In Silico Evidence of a TPP-Binding Protein Family in Methanococci Archaeal Species

Laura K. Harris, Rutgers University, PhD candidate, Davenport University, Faculty, Lansing, MI, 48933

Creative learning approaches for geomicrobiology activities for K-12 students

Zahra M. Ahmad, Dean Horton, Scott Leppert2, Deric R. Learman, Central Michigan University, Mount Pleasant, Michigan 48859 and 2Beal City High School, Mount Pleasant, Michigan 48858

Starting with the first spark of life on Earth, microbes, minerals, and the environment have co-evolved to play a key role in transforming elements that are required for life as we know it.  While the interactions between microbes, minerals, and the environment (called Geomicrobiology) is important, these interactions are difficult to K-12 students as elements and microbes are typically invisible to the naked eye.  An additional issue is that geomicrobiology may seem too complex for younger students.  One way to engage students on this topic is redirecting the classroom in which students learn from a traditional lecture, to more a critical thinking approach with collaborative learning which will promote the student’s interest and ability to grasp concepts beyond simply memorizing terms. This poster presents alternative methods to teaching microbial relationships in the environment through creative labs in combination with classroom approaches. Teachers can incorporate these age specific labs in their own classrooms to excite students about the microbial world. We created novel activities that combine with learning approaches that not only present content to students, but deliver them in a memorable and creative way.

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Development of Reagents for Chikungunya Virus Vaccine-related Research

Rupsa Basu, Justin Workman and Ebenezer Tumban, Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931

Chikungunya virus (CHIKV) infection is raising concerns across the globe. The virus is transmitted to humans by mosquitoes and it is associated with musculoskeletal pain. Despite the fact that neutralizing antibody epitope-targets have been identified on the virus, there are no vaccines to protect against CHIKV infections. In order to develop reagents that could be used to assess immune responses against CHIKV infection, we explored the possibility of genetically displaying a CHIKV neutralizing epitope, E2EP3, on the surfaces of highly immunogenic platform, such as bacteriophage AP205, Qβ and MS2 virus like particles (VLPs). Genetic insertions of E2EP3 on the coat proteins of these bacteriophages did not affect the expression of the coat proteins. However, the insertions affected the assembly of the recombinant E2EP3-coat proteins into VLPs suggesting that E2EP3 cannot be genetically displayed on some sites on these VLPs. To explore if the peptide can be displayed by an alternative approach, we used a crosslinker (SMPH) to chemically conjugate E2EP3 to Qβ VLPs. Our results show that E2EP3 can be displayed on Qβ VLPs by chemical conjugation. Studies are ongoing to assess the immunogenicity of E2EP3 on these Qβ VLPs.

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Phenotypic Characterization of Soil Isolates Paenibacillus sp., strains A1 and A3

Crisha Barrett, Sarah Place, and Anne M. Spain, Ferris State University, Big Rapids

Survey of Small Hypothetical Proteins in S. aureus

Kuana School and Harris, Laura K., Davenport University, Lansing, MI, 48933

Antibiotic Resistance Patterns Among Bacterial Isolates Obtained from Clinical vs. Community Settings

Ashley A. Beaudry and Spain, Anne M., Ferris State University, Big Rapids, MI  49307

An increasing public health concern is the prevalence of multidrug resistant bacteria and their ability to cause life-threatening infections. These microbes do not respond to frequently used antibiotics as a result of continuous exposure and are at a higher risk of developing resistance to last-line antibiotics; thus, treating related infections can be extremely difficult and at times impossible. Being aware of where multidrug resistant microbes exist is crucial in the prevention of acquiring such infections. The objective of this experiment was to demonstrate that multidrug resistant bacteria would be more prevalent on surfaces in a clinical setting vs. surfaces in a non-clinical built environment.  Bacterial isolates were obtained from three doorknobs in the long-term care section of a nursing home and three doorknobs from a college campus building and tested for antibiotic resistance using Kirby Bauer Assays. As expected, isolates from the nursing home were more resistant to antibiotics than those isolated from campus. Of the 24 clinical environment isolates obtained, eight were susceptible to all antibiotics tested compared to ten of the 20 community environment isolates. While none of the campus isolates were resistant to more than one antibiotic, six nursing home isolates were resistant to two or three antibiotics.  One of these stained gram-negative and was resistant to penicillin, ampicillin, and cephalothin, while two stained gram-positive and were resistant to penicillin, ampicillin, and erythromycin.  While results indicated that multidrug resistant bacteria are more prevalent in a clinical environment compared to a non-clinical environment, future research should survey more types of surfaces and include a larger number of isolates, which should be identified and further characterized.

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Hydrogen peroxide is a major environmental driver of microbial community composition and toxicity during a Lake Erie cyanobacterial harmful algae bloom

Derek J. Smith1, Michelle A. Berry1, Rose M. Cory1, Vincent J. Denef1, Kevin A. Meyer1, Timothy W. Davis2, Gregory J. Dick1 ,1: University of Michigan, Ann Arbor, MI, 48109, 2: NOAA-Great Lakes Environmental Research Laboratory, Ann Arbor, MI, 48108

Anthropogenic eutrophication and annual cyanobacterial harmful algae blooms (CHABs) have had a deleterious impact on Lake Erie, a system of both ecological and socio-economic importance. CHABs are complex, involving diverse groups of microorganisms. The main genus of concern is Microcystis sp. because of its abundance and potential to produce microcystins, a class of hepatotoxins. Chronic exposure to microcystins has been linked to increased rates of liver cancer in humans, and these compounds led to the 2014 Toledo, Ohio drinking water crisis that forced roughly 500,000 people to be without tap water for over 48 hours. Because of this, the contamination of drinking water supplies by microcystins has become an increasing public health concern. Experimental evidence has implicated that reactive oxygen species (ROS), in particular hydrogen peroxide (H2O2), may play an important role in determining the toxicity of CHABs. Therefore, it is critical to understand the sources and sinks of H2O2 in the environment in order to understand and predict CHAB toxicity. Historically, the main source of H2O2 in natural waters was thought to be photochemical reactions with chromophoric dissolved organic matter (CDOM). However, substantial dark production of H2O2 in natural waters has been recently reported, and this production is hypothesized to be from biological activity. Indeed, ROS (including H2O2) are known byproducts of both aerobic respiration and oxygenic photosynthesis. Interestingly, ROS can cause extensive damage to lipids, protein, and nucleic acids due to their highly reactive nature; therefore, aerobically growing microorganisms require strategies to quench ROS and repair ROS-mediated damage. From this evidence, we hypothesize that microbial activity and community structure both influence and are influenced by seasonal variation in H2O2 concentrations in the environment.
To investigate the dynamics between H2O2, algal and microbial growth, and bloom toxicity, we performed weekly sampling of a western Lake Erie CHAB during the summer of 2014 at three stations (WE12, WE2, WE4) in tandem with NOAA-GLERL’s HAB monitoring program. 20 L water was collected from 0.5 m below the surface – 1 m from bottom to form a depth-integrated sample, and measurements of hydrogen peroxide, algal pigments, community genetics, and microcystins were taken. To study the microbial community, a size fractionating filtration was performed to separate the “Microcystis-associated” and “full community” fractions. The “Microcystis-associated” fraction was collected by filtering 2 L of depth-integrated water through a 100 μm mesh, then the filtered biomass was backwashed into a falcon tube and collected on a 1 μm Glass Fiber Filter with a syringe. “Full community” samples were obtained by syringe-filtering 150 μL of water onto a 0.22um Millipore Express Plus filter. All collected filters were stored in RNAlater and frozen at -80 °C until DNA extraction was performed back at the lab. Shotgun DNA sequencing was performed on a subset of the samples using Illumina HiSeq PE125 v4 reagents. The ratio of toxic:non-toxic Microcystis was measured by comparing toxin gene (mcyD) copy number to Microcystis 16S rRNA copy number via qPCR and/or BLAST search against the shotgun data. A pilot metagenomic analysis was performed on two samples collected on July 8th and August 4th respectively based on the observed trends in hydrogen peroxide concentration, pigment, and toxic:non-toxic Microcystis ratio. Genomic bins were constructed from the resulting dataset by clustering the assembled scaffolds by tetranucleotide frequency in an emergent self-organizing map (ESOM), and the scaffolds were mined for genes encoding catalase (CAT) and bifunctional catalase-peroxidase (katG), two of the main enzymes recognized for detoxifying exogenous hydrogen peroxide.
Our data support that H2O2 corresponds to CHAB toxicity and influences the composition and function of the microbial community. Phycocyanin concentrations peaked at all stations in early August and corresponded to peaks in particulate microcystin concentrations. Interestingly, peak hydrogen peroxide concentrations occurred at each station one to two weeks before the initial peaks in phycocyanin and microcystin. Stations WE12 and WE2 also exhibited peaks in phycocyanin in late September, but these did not coincide with substantially elevated levels of microcystin or H2O2. The highest ratios of toxic:non-toxic strains of Microcystis were observed immediately preceding the peak in H2O2 at stations WE12 and WE2, indicating that H2O2 could favor the dominance of toxic strains of Microcystis over their non-toxic counterparts in the environment. Finally, our metagenomic analysis revealed that the abundance of CAT and katG gene copy number, as well as the number of bacterial groups that contain the gene, increased at WE12 immediately after the peak in H2O2. This indicates that high concentrations of H2O2 favor the proliferation of microbial strains that have mechanisms to dismutate exogenous H2O2. Furthermore, the proliferation of these strains simultaneously may lead to the decline of H2O2 back to baseline levels. Overall, our results provide evidence for the importance of H2O2 as a determinant of microbial community composition and CHAB toxicity.

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Pathogenic Bacteria S. aureus on the Footwear of Healthcare Workers

Carissa L. Paiz, Lake Superior State University, Sault Ste. Marie, MI 49783

Millions of people every year stay in the hospital for an infection. 1 out of 25 of these people will contract another infection while being treated for the first infection. These infections, known as healthcare associated infections (HAI’s), are caused by the transfer of an infectious bacteria from one patient to another. Staphylococcus aureus (S. aureus), a bacteria that becomes harmful when it enters the body, has been found to cause several of these infections. If these infections are methicillin-resistent Staphylococcus aureus (MRSA), it is more dangerous to patients because it is resistent to antibiotics.  S. aureus can spread on the clinical footwear of healthcare workers, and it has the potential to cause HAI’s because of the lack of infection control protocols for footwear, and how they are commonly worn between patients without cleaning. For this experiment, the clinical footwear of LSSU Nursing Students were swabbed and cultured to observe for S. aureus. The distance bacteria could travel on footwear plays a role in its transmission from one patient to another and produces HAI’s. Therefore, the distance bacteria could travel on footwear was also tested. After culturing the swabbing samples, no S. aureus was observed on any of the samples. After four replications, the average distance that a bacteria traveled was 37.8m thus demonstrating a potential issue with infection control should a pathogenic bacteria come into contact with healthcare worker’s footwear.

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Survey of Hypothetical Proteins with High Number of Cysteine Residues

Jessica Marklevitz and Laura K. Harris, Davenport University, Lansing, MI, 48933

An analysis was conducted on Staphylococcus aureus hypothetical proteins in an effort to predict potential functions. Thirteen hypothetical proteins were selected at random. These proteins were then analyzed using the following programs respectively: Expasy, BLASTP, PSI-BLAST, Pfam, (PS)2, STRING, STRING, and Disulfind. ExPASy analyses examined the physiochemical features of each protein.  It deemed seven proteins unstable in a test tube, making future wet bench experiments troublesome, so they were removed from further consideration. BLASTP and PSI-BLAST are performed to identify the best-fit proteins based on sequence and structure. Proteins without a BLAST or PSI-BLAST were removed from further study because insufficient information is available to make predictions on their function. The remaining proteins were: SAOUHSC_00219; SAOUHSC_00455; SAOUHSC_00766; SAOUHSC_01931; SAOUHSC_02435; and SAOUHSC_02911.  Protein domains, tertiary structure, potential functional partners, and disulfide bridges were examined. From these results, SAOUHSC_00219 appears to be involved in sugar metabolism.  SAOUHSC_00455 is an enzyme, but its specific function is unclear.  Protein SAOUHSC_00766 is a competence-related protein.  Protein SAOUHSC_01931 may be an NTPase, but most programs could not find information in this protein based on current knowledge, so it will not be examined further.  SAOUHSC_02435 is a member of the Major Facilitator Superfamily.  SAOUHSC_02911 has uncharacterized domains and inconsistent BLAST and (PS)2 findings.  Disulfind did not find any disulfide bridges in this protein set, regardless of cysteine composition.  Further analysis using CDD-BLAST for domain identification and fpocket for active site characterization will be performed.

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Evaluating Codon Usage in Lactobacilli Species

Jacob Lopez and Laura K. Harris, Davenport University, Lansing, MI, 48933

Since the time of Louis Pasteur, Lactobacilli organisms have been of research interest.  Even though this broad group of lactose fermenting bacteria has divided over the centuries, over 120 species still belong to the genus Lactobacillus, a sub-group of Lactobacilli.  This subgroup has members that assist in fermentation and digestion, making them a focus for probiotic therapy.  Eighteen of these species have fully sequenced, publicly available National Center for Biotechnology Information (NCBI) genomes with a wide range of %(G+C) values; 32.9% for L. salivarius to 51.5% for L. fermentum.  The %(G+C) range should be tighter 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 13 of these 18 organisms.  Frequencies were calculated using Python programs and chi-squared analyses performed using MiniTab statistical algorithms.  Based on the nucleotide and absolute codon frequency data, Lactobacillus species seem to group with other species with similar fermentation type though this method did not clearly distinguish between groups statistically.  While organisms tended to fall along fermentation preferences when examining amino acid frequencies, none was statistically different from another.  These findings were similar to those presented earlier.  These findings potentially support the separation of the genus into obligate homofermenter and heterofermenter subspecies, though this approach is not useful in differentiating facultative heterofermenters.  New methods of evaluating coding bias are being investigated.

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Microbial Community Response to Lake Ice Freezing

Timothy M Butler, Anna-Catharina Wilhelm, and Stephen M Techtmann, Michigan Technological University, Houghton MI, 49931

Genome sequence of a sulfate-reducing bacterium associated with cyanobacterial mats reveals metabolic versatility in a redox-stratified sulfidic environment

Matthew J. Medina, 2Kinsman-Costello L., 3Voorhies A.A. and 1Dick Gregory J., 1University of Michigan, Ann Arbor, MI, 2 Kent State University, Kent, OH, 3J. Craig Venter Institute, Rockville, MD

The Middle Island Sinkhole lies 20 meters below the surface of Lake Huron and hosts cyanobacterial mats that live under low-oxygen and sulfidic conditions due to groundwater intrusion. Because of these conditions, the MIS microbial mats represent a modern analogue of Proterozoic microbial mats ecosystems. Arguably, one of the important groups of microorganisms that occur in this mat are the sulfate reducing bacteria (SRB) because they play a key role in the biogeochemistry. As producers of sulfide, they influence the balance of oxygenic versus anoxygenic photosynthesis, both of which can be conducted by cyanobacteria at the MIS. SRB are also responsible for sulfur isotope fractionation, which may provide unique signatures of Proterozoic cyanobacterial mats. SRB are important but their genomes are poorly represented in this and other environments. The goal of this research is to reconstruct the genomes of SRB from environmental samples with the intent of understanding the MIS microbial ecology.   Metagenomic and metatranscriptomic methods were used to determine the functional components of genomes. Through genomic binning of metagenomic data, nearly complete genomes of a number of sulfate reducing bacteria were identified. Through genomic binning of metagenomic data, we identified a nearly complete genome of a member of the order Desulfobacterales. Annotation of this genomic bin revealed genes that indicate cell motility and signaling (chemotaxis and phytochromes), consistent with environmentally-directed migration. Additionally, genes for use of diverse electron donors (various organic compounds) and acceptors (oxygen, nitrate, sulfate and polysulfide) suggest a versatile energy metabolism. The genomic content of the Desulfobacterales bin suggests that cell migration and metabolic versatility are adaptions to a redox-stratified environment where the redox front moves on a diurnal cycle. This research promises to enhance our understanding of the microbial ecology in anoxic environments and early Earth geobiology.
**Poster has been presented at another meeting—Midwest Geobiology

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Coaggregation Of Gut Bacteroides & Parabacteroides With Probiotic Lactobacillus Rhamnosus GG

Samuel Schotten, Gregory Kruse, Kasem Ghannam, Radhika Hulbatte, and Daniel Clemans, Eastern Michigan University

Coaggregation is the interaction between genetically distinct bacteria and has been indicated as a key mechanism in the formation of microbial biofilms.  Bacteroides sp. are one of the most abundant clades of bacteria in the human gastrointestinal tract (GI) and has the potential to coaggregate with a variety of probiotic bacteria within the microbial community.  This research sought to characterize the coaggregative interactions between several gastrointestinal Bacteroides sp.and Parabacteroides sp. and the probiotic strain Lactobacillus rhamnosus GG (LGG) using in vitro coaggregation assays and assess the influence of growth medium on coaggregative ability.  Mixtures of bacterial suspensions were assayed for coaggregation using qualitative scoring scale with controls for autoaggregation.  The survey revealed a relatively wide spectrum of moderate to strong coaggregation with the most prominent interactions occurring with LGG grown in Todd Hewitt medium and nine of the 21 Bacteroides/Parabacteroides strains tested.  A narrower range of interactions was seen with LGG cultured in other media, including deMn, Rogosa, and Sharpe and Brain Heart Infusion.  Bacteroides oleiciplenius DSM 22535 was the only partner to show consistent coaggregation with LGG grown in all three media.  Bacteroides eggerthii DSM 20697 emerged as the strongest partner of LGG.  In addition, LGG showed selective ability to coaggregate consistently with B. thetaiotaomicron capsular type CPS 2 and 8, and a capsule-deficient null mutant.  Use of scanning electron microscopy has revealed that a morphologically-based variation in coaggregation may be occurring amongst the growth medium types of LGG.  Taken together, LGG shows a culture-medium-dependent ability to coaggregate with a variety of human GI tract Bacteroides sp. and Parabacteroides sp.

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Ultraviolet radiation as a ballast water treatment strategy: Inactivation of viruses measured with plaque assay

Vanessa Reichel-Deland, Yiseul Kim1, Samuel Snow2, Joan B. Rose1
1Department of Fisheries and Wildlife, 2Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI

Ballast water is used by cargo ships to improve their stability during movement. The water that is used is typically taken up in one port and discharged at the port of final destination. Transport of the water leads to disturbance of the native biodiversity and the ecosystem. Ballast water is known to contain variety of microorganisms, including viruses, which exist through parasitic relationships. Public health problems might occur with the transport of ballast water including biological materials. To prevent the introduction and spread of aquatic invasive species through the ballast water, there is a need to treat/inactivate organisms prior to ballast water discharge. Ballast water treatment, however, is one of the most significant challenges for the shipping industry, as technologies for ballast water treatment are still in the research and development phase. Therefore, this study evaluated UV irradiation in reducing viruses present in different types of water. Four different viruses seeded in four types of water were tested in this study to examine effectiveness of UV irradiation in reducing virus concentration. Different viruses showed different resistance to UV irradiation, while the type of water does not play a major role in the UV effect of virus removal. The study shows that UV irradiation has potential for virus removal as a ballast water treatment method.

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Characterization of Coaggregation Of Gut Bacteria With Lactobacillus Rhamnosus GG

Kasem Ghannam, Radhika Hulbatte, Samuel Schotten, Gregory Kruse, & Daniel Clemans, Department of Biology, Eastern Michigan University

Bacteroides sp. are one of the most abundant clades of bacteria in the human gastrointestinal tract (GI) and has the potential to coaggregate with a variety of probiotic bacteria. Coaggregation is defined as the cell-cell interactions between genetically distinct bacterial strains. Previous studies showed that Lactobacillus rhamonosus GG (ATCC53103) (LGG) shows extensive coaggregation between several representative human GI tract strains of Bacteroides sp. and Parabacteroides sp. We characterized the coaggregation interactions between LGG and representative GI tract Bacteroides sp. and Parabacteroides sp. by protease inhibition assays, carbohydrate inhibition assays, and hydrophobicity assays. Using protease inhibition, it was confirmed that the presence of a novel adhesin(s) occur on LGG, mediating coaggregation to moderate strength with a variety of Bacteroides sp. and Parabacteroides sp. strains, and with certain capsular-deficient strains of B. thetaiotaomicron VPI-5482.Carbohydrate inhibition was used to determine if these interactions were mediated by lectin-like adhesins. Two different pools of monosaccharides and disaccharides were made in coaggregation buffer each at a concentration of 160mM: Lactose, D(+)-Glucose, L(+)-Arabinose, D(-)-Arabinose and L(-)- sorbose in pool 1; and D(-)-Mannose, Sucrose, Maltose and α-Rhamnose in pool 2. The LGG grown individually in Todd Hewitt (TH), deMan, Rogosa, and Sharpe (MRS) and Brain Heart Infusion (BHI) media were resuspended in the carbohydrate pools and tested for coaggregation with GI tract Bacteroides sp. and Parabacteroides sp. The survey revealed broad spectra of results with few bacteria ranging from complete inhibition of coaggregation to no inhibition of coaggregation. Finally, hydrophobicity was measured on all cultures using a hexadecane partition assay. This assay revealed that Bacteroides sp. with higher hydrophobicities tend to have stronger interactions with LGG. In particular, Bacteroides eggerthii, B. fragilis, and B. stercoris have the strongest interactions with LGG. It was found that LGG had a very low hydrophobicity that did not appear to be media dependant. Taken together, these data indicate that LGG contains at least one surface adhesin that mediates coaggretation with various Bacteroides sp. and Parabacteroides sp. Some of these interactions may be mediated by cell-surface lectins.

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