Esiobu, Nwadiuto

Emerging insights on the role of microbiomes in the sustainability of ecosystems and plant cover are transforming knowledge-driven agro-environmental management practices. For more than a century, the Brazilian pepper tree -BP (Schinus terebinthifolius), a category 1 invasive plant in Florida has defied numerous conventional control measures directed at its well-known ecology. This dissertation is one of the pioneer studies designed to determine whether microorganisms play a role in the aggressive invasion of BP in Florida and examine potential mechanisms with the goal of creating supplemental restoration tools. To test the hypothesis that enhanced mutualism of Brazilian pepper tree with microbes, compounded by relatively low biotic resistance of Florida soils is a critical driver of its invasion, plant biomass indices, metagenomics analysis of microbial community shifts, electron microscopy of endomycorrhizal infection and qPCR of key rhizobacterial taxa were measured. A multifactorial grow-room experiment was conducted simulating invasion with BP and two Florida natives (Pinus elliottii and Bidens alba) in a sterile, bioinoculant supplemented, and non-sterile control soils with various plant combinations.

Bacteria are inarguably the most ubiquitous and adaptive organisms on the planet. The vast, diverse community of microbes residing in soil are mostly studied using sequencing technologies because over 99% of them are currently uncultivable in the laboratory. This lack of diverse bacterial cultivation presents a serious challenge for modern microbiological and medical science where the discovery of novel antibiotic producers and microbial products has been outpaced by the rise in drug resistance. This study designed and tested two new cost-effective culture systems called the “Dynamic Soil Environment” and Soil Extract Systems with the goal of increasing the cultivable communities of diverse bacteria in a soil sample over standard methods. Illumina MiSeq sequencing and DADA2 pipeline protocols were used to analyze community DNA from cultivated samples and source soil metagenomes. Autoclaved soil extract media in the Soil Extract Experiment yielded a statistically significantly greater Shannon’s (p = 0.008) and Simpson’s diversity (p = 0.007) of bacteria over pH modified (6.4) nutrient agar media over 30 days of incubation. Autoclaved soil extract media was also able to cultivate, on average, 33% of species in bulk soil sequences compared to 27% from standard nutrient agar however these differences weren’t statistically significant. The length of incubation had a lesser effect than media type on yield of bacteria over 30 days in batch culture conditions. Species richness and diversity generally decreased over time except in soil extract samples. In the Dynamic Soil Environment experiment, membrane plates placed on a live soil environment produced a slightly higher diversity than autoclaved membrane plates and control plates without soil, however, these differences were not statistically significant except when analyzed with Chao1 diversity (0.041).
Cultivated bacterial diversity and communities differed more according to media type than soil environment with statistically significant differences between standard and pH modified nutrient agar. Media with a 5.8 pH buffer produced a significantly higher relative abundance of the well-known antibiotic-producers, Actinobacteria (t(10) = -5.715, p < .000) and also Proteobacteria (t(10) = -10.127, p < .000). This study establishes cost-effective methods of cultivating more diverse bacterial communities for low-funded laboratories. Culture conditions for the reliable cultivation of higher relative abundances of bacterial groups belonging to Actinobacteria and Proteobacteria are also established with the Dynamic Soil Environment Experiment.

Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of
lives in Africa and could become a global health threat. Because of the danger of
infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress
due to both the cost and expertise required to operate these laboratories. The development
of a safe surrogate would speed research and reduce risk to researchers.
Two highly conserved Ebola gene segments—from the glycoprotein and
nucleoprotein genes—were designed with modifications preventing expression while
maintaining sequence integrity, spliced into high copy number plasmids, cloned into
E.coli, and tested for stability, safety, and potential research applications. The surrogates
were stable over 2-3 months, had a negligible mutation rate (<0.165% over the
experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL.
These protocols could be used to safely simulate other pathogens and promote infectious
disease treatment and detection research.

Background: Prostate Cancer, in the absence of skin cancer, is the most prevalent type of cancer found
in the male population. Reactive Oxygen Species (ROS) can promote cancer cell proliferation when
they are at elevated levels. Vitamin C is a water-soluble antioxidant capable of inhibiting the formation
of ROS. Genistein, an isoflavone found in plants, also possesses the ability to inhibit ROS formation.
Objective To determine the potential therapeutic synergy between genistein and vitamin C and
investigate mechanism of action of genistein and/or vitamin C. Methods: Trypan blue assay was carried
out to know the % of viable cells. Varying concentrations of genistein with a constant concentration of
Vitamin C was used to treat LNCaP cells. After treatment of the cells with genistein and Vitamin C, MTT
assay of the cancer cells was performed and absorbance read through an ELISA reader. This gives the
values needed for interpreting cell viability after treatment. A statistical analysis performed to determine
whether the obtained results are statistically significant. Results: The results obtained from our
experiments are inconclusive with regards to the impact of Vitamin C on apoptotic cancer cell death
following genistein treatment. However the combination of genistein and vitamin C was more efficient in
tumor suppression than when the drugs were given separately. Conclusion: This study suggests that
treatment of prostate cancer using genistein can be enhanced by adjuvant treatment with vitamin C.
This study is of potential clinical success in reducing the cell death by necrosis.

Defining the bacterial communities associated with healthy status will permit rapid
detection of shifts associated with disease and foster effective probiotic intervention. This study
compares the PCR-RFLP of oral metagenomes and cultured bacterial community, as well as the
bacterial diversity profile of smokers, non-smokers and oral disease patients. The goal was to
evaluate the stability of bacteria associated with health and capture possible shifts potentially
diagnostic of smokers and oral disease.
Oral wash samples were obtained from 5 healthy and 5 smokers twice, 3 months apart. Samples
from 5 oral disease patients were also collected. Metagenomic and the genomic DNA of a
cultured subset were amplified using primer 1492R and 27F.The generated 16SrRNA gene
amplicons were used for arestriction digestion assay and bcloning with the Gene Hunter PCRTrap
vector and pCR 4- TOPO cloning kits. The restriction fingerprints were statistically tested
using Gel Compare II. The cloned 16S genes were sequenced using the ABI GeneAmp 9700
Thermal cycler. Sequences were analyzed by BLAST on the RDB II database and the HOMD.
Sau 3AI and Alu I produced the best distinctive markers of 300 bp in healthy nonsmokers and
500bp in smokers in plate wash communities. The 16S sequence data suggest the presence of a
core microbiota in all subjects mostly Streptococcus, Gamella, Candidatuse spp and confirmed
that the smokers harbored highly diverse and distinct community Neisseria pharynges, Rothia
mucilaginosa. Remarkably, there was a high stability of the fingerprints and diversity profile for
smokers and nonsmokers after 3 months.

The overuse and misuse of antibiotics in environmental and clinical settings have been a
driving force for the prevalence of bacterial resistance. In constant interaction with these
chemicals which can harm them, adaptively and inherently, bacteria have devised resistance
mechanisms to combat the deleterious effects posed. In the presence of a particular antibiotic, it
is expected there will be selection of resistant micro-organisms and their associated resistance
genes if present. In this study, a set of 10 samples were taken from recreational beaches in Ft.
Lauderdale, Miami and Hollywood and four different agricultural soils. These soils were
enriched after being collected aseptically with three commonly used antibiotics; Ciprofloxacin,
Tetracycline and Vancomycin to select for resistant organisms, which produced 29 total samples.
A metagenomic analysis was done with 16S rDNA amplification with primers 27F and 1492R
which produced 14 out of 29 amplicons producing the expected ~1400 bp fragment from the
conserved SSU 16S rDNA region using Agarose gel electrophoresis. From these 14 samples
amplified, a second PCR would be run from each enriched antibiotic sample with their respective
antibiotic resistance primers eg. vanA-D, tetO and qnrA to identify the resistance genes present
expecting that the antibiotic used for enrichment would select for the resistant organisms. Future
work includes the sequencing of the amplified resistance genes to help identify novel genetic
alterations indicative of new adaptive mechanisms.

Purpose: This study was designed to define the antibiotic resistance index of the
cultivable oral microbiome to Amoxiacilin Clavulanic acid, Vancomycin, Ciprofloxacin,
Clarithomycin, Chlorotetracyclin, Bacitracin, Kanamycin and Tobramycin using a new method
adapted from the Kirby Bauer assay.
Method: Oral wash samples were collected from 2 current smokers and 2 nonsmokers. Bacterial
community were pelleted by centrifugation and used to create a lawn for the assay employing
standard disk diffusion assay. Zones of inhibition and number of colonies in the zone were
recorded. Mean values of inhibition zones were compared to established databases to draw
conclusions.
Result: The zones of inhibition of Bacitracin antibiotics shows that several bacteria from one of
the non smokers were resistant to Bacitracin, while the smokers showed marked susceptibility.
Conclusion: The new method developed in our lab yielded consistent set of data which serve as
criteria for determining resistance of the oral microbiome to antibiotics. Quite remarkably, it is
known that pathogenic beta Streptococci are susceptible to Bacitracin while non-pathogens are
not; confirming that healthy persons harbor the healthy strains of streptococci. However the
unanswered question is …. Could these normal biota pick up genes and become resistant too?
Only time and human habits will decide but we have developed a baseline and an easy method
for testing.

The interphase (littoral zone) of the Florida Everglades
and surrounding marine environments are
dominated by mangrove forests and mudflats that
transition into freshwater communities. As salinity
levels change, plant cover of the freshwater community
will be adversely affected, but perhaps more
importantly, their microbial symbionts responsible for
their tolerance to high salinity will be impacted as
well. Microbes respond quicker to changing environments
and could provide an early warning of the
loss of resiliency of the Florida coastline ecosystem.
For 10 weeks, soil samples from Biscayne National
Park will be placed in standardized mesocosms and
subject to local conditions like sunlight, tides, plants,
increased sea levels, higher salinities, and variant
inundation. Standard and variant conditions will
be employed on different mesocosms, and weekly
DNA extractions of the various soil samples will
be analyzed via metagenomic analysis, creating a
library of the 16S rRNA community structure over the
10 week period.

The composition of marine bacterial communities from South Florida beaches
were characterized using 16S rRNA sequence analysis. To compare cultivable and noncultured
populations, community genomic DNA was extracted directly from sand and
seawater samples and from two cultured equivalents. Only two ofthe 86 (2.3%) direct
extracted sequences and 79 of 150 (52.6%) culture sequences belong to known isolates in
Ribosomal Database (version 9.0) at 95% confidence level. At low stringency (p=0.70),
the populations cluster into several unknown clads with early divergence, indicating the
presence of novel well established bacterial groups. Members of phylum Bacteroidetes,
Firmicuites and Proteobacteria were identified, with the latter being the most prevalent in
culture. Diversity indices rank Hollywood beach > Fort Lauderdale > Hobie beach.
Taxonomic representation indicates marine water as more diverse compared to dry sand
and wet sand. A combination of phylogenetic markers will be needed to define the
immense diversity of this niche.

The Brazilian pepper tree (BP, Schinus terebinthifolius), introduced to the United States
in the 1800s, has since become a category one invasive plant in Florida, aggressively
spreading to 3000 km2 of prime habitat. There is a serious dearth of knowledge on
whether the rhizobiome plays any roles in the displacement of native flora and the range
expansion of BP. This thesis discusses the well-established plant invasion mechanisms of
the BP and highlights key emerging mechanisms and gaps in (a) the current
understanding of the molecular, below-ground processes of BP invasion and (b) studies
on the potential role of microbial interactions in the success of BP invasion already
established for other select invasive species, and the intervention of soil metagenomic
studies to elucidate plant invasive mechanisms. These poorly studied mechanisms could further explain the aggressive spread and resilience of BP and contribute significantly to
the development of effective and sustainable control measures, enabling appropriate
strategies for restoring native plants.