 
Results
Twelve of the clinically isolated Pseudomonas spp. grouped through MLSA within previously defined subclades (I - III) of the P. fluorescens species-complex. However, 10 strains clustered as a unique fourth subclade. Subclade IV displayed marked reductions in genome size, gene diversity and GC content, as well as containing all genetic elements for a type III secretion system that was extremely similar to that found in P. aeruginosa.  Furthermore, the subclade IV strains, collected from geographic locations across the U.S., had high levels of shared nucleotide identity and a small accessory genome, suggesting that they may have evolved from living in multi-trophic environments to a life in a much narrower niche, perhaps human airways.
Conclusion
Altogether, these studies revealed that P. fluorescens is an under-appreciated colonizer of humans, particularly in the context of lung disease, and laid the groundwork for future studies delineating the contribution and molecular mechanisms of this host-microbe interaction.

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TosR (PapB homolog), Lrp, and H-NS Induce and Repress Expression of the Uropathogenic Escherichia coli RTX Adhesin, TosA

Michael D. Engstrom and Mobley, Harry L. T., University of Michigan, Ann Arbor, Michigan, 48109

Uropathogenic Escherichia coli (UPEC) form a diverse group of extraintestinal pathogenic E. coli (ExPEC) isolates and are responsible for most uncomplicated urinary tract infections (UTIs) in humans. In accordance with UPEC diversity, no core set of virulence factors exists among different strains. Therefore, the focus of uropathogenesis has shifted to studying broad classes of virulence factors, including many distinct and novel adhesins. For instance, the E. coli CFT073 repeats-in-toxin (RTX) non-fimbrial adhesin, TosA, is expressed during experimental UTI and promotes adherence to host cells derived from the upper urinary tract. However, tos operon regulation has not been fully elucidated. Our earlier work identified TosR (a PapB homolog) and TosEF (LuxR family members) as negative regulators of the tos operon and motility, respectively. Inducers of the tos operon, however, remained unknown. To determine whether TosR and TosEF also induce expression of the tos operon and to map the precise location of the tos operon promoter, we used a combination of western blots of key overexpression constructs in wild type CFT073 and CFT073 mutant strains and a 5’ rapid amplification of cDNA ends (RACE) assay. We found that TosR is both an inducer (at low levels) and repressor (at high levels) of tosA. In mutant strains of CFT073, TosR regulatory activity is modified by the nucleoid structuring proteins Lrp and H-NS. Intriguingly, TosR regulatory activity appears independent of a cognate PapI homolog, which is not the case for many other adhesin operons regulated by PapB homologs. Consistent with recent RNA-Seq experiments, we also determined that the tos operon promoter is at least 30 bp upstream of tosR and approximately 160 bp downstream of a previously identified TosR DNA binding site. Our work expands the current knowledge of adhesin operon regulation by PapB family members, including the contribution of differential nucleoid structures.

This work was previously presented at the ASM 2015 General Meeting.

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The Amylolytic Systems of Eubacterium rectale and Ruminococcus bromii Rely on Uniquely Adapted Enzymes

Darrell Cockburn, Krizia Perez Medina, Ryan Kibler and Nicole Koropatkin, Department of Microbiology and Immunology, University of Michigan

Starch is the most frequent nutritious polysaccharide consumed in the typical human diet and thus has a major impact on human health. However, not all the starch we consume can be degraded by human derived enzymes. The remaining fraction is termed resistant starch (RS) and serves as an important substrate for the gut microbiota, which returns some of the stored energy to their human host in the form of short chain fatty acids (SCFA) such as butyrate. The ability to utilize starch or its derived components is predicted to be widespread among members of the gut microbiota, but little is known about the degradation of RS. Among the organisms known to flourish on diets rich in RS are the cluster XIVa and cluster IV clostridia. The cluster XIVa clostridia contain several of the most important butyrate producers in the gut and butyrate is thought to be the most beneficial of the SCFA. Eubacterium rectale is a key member of the cluster XIVa group and serves as an important model. Cluster IV includes Ruminococcus bromii, an organism that has been found to be particularly adept at breaking down RS. We have undertaken the study of the amylolytic systems of these two key organisms in order to gain insight into how RS is utilized in the gut and how the various players interact with one another. One thing these organisms have in common is a series of adaptations in their key extracellular enzymes, including previously unrecognized carbohydrate binding modules (CBMs). In E. rectale there are two enzymes and two transport systems that are upregulated in the presence of starch. The two enzymes are both amylases which are readily able to degrade substrates with α-1,4 linked glucose, but the lack of an extracellular α-1,6 linkage specific enzyme places limits on the organism’s ability to efficiently degrade starch. In contrast R. bromii contains a more robust complement of enzymes for starch degradation, including a predicted 4 pullulanases (α-1,6 linkage specific enzymes). Here we present the structural and biochemical characterization of these unique enzymes and their CBMs, providing a first look at how these key organisms interact with and degrade starch at the molecular level.

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MapA as Potential Lipoprotein Sorting Factor in lol System of Campylobacter jejuni

Marisa R. Fallone, Jeremiah G. Johnson, and Victor J. DiRita, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Campylobacter jejuni is a major cause of gastroenteritis in the United States, often by infecting those who consume undercooked poultry or other food that has contacted a contaminated surface. This is due to C. jejuni asymptomatically colonizing the intestinal tract of chickens, where it can be released during processing, contaminating the meat and becoming potentially infectious to humans. Using INseq, the mapA gene was identified as contributing to the fitness of C. jejuni during colonization of the chicken cecum. We hypothesize that MapA plays a role in lipoprotein sorting to the inner and outer membranes of this gram negative bacterium. In Escherichia coli and other gram negatives, the lol system is responsible for shuttling lipoproteins from the cytoplasm to their appropriate membranes. In this system, LolCDE in the inner membrane releases lipoproteins destined for the outer membrane to LolA, in the periplasm. LolA passes these lipoproteins to the outer membrane protein LolB. Annotated orthologues of all lol genes except lolB are encoded within the C. jejuni genome. Our analysis of MapA shows that it shares homology to LolB, suggesting that MapA substitutes for LolB. We have found that mapA mutants are hypersensitive to the membrane perturbing antibiotic, polymyxin. Additionally, we have been successful at generating a FLAG-tagged derivative of MapA, which has been shown to be expressed in a mapA mutant.  Currently, we are using this tagged version of mapA to determine membrane localization of the protein as well as to examine for interactions with LolA.

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Genome-wide Identification of Fitness Genes in Acinetobacter baumannii During Bacteremia

Sargurunathan Subashchandrabose, Sara Smith, Valerie Forsyth, Sebastien Crepin, Monica Kole, Carina Zahdeh, and Harry LT Mobley, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI

Acinetobacter baumannii, a Gram-negative bacterium, is emerging as a leading antibiotic resistant nosocomial pathogen, both in the United States and around the world. However, little is known about A. baumannii fitness and virulence mechanisms that are involved in pathogenesis in a mammalian host. We used transposon directed insertion-site sequencing (TraDIS) for genome-wide identification of A. baumannii genes that contribute to fitness during bacteremia in a murine model. Using a saturating transposon library of 109,000 Tn5 mutantsin A. baumannii strain ATCC 17978, a leukopenic mouse model of bacteremia, and high-throughput DNA sequencing, we identified novel fitness genes in A. baumannii. Unique insertion sites (2,614) were identified in the input and output libraries and fitness factors were calculated for each insertion site. Eighty nine transposon mutants that exhibited a fitness factor ≥ 6.3 (mean + 2 SD) were selected for further investigation. Genetically defined mutants were generated by recombineering in A. baumannii strain ATCC 17978. Defined mutants lacking feoB (ferrous iron import), ddc (D-ala-D-ala-carboxypeptidase), and pntB (pyridine nucleotide transhydrogenase subunit) exhibit a fitness defect during systemic infection resulting from bacteremia. In vitro, these mutants are defective in survival in human serum, within macrophages, and are hypersensitive to killing by antimicrobial peptides. In summary, we have determined novel A. baumannii fitness genes involved in mammalian infection and propose mechanisms by which these genes contribute to A. baumannii pathogenesis.

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The endoplasmic reticulum stress sensor IRE1α is a key regulator of neutrophil effector function

Basel H. Abuaita, Denise N. Bronner, and Mary X.D. O’Riordan, Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI

Neutrophils are a critical first line of defense against bacterial infection. We recently demonstrated that the inositol-requiring enzyme 1-α (IRE1α) sensor of the unfolded protein response (UPR) is required for clearance of methicillin-resistant Staphylococcus aureus (MRSA) in a skin abscess model. However, the specific role of IRE1α in neutrophil effector function is unknown. Here we show that infection-induced IRE1α activation drives neutrophil bactericidal activity against MRSA, and is essential for cytokine production. Neutrophils treated with an IRE1α inhibitor exhibit a strong defect in MRSA killing, which is independent of the major phagocyte oxidase, NADPH oxidase 2 (NOX2). Instead, IRE1α inhibition markedly decreased production of the cationic antimicrobial peptide, cathelicidin. IRE1α inhibition also caused a substantial decrease in neutrophil-derived cytokine production during MRSA infection both in vitro and in vivo. Our data show that IRE1α is a major driver of neutrophil immunity against MRSA infection, enhancing production of antimicrobial peptides and inflammatory cytokines. Evidence of IRE1α activation is associated with many human chronic and inflammatory diseases, and our results now reveal the importance of IRE1α in neutrophils, which play a key role in shaping inflammatory responses.

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SIRT3 controls infection-induced inflammation and adaptive stress response programs

Denise N. Bronner, B.A. Abuaita, J.A. Myers, J. Park, D.B. Lombard, Mary X. O’Riordan, Experimental Immunology T32 Postdoctoral Trainee, University of Michigan, Ann Arbor

Cellular stress pathways are responsive to perturbation of homeostasis, and can be triggered by microbial infection. We previously showed that infection-induced ER stress promotes robust production of reactive oxygen species (ROS), which can initiate inflammatory signaling. ROS also damage biomolecules; therefore, it is essential to coordinate mechanisms that trigger their generation while limiting toxicity. Within the mitochondrial network, the SIRT3 deacetylase, regulates mitochondrial redox balance by activating ROS detoxification mechanisms. How SIRT3 is regulated during infection, and what its effects are on infection-induced ROS and inflammation are known. We find that upon infection, SIRT3 is translocated out of the mitochondrial network. Upon translocation, SIRT3 is localized in the nucleus and also associates with cytosolic aggresome-like induced structures (ALIS), which are characteristic of proteotoxic stress.  We observed SIRT3-dependent upregulation of the proteotoxic stress response proteins, Hsp70 and p53. We hypothesized that SIRT3 deficiency might increase bactericidal activity due to higher ROS production, however Sirt3-/- macrophages and mice failed to control methicillin-resistant Staphylococcus aureus (MRSA) in a skin abscess model of infection. SIRT3 deficiency led to increased apoptosis, and inhibition of caspase-3 increased viability and bactericidal activity in Sirt3-/- macrophages. Taken together, these observations suggest that infection-induced SIRT3 translocation permits a transient ROS spike, driving controlled inflammation and bacterial killing.  In the absence of SIRT3, ungoverned ROS production causes lethal host cell damage during infection. We propose that SIRT3 translocation protects phagocytes from self-destruction by initiating adaptive proteotoxic and oxidative stress responses, thus “pre-conditioning” these cells to better survive damage from immune effector mechanisms. Overall, our work suggests a novel role for SIRT3 in modulating immunity by triggering adaptive stress responses during infection.

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Regulatory Control of a Broad Spectrum Lantibiotic in Streptococcus pneumoniae is Controlled Through the Concerted Action of Three Peptides and an Immunity Transporter

Natalie Maricic, Erica Anderson, and Suzanne Dawid, University of Michigan, Ann Arbor, Michigan 48109

Streptococcus pneumoniae utilizes a number of strategies to outcompete other bacterial strains found in the nasopharynx including the secretion of antimicrobial peptides. We have identified a clinical isolate (P174) that secretes a novel lantibiotic that is characterized by a broad range of inhibitory activity. The locus encodes the typical components required for lantibiotic production including proteins involved in regulation, modification, transport and immunity.  Interestingly, the locus is characterized by an unusual tandem array of four ORFs predicted to encode four homologous but distinct lantibiotic peptides, (PldA1-4). Individual peptide deletions showed that the first three of these peptides were all absolutely required for self-activation of the locus.  Further characterization of the role of the individual peptides was initially hampered by the lack of activity in broth culture, however, in the course of our studies we identified a novel mutant of P174 (P174act) that had an over-producer phenotype demonstrating locus activation in broth culture.   Linkage analysis confirmed that the mutation responsible for the P174act phenotype was located in the intergenic region between the peptides and the immunity transporter, pldFE.  Using pld reporter fusions, we found that broth grown P174act activated pld transcription in response to Pld peptides whereas no stimulation was seen in 174 reporters.  A pldFE reporter confirmed that the mutation decreases expression of the immunity transporter following peptide stimulation.  This observation suggested that, in addition to immunity, the transporter plays a novel role in signal recognition or sequestration.  Given the lowered requirements for peptide stimulation in P174act, individual peptide deletions were moved into this background.  In contrast to P174, each of the deletions in P174act was characterized by a distinct phenotype, suggesting distinct roles for the peptides. The pld locus in P174 represents a unique strategy in bacterial competition that requires the presence of three homologous but distinct peptides that potentially interact with an immunity protein/regulator complex.

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Average Frequency Vector Analyses of Hypothetical Open Reading Frames

Jessica Marklevitz, Amberlee Hicks-Marshall, and Laura K. Harris, Davenport University, Lansing Campus, Michigan, 48933

Since the advent of nucleotide sequencing, there is an explosion of newly sequenced organisms.  Doing bench experiments to establish the function of each potential open reading frame (ORF) for each organism is time-consuming and expensive.  Computerized algorithms, like the Basic Local Alignment Search Tool (BLAST), allow some ORF function predictability based on sequence homology and alignment on an individual protein basis.  This approach to predicting the functions of hypothetical ORFs individually is still time-consuming and expensive, often requiring experimental validation particularly for proteins with uncommon functions.  As a result, for many organisms including most archaea, the functions of approximately one third of their potential ORFs remain unknown.  If the list of hypothetical ORFs narrowed to just protein-encoding ORFs with some quick computational analyses, annotation would be easier.  Ergo, this work examines if the average nucleotide, absolute codon, and amino acid frequencies for hypothetical ORFs differ from those of ORFs with established function.  Examined ORF averages from 26 bacterial and 17 archaeal species with variation in their genome annotation completeness showed no statistical difference between hypothetical ORFs and ORFs with predicted function.  This indicates that nucleotide, absolute codon, and amino acid frequencies are not indicators for the presence or absence of protein-encoding ORFs though a trend that hypothetical ORFs are more dissimilar than frequencies from genes with established function was observed.

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Oncolytic Tanapoxvirus: Experimental Immuno-virotherapy of triple negative breast cancer

Yogesh Suryawanshi, John Christie and Essani Karim, Western Michigan University, Kalamazoo, MI 49008

Oncolytic virotherapy (OVT) is an emerging therapeutic option for cancer that uses replication competent viruses that can selectively or preferably infect cancer cells and kill them by lysis, with minimum damage to normal cells. Many solid tumors remain resistant to traditional anti-cancer approaches. For example, triple-negative breast cancer (TNBC) is one of the subtypes of breast cancer (BC) that is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human-epidermal growth factor-2 (HER-2). It is a relatively aggressive type of BC, with high rate of mortality and recurrence as compared to other BC subtypes. The heterogeneous nature of this cancer type is a big hurdle in using most of the current standard treatment options, such as chemotherapy and radiotherapy. A central tenet of our approach is that TNBC and other resistant cancers may be susceptible to virotherapy based upon Tanapox virus (TPV). A panel of TPV recombinants has been generated in our lab earlier by ablation of one or more of the specific genes from the viral genome such as the 66R and 2L. Some recombinant viruses carry the immune-stimulatory transgenes, inserted in their genome, such as the mouse granulocyte monocyte colony stimulating factor (mGMCSF), the mouse macrophage inflammatory protein (mCCL2) and bacterial flagellin protein (FliC). Recombinants of TPV with one or more specific gene ablations (66R and 2L) are expected to selectively target cancer cells, while the immune-regulatory transgenes are expected to harness benefits of virotherapy and immunotherapy. Replication ability of TPV recombinants was studied in a panel of TNBC cells, in vitro. TPV recombinants were able to replicate efficiently in all cell lines in the TNBC panel, except MDA-MB 453. TNBC cell lines that supported viral replication well are also known to be tumorigenic and will be used to induce tumor xenografts in athymic nude mice.  Oncolytic potential of TPV recombinants will be tested in vivo by intra-tumoral injection of virus particles during future studies which will allow exploring the oncolytic potential of TPV recombinants comprehensively. This work was supported by a grant from OVPR, WMU.

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Prioritization of Polysaccharide Utilization by Bacteroides ovatus

Yao Xiao and Eric Martens, University of Michigan, Ann Arbor, Michigan 48109

The hundreds of species of bacteria that compose the human gut microbiota experience dynamic and chemically complex nutrient environments, in which the presence of polysaccharides plays a major role. Bacterial responses to these nutrients are therefore a major factor that influences the physiology and composition of the microbial community. Members of the Bacteroidetes, one of few dominant phyla in the gut, possess the abilities to degrade a wide range of complex dietary and host polysaccharides (glycans) through the functions of a series of gene clusters termed polysaccharide utilization loci (PULs).  One species of human gut Bacteroides, Bacteroides ovatus, possesses broad capacity in degrading chemically diverse polysaccharides including plant cell wall hemicelluloses, which are found in common vegetables and whole wheat. To understand how gut commensal ­bacteria response to their complex and dynamic nutrient environment, we investigated the response of B. ovatus to a polysaccharide mixture (PSM) that consists of 13 common plant- or host- derived polysaccharides. We found that B. ovatus elicits polyauxic growth in PSM. This is likely due to prioritized utilization of certain polysaccharides, which is analogous to the classic E. coli diauxie on glucose and lactose. A time-course transcriptional analysis (using qRT-PCR and RNAseq) of B. ovatus PULs responsible for degrading the 13 different glycans revealed both sequential activation and repression of PUL gene expression during a 12-hour growth period. This is consistent with B. ovatus prioritization of various carbohydrates as it grows on this dynamic mixture. High-throughput screening of B. ovatus growth on pairs of polysaccharides revealed wide-spread prioritization of polysaccharide metabolism. A detailed glycan hierarchy model was generated and two dietary fiber hemicelluloses were found to be low in the hierarchy. We are currently investigating the biological roles of glycan prioritization on Bacteroides physiology and competition in vivo. This work was presented at the Microbiome Symposium at the University of Michigan.

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A Novel and Rapid Staphylococcus aureus Bacterial Identification Method Utilizing ImmunoMagnetic Beads and Single Cell Laser Scattering

Kaylagh R. Hollen  and Sharp, Josh S., Northern Michigan University, Marquette, MI, 49855

Staphylococcus aureus is one most common cause of infections in hospitalized patients and is responsible for nearly 500,000 hospitalizations per year in the United States. In 2011, the United States had 80, 461 cases of invasive Methicillin-resistant S. aureus (MRSA) infections and 11,285 related deaths. With rising healthcare costs, fast, accurate, and cost effective methods of pathogen identification are needed.  Technology developed by Micro Identification Technologies (MIT) identifies specific bacteria by measuring laser light scattering patterns of single cells.  Light scattering patterns, recognized by photo detectors, generate identifiers for known bacteria species.  The MIT 1000 can specifically identify Staphylococcus at the genus level and an identifier for S. aureus is in development. Faster pathogen identification leads to faster implementation of appropriate treatment, and is linked to improved patient outcomes. Developing a rapid method to isolate and amplify Staphylococcus species, combined with laser light scattering identification could potentially reduce the average length of hospital stay, quicken patient recovery time, and decrease mortality rates.  The focus of this study is to develop an ImmunoMagnetic Separation (IMS) technique that can isolate S. aureus from liquid samples using streptavidin-coated magnetic Dynabeads and biotinylated anti-Protein Aantibodies. A combination of IMS and light scattering analysis of captured cells could be utilized to rapidly identify the bacteria. We aim to complete identification in 4-8 hours as opposed to current culture methods which take up to 18-48 hours.  Preliminary data suggests that S. aureus can be successfully isolated and identified with single cell light scattering in 4 hours after swab specimen collection with an average initial input of 1.37 x 108 CFU/mL.

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Experimental virotherapy for ovarian cancer

Jonathan D. Hoerger, Yogesh Suryawanshi, Emily Byers, and Karim Essani, Western Michigan University, Kalamazoo, MI 49008

Ovarian cancer (OC) is the leading cause of death among all gynecological cancers with an estimated 14,270 deaths in the U.S. in 2014.  The 5-year survival rate for OC is 44.6%, with the current standard treatment options.  A promising option of treatment termed, oncolytic virotherapy (OV), is being developed for many different types of cancer including OC. Tanapoxvirus (TPV) is an antigenically distinct poxvirus, and is limited to equatorial Africa. Most patients have no prior immunity to TPV, unlike most OVs currently under development.  TPV has been tested against human colorectal cancer (hCRC) and has demonstrated an ability to regress cancer growth in nude mice xenografted with hCRC. 
TPV was genetically modified to target carcinomas, which typically have increased levels of thymidine monophosphate (TMP) the product of thymidine kinase (TK), by deleting the viral TK gene (66R).  In this study, we have tested a number of TPV recombinants (TPV/egfp, TPV/∆2L, TPV/∆2L/∆66R, TPV/∆2L/∆66R/flic, TPV/∆66R/flic, TPV/∆66R) for their ability to replicate in the human OC cells (including the cell line A2780).  These viruses were also tested for their ability to decrease OC cell viability in OC cell lines (including the cell line A2780).  The positive results of the in vitro work lead logically to test the same viruses against the same cell line (A2780) in induced xenografts in nude mice.  Once the tumors reached 75 mm3, mice were randomly assigned to groups for treatment (mock or recombinant TPV at a concentration of 5 x 106 pfu/100 µL intratumorally).  The tumor volume was measured every 3 days.
The cell viability assay showed that TPV decreased cell viability by 44.1% on average, in an 8 day period.  The viral replication assay showed that on average the viruses replicated 15.1 fold in 8 days.  The mouse xenograft model showed that TPV/∆66R, and to a greater extent TPV/∆2L, were capable of significantly decreasing tumor growth rates, with TPV/∆2L/∆66R trending towards significance.  Our results suggest that TPV is an effective OV in vitro and TPV/∆2L shows tumor burden reduction in vivo.  Support for this study was provided by a FRACAA grant from WMU.

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Evaluating Genomic Patterns in Lactobacilli Species

Katelyn Wormmeester and Harris, Laura K., Davenport University, Lansing, MI, 48933

Over 125 species belong to the genus Lactobacillus, whose members assist in fermentation and digestion.  Eighteen of these species have fully sequenced, publically available National Center for Biotechnology Information genomes with a wide range of %(G+C) values; 32.9% for L. salivarius to 51.5% for L. fermentum.  One would expect a tighter range of %(G+C) for genomes in the same genus.  Ergo, this project aims to understand better the taxonomy of this large, diverse group of organisms by evaluating the nucleotide, absolute codon, and amino acid frequencies for 12 of these 18 organisms and 29 of their strains.  Frequencies were calculated using Python programs and chi-squared analyses done using MiniTab statistical algorithms.  Out of the strains examined, only Lactobacillus acidophilus 30SC had statistically different absolute and amino acid frequencies compared to the other three L. acidophilus strains (p-values = 0.000).  Lactobacillus acidophilus 30SC also had a different nucleotide frequency but it was not statistically significant with around 0.098 p-values.  Similarly, L. acidophilus 30SC had statistically significant amino acid frequency (p<0.005) for all 12 organisms examined whereas no other species had a statistically significant difference when compared to the others.  Based on the nucleotide and absolute codon frequency data, L. amylovorous, L. gasseri, L. helveticus, L. kerfiranofaciens, L. salivarius, and L. johnsonii were similar to each other but not to L. brevis, L. buchneri, L. fermentum,and L. casei, who look similar to each other.  Though findings support the separation of the genus into homofermenter and heterofermenter subspecies, more data are needed to determine if this approach is useful in differentiating facultative and obligate heterofermenters.

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Dynamics of Clostridium difficile and the Gut Microbiota in Recurrent C. difficile Infection

Anna M. Seekatz, Krishna Rao, Kavitha Santhosh, and Vincent B. Young, University of Michigan Medical School, Ann Arbor, MI 48103

Clostridium difficile infection (CDI) is a leading nosocomial infection, resulting in up to $3B dollars of excess healthcare costs annually. Disruption of the gut microbiota is correlated to initial CDI susceptibility; however, it is currently unknown why 20-30% of CDI patients experience disease recurrence following treatment. This study investigates the potential microbial factors that may impact susceptibility to recurrence in CDI patients. To identify the microbial factors involved in developing recurrence, we used 16S rRNA sequencing to compare changes in the gut microbiota during recurrent CDI. We compared the gut microbiota of patients with recurrent and nonrecurrent CDI over time. Additionally, we compared these to the gut microbiota of 14 recurrent patients treated with fecal microbiota transplantation (FMT) and their healthy donors. We observed that the composition of recurrent patients was dominated by members of the Enterobacteriaceae family. Conversely, the microbiota of patients treated with FMT resembled that of the donor rather than the community before FMT. Although we observed variability among all comparisons, the overall diversity of both Bacteroidetes and Firmicutes was highest in healthy donors. To explore whether pathogen variability impacted susceptibility to recurrence, we isolated C. difficile strains from recurrent CDI patients and compared strain type (ribotype). C. difficile ribotype F014-020 was the most common ribotype found in both recurrent and nonrecurrent patient populations. These results are part of an ongoing study on the development of recurrent CDI. Future prospective studies in patients that develop recurrent CDI may be helpful in identifying specific community members that confer colonization resistance against C. difficile. This poster was presented at the 2015 ClostPath conference in Freiburg, Germany.

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Isolation of Bacteriophage against Pseudomonas aeruginosa Resistant to Antibiotics in Undergraduate Research

James F. Graves (F/S), Adam S. McDonald (UG), Hung M. Nguyen (UG), and Alina Ivaniv (UG), University of Detroit Mercy, Detroit, MI 48221

Pseudomonas aeruginosa is an important cause of opportunistic infections and is highly antibiotic resistant.  The purpose of this investigation was to isolate bacteriophage against  antibiotic resistant P. aeruginosa and examine the antibacterial activity.  Antibiograms produced  by the disk diffusion technique on LB (Luria-Bertani) agar showed that P.  aeruginosa strains examined had sensitivity to ciprofloxacin and gentamicin, but some grew up to disks of chloramphenicol, erythromycin and tetracycline.  Bacteriophage were successfully isolated from municipal sewage and the sediment of a tributary of the Detroit River in contrast to other selected sites by a host strain enrichment technique.  Propagation of a bacteriophage isolated from sediment on strain ATCC 13388 (suitable for pathogen research and biodegradability testing) in agar overlay produced eluent with a titer exceeding 1011 plaque forming units (PFU)/ml.  Plaque diameter in a .4% agar overlay was only about 1 mm.  Addition of a divalent cation (calcium or magnesium) did not appear to enhance bacteriophage replication.  Routine test dilution (RTD) assay showed complete zones of lysis on lawns of cells on agar for dilutions up through 10-6.  The zone for the 10-8 dilution showed incomplete lysis.  The bacteriophage demonstrated lytic zones for 3/5 strains tested, but would not lyse Pseudomonas cepacia or Pseudomonas stutzeri.  The effect of bacteriophage on growth of P. aeruginosa in broth, in absence of other antibacterial factors, was assessed by measurement of culture optical density (OD) with a Klett-Summerson colorimeter.  Inclusion of bacteriophage at a multiplicity of infection (MOI) of approximately 1.0, after 24 hr. incubation, resulted in a bacterial culture with OD 24, in contrast to a culture without bacteriophage with OD 127.  Electron microscopy revealed that some P. aeruginosa bacteriophage have combined capsid symmetry.  Bacteriophage should be investigated as an antibacterial agent for some types of P. aeruginosa infections.

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Insertion of HPV16 E7 T Cell Epitope 10-21 into the Coat Protein of Bacteriophage PP7 does not affect the Assembly of the Coat Protein into Virus-like Particles

Rupsa Basu, Justin Workman, and Ebenezer Tumban, Department of Biological Sciences, Michigan Technological University, Houghton, Michigan

Available prophylactic vaccines against human papillomaviruses (HPVs) do not have any therapeutic effects in individuals who are already infected with HPVs or have HPV-associated cancers. Current treatments for HPV-associated cancers rely on surgery, chemotherapy, and radiation. Although chemotherapy treatment can be an effective treatment option, side effects from cytotoxicity and resistance to drugs can emerge. Immunotherapy using HPV E6 and E7 peptides or proteins targeting E6 and E7 oncogenes, respectively, can be an effective and safe approach for targeted treatment of HPV-associated cancers; immunizations with E6 or E7 peptides or proteins elicit cytotoxic T cells responses. However, these responses are effective in regressing HPV-associated tumors only to a certain extent; E6 or E7 peptides or proteins are less immunogenic. The immunogenicity of E6 or E7 peptides can be enhanced by immunizing the peptides in the context of an antigen (or complex) that mimics microorganisms in terms of size, morphology, and adjuvanticity. Virus-like particles (VLPs) have these features; they are empty viral coat proteins (capsid) that resemble the virus from which they are derived in terms of size, morphology, and T cell responses (they lack viral genomes and are absolutely noninfectious). Thus, VLPs can be used as platforms or vehicles to engineer tumor antigens/T cell epitopes in order to enhance cytotoxic T cell responses against oncogenes of interest. To assess if HPV16 cytotoxic T cell epitopes can be engineered into PP7 VLPs (which could be used as a reagent in the future for HPV-associated cancer studies), we genetically inserted T cell epitopes from HPV16 E6 and E7 into the coat protein of PP7 bacteriophage. We assessed the ability of the recombinant E6/E7 PP7 coat proteins to assemble into VLPs by SDS PAGE and transmission electron microscopy. The insertion of E7 epitope 10-21 (but not other E6 or E7 T cell epitopes) did not prevent the coat proteins from assembling into VLPs. E7(10-21) PP7 bacteriophage VLPs can be used as a reagent to assess cytotoxic T cell responses against E7 oncogene.

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A Bivalent Epitope L2 MS2 Bacteriophage VLP to Enhance Antibody Responses against HPV L2 Capsid Protein

Lukai Zhai, Damon Stafford, and Ebenezer Tumban
Department of Biological Sciences, Michigan Technological University, Houghton, MI

Human papillomaviruses (HPVs) cause about 500,000 cases of cervical cancer and ~250, 000 deaths each year worldwide. Three prophylactic vaccines have been approved to protect against HPV infection; however, these vaccines protect only against the HPV types included in the vaccines; those that cause 70-90% of cervical cancers. Infections with more than 2 HPV types have been reported in more than 50% of patients infected with HPV thus suggesting that a small percentage of immunized patients can still be infected with HPV types not included in the vaccines. To develop vaccines that protect against more HPV types associated with cancer, we targeted the minor capsid protein, L2, that is conserved among many HPV types. In our previous study, we demonstrated that immunization with MS2 bacteriophage VLPs displaying a single conserved epitope derived from HPV16L2 provided protection from vaginal infection against a broad range of diverse HPV types associated with cancer. However, protection against some HPV types was suboptimal. To enhance immune responses targeting the L2 capsid protein, we have developed a bivalent epitope L2 MS2 bacteriophage VLP targeting L2 epitopes from both HPV16 and HPV31. Our preliminary data shows that immunization with the bivalent epitope L2 bacteriophage VLPs slightly enhances the immune response against HPV16L2 and HPV31 L2 peptides compared to immunization with bacteriophage VLPs display a single epitope from HPV16 L2.

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Quantitation of toxin-induced diarrhea in zebrafish infected with Vibrio cholerae

Kristie C. Mitchell, Melody N. Neely, and Jeffrey H. Withey, Wayne State University, Detroit, MI

Vibrio cholerae is a Gram-negative aquatic bacterium that is responsible for cholera, an acute intestinal infection in humans characterized by profuse watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the infrastructure for proper water and sewage treatment. There is mounting evidence that fish in the wild serve as reservoirs for V. cholerae between human outbreaks of cholera. Our lab has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization and transmission.
Within six hours of infection with V. cholerae, zebrafish develop diarrhea. Prior work in our lab has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. This could be an overlooked method of V. cholerae transmission in the environment. Despite some initial difficulty in devising a reliable method of quantifying fish excretion, we have made recent progress. We have adapted use of OD­600 cell density readings, protein and mucin assays. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish, and consequently, which toxins may play a role in environmental transmission. This poster was also presented at the 22nd Annual Midwest Microbial Pathogenesis Conference (2015).

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Characterization of anti-viral elements in Vibrio cholerae

Brendan J O’Hara, Amelia C McKitterick, Zachary K Barth, Kimberley D Seed Department of Molecular, Cellular and Developmental Biology, University of Michigan Ann Arbor, MI, 48109

The bacterium Vibrio cholerae has long burdened humanity as the causative agent of the disease cholera. In the aquatic environment and within the human intestinal tract V. cholerae must evolve to defend against predatory bacterial viruses (phages). One particularly prevalent phage, ICP1, was recently shown to encode a CRISPR-Cas system. The phage-encoded CRISPR-Cas system is used to target a region of the V. cholerae chromosome, the phage-inducible chromosomal island-like element (PLE). Initial studies showed that in the absence of CRISPR-Cas activity, PLE prevents the ability of ICP1 to form plaques. We performed experiments towards a quantitative and mechanistic understanding of PLE activity. We identified five PLEs, which share eleven core genes and exhibit a similar phenotype: upon phage infection PLE is induced to excise and replicate, killing the infected cell before progeny phage can be released. These five PLEs were identified by whole genome sequencing of V. cholerae isolates collected between 1949-2011. Despite the homology between PLEs, we found that each PLE is protective against different CRISPR-Cas deficient ICP1 isolates. All five PLEs are specifically induced by ICP1 infection, and are not responsive to infection by other unrelated phages or by the SOS response. PLEs completely block phage replication compared to otherwise isogeneic PLE- V. cholerae, which permits robust phage replication (~100 particles per cell).  Using qPCR we determined that PLE copy number increases nearly 1000-fold following ICP1 infection. We hypothesize that this replication is in direct competition with the infecting phage, depleting the necessary machinery and metabolites needed for phage replication. Additionally, we discovered that following induction PLEs can be packaged and transduced to V. cholerae, where they integrate into the bacterial chromosome in a site-specific manner. Comparative analysis of these elements establishes PLEs as intriguing mobile viral defense islands and demonstrates that the evolution of V. cholerae has been specifically impacted by ICP1 predation.

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