12 research outputs found
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What is killing the corals? Viral and bacterial interrogations using metagenomics and microscopy
Corals have multiple roles in maintaining ocean health and are some of the world’s most diverse ecosystems. The coral animal is host to a multitude of taxa, including symbiotic dinoflagellate algae, fungi, bacteria, protists, and viruses. Environmental stressors and disease agents can perturb the delicate balance of the coral host and its microbiota, which can lead to disease. This ultimately results in reduced fitness and/or mortality. There are over 30 described coral disease signs, but with a few exceptions, little is known about their potential etiological agents. Without knowledge on the causes of these diseases, little can be done to mitigate future outbreaks. Chapter 1 of this dissertation provides examples for studying novel diseases in difficult to study organisms with the use of metagenomic tools. Armed with these metagenomic tools and electron microscopy, this dissertation aimed to determine potential pathogens in two coral diseases, White Plague disease (WP), and Growth Anomalies (GAs). First, I set to determine whether viruses may be involved in WP disease from a 2010 outbreak in the US Virgin Islands (USVI). In chapter 2, I compared viromes from 21 Montastraea annularis samples (7= Diseased, 7= Diseased=Bleached, 5= Bleached, 2= Healthy) and surrounding seawater (n=2). After comparing the viromes, I found that small circular REP- encoding eukaryotic ssDNA viruses (SCSDv) similar to circo and nano viruses were associated with WP diseased tissue, and thus potential pathogens. Electron microscopy confirmed the presence of viral particles, and absence of bacterial infection in WP diseased tissue. Even though it was likely that viruses are involved with WP it was still important to understand the changes in bacterial community and the roles of bacteriophages during WP coral infection to characterize opportunistic microbes. These questions were addressed in Chapter 3 where I described the bacterial communities and bacteriophage consortia associated with the USVI corals. I also constructed phage-bacteria networks to understand which phages may interact with bacteria of interest (those shown to be differentially abundant in Montastraea annularis of different health states). I determined that there was a range of interaction specificity across the different phages and bacteria. Chapter 4 aimed to elucidate a potential pathogen responsible for Growth Anomalies, a chronic disease resulting in skeletal deformities, loss of symbiotic algae and reduction of fitness in Hawaiin Porites lobata. By comparing microbial and viral metagenomes across health states I was able to determine any changes in composition from healthy and diseased corals. Overall, microbial communities and viral consortia did not vary across health states. The hypothesized Porites spp. symbiont Oceanspirillales was dominant in all libraries. However, relative abundances of taxa in the orders Vibrionales and Verrucomicrobiales were elevated in diseased compared to healthy corals and healthy appearing tissue of GA infected corals. In addition, bacterial functional pathways remained stable across health states, while signatures of virulence factors were elevated in diseased viromes from healthy. Lastly, chapter 5 summarized overall trends of microbes and viruses determined through the studies. In addition, suggestions for future studies were outlined. Overall, this dissertation revealed a potential viral pathogenic group for white plague disease, explored how phages can influence bacterial opportunists in corals of different health states, and determined bacterial orders and virulence factors that are associated with Growth Anomalies. This dissertation includes the first study to find an association between a viral group and coral disease. In addition, this dissertation contains the first (to date) phage-bacterial inferred network constructed from paired phage-bacteria metagenomes
A bacteriophage cocktail targeting <i>Escherichia coli</i> reduces <i>E. coli</i> in simulated gut conditions, while preserving a non-targeted representative commensal normal microbiota
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Practical Applications for Symbiodinium Grown on Solid Media: Culturing, Fluorometry and Transformations
Symbiotic dinoflagellates in the genus Symbiodinium are critical to the success of scleractinian reef corals in shallow tropical seas. These symbionts are commonly isolated from hosts and cultured separately in liquid media (f/2 or ASP8a), but initial isolations can be prone to abundant contaminants that can persist long-term in culture. To help remove these contaminants, I developed a solid growth substrate composed of 1% f/2 medium in agar, supplemented with a variety of antibiotics, to help isolate individual clones and establish new ?axenic? cultures. I found that an antibiotic cocktail of kanamycin (50 µg/mL), ampicillin (100 µg/mL) and streptomycin (50 µg/mL) was the most effective at eliminating visual signs of contamination without apparent harm to a variety of Symbiodinium in culture. Photophysiological measurements of Symbiodinium grown on f/2 agar plates, taken using an Imaging Pulse Amplitude Modulated (I-PAM) fluorometer, were comparable with those grown in liquid f/2, both with and without antibiotics. Eight types of Symbiodinium in clades A-D grown on f/2 agar plates at low irradiance (19-46 µmol photons m-2 s-1) were exposed to higher irradiance conditions (50-90 µmol photons m-2s-1) for 13 days and their photosynthetic efficiencies (Fv/Fm) were compared using the I-PAM. There were significant differences among and within clades, except for two types in clade C (C2 and C3) which did not perform differently from eachother. All types showed reduced Fv/Fm after 12 days in higher light. Type D1a showed high mortality after 13 days of higher light stress. Finally, preliminary work to fluorescently label Symbiodinium determined that available vital stains were not generally practical for symbiosis studies. Attempts to transform Symbiodinium with a variety of plasmids containing fluorescent reporters and/or genes for antibiotic resistance were not successful, but did provide a baseline for future work. In summary, Symbiodinium cultures grown on solid substrates supplemented with antibiotics are useful for: (1) isolating individual cells or clones for subsequent applications and establishing and maintaining ?axenic? cultures that are free of observable contaminants; (2) directly comparing the photophysiology of different cultures using an I-PAM fluorometer; (3) quantifying cells on agar plates using the I-PAM and (4) selecting possible transgenic symbionts for symbiosis studies.</p
Bacteriophage preparation lytic for Shigella significantly reduces Shigella sonnei contamination in various foods.
ShigaShield™ is a phage preparation composed of five lytic bacteriophages that specifically target pathogenic Shigella species found in contaminated waters and foods. In this study, we examined the efficacy of various doses (9x105-9x107 PFU/g) of ShigaShield™ in removing experimentally added Shigella on deli meat, smoked salmon, pre-cooked chicken, lettuce, melon and yogurt. The highest dose (2x107 or 9x107 PFU/g) of ShigaShield™ applied to each food type resulted in at least 1 log (90%) reduction of Shigella in all the food types. There was significant (P<0.01) reduction in the Shigella levels in all phage treated foods compared to controls, except for the lowest phage dose (9x105 PFU/g) on melon where reduction was only ca. 45% (0.25 log). The genomes of each component phage in the cocktail were fully sequenced and analyzed, and they were found not to contain any "undesirable genes" including those listed in the US Code for Federal Regulations (40 CFR Ch1). Our data suggest that ShigaShield™ (and similar phage preparations with potent lytic activity against Shigella spp.) may offer a safe and effective approach for reducing the levels of Shigella in various foods that may be contaminated with the bacterium
Reduction in the <i>Shigella</i> levels in various foods treated with bacteriophages vs. water- or PBS-treated controls.
<p>Reduction in the <i>Shigella</i> levels in various foods treated with bacteriophages vs. water- or PBS-treated controls.</p
Effect of ShigaShield on the <i>Shigella</i> levels in various foods.
<p>Mean CFU recovered per gram of food (±SEM); for each food, means with different letters are significantly different (<i>P</i><0.01). P-values are based on Tukey-Kramer multiple comparison post-hoc tests.</p
List of new <i>Shigella</i> isolates tested for susceptibility to ShigaShield<sup>™</sup>.
<p>List of new <i>Shigella</i> isolates tested for susceptibility to ShigaShield<sup>™</sup>.</p
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Relevance of Alternaria to asthma symptoms and exhaled NO among NYC children
The importance of domestic Alternaria exposure to allergic disease in urban communities is underrecognized relative to cockroach, dust mite, and mouse exposures. Concentrations of Alternaria are higher in outdoor than in indoor air; however, dampness, leaks, and resident behaviors can influence fungal penetrance and secondary growth indoors. Moreover, given the amount of time children spend indoors, domestic exposure may contribute more than outdoor exposure to asthma morbidity.
Byproducts from fossil fuel combustion are common in urban air and are the most well-established anthropogenic environmental adjuvants of allergic sensitization. Black carbon and elemental carbon (EC), indicators of combustion exposure, vary across cities such as New York (NYC) because of vehicle and residential heating sources. Among NYC children, we previously demonstrated an interaction between exposure to combustion byproducts and cockroach allergen on cockroach sensitization and an association between black carbon measured inside homes and fractional exhaled nitric oxide (Feno), a marker of airway inflammation. Therefore, combustion byproducts might enhance the effect of fungal exposure on allergic disease outcomes among NYC children.
We hypothesized that, among NYC children, (1) sensitization to Alternaria alternata would be associated with increased asthma symptoms, (2) domestic exposure to A alternata would be associated with increased Feno, and (3) the association with Feno would be modified by neighborhood EC concentrations