Browsing by Subject "Biofilm"

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that accumulate in soils and sediment due to their physicochemical properties. In these environmental matrices, PAHs are predominantly transformed and degraded by the native fungal and bacterial communities. However, microbial degradation of PAHs is a slow process that requires engineered approaches to improve degradation rates to meet remediation criteria.

Engineered bioremediation approaches consist of altering the microbial community by either increasing cell concentrations of specific, targeted organisms or by introducing catabolic genes that confer for a phenotype that can degrade the target contaminant. This approach is called bioaugmentation and is generally applied using the former strategy. Biostimulation is another method, which includes the addition of nutrients that may be limited to microorganisms and can help grow the indigenous microbial community and accelerate contaminant degradation. However, biostimulation is not a targeted approach and may stimulate the entire microbial community, not just organisms capable of degrading the target contaminant.

Bioaugmentation of sediments is challenging due to constraints surrounding the longevity, stability, and delivery of microorganisms. To address the limitations of this remediation approach, the work within this dissertation outlines methods for developing a consortium of PAH-degrading bacteria coordinated within a stable community, as well as a technology for delivering this consortium to creosote contaminated sediments.

The first objective was to identify and isolate PAH-degrading bacteria from creosote contaminated sediment. Sediment was collected from sites along the Elizabeth River, VA and a 16S rRNA amplicon library of sequences was analyzed to generally evaluate the influence of chemical contamination on the bacterial community structure. To detect PAH-degrading organisms within sediment communities, DNA-SIP using uniformly labeled stable isotopes of phenanthrene and fluoranthene were prepared in incubations with Republic Creosoting site sediment. Clones derived from this experiment revealed one prominent degrader of phenanthrene and two prominent fluoranthene degrading bacteria. In an attempt to isolate these and other PAH-degrading organisms for laboratory evaluation, culture-based methods were employed and resulted in the successful isolation of 6 unique bacteria, including one strain which was detected in the DNA-SIP experiments. Overall, it was determined that PAH-degrading bacteria exist in Republic Creosoting site sediments, although not in significant relative abundance compared to other bacteria. This finding suggests that these contaminated sediments could be a good candidate for a bioaugmentation approach.

Most of the research on bioremediation has focused on organisms in isolation and existing in a free-floating, or planktonic, cellular state. The second objective of this dissertation was to confirm the PAH-degrading capabilities of isolated bacteria and to coordinate these organisms into a biofilm structure, which provides protection and additional community benefits to participating microorganisms. To this end, we employed a high-throughput, reproducible assay to confirm whether or not isolated bacteria are capable of coordinating within a biofilm. We also used culture-based methods and performed incubations with multiple types of PAHs to determine if the isolated organisms can interact with PAHs of various size and ring number. Finally, we used a metabolic assay for the novel application of assessing the respiration capacity of the isolated PAH-degrading bacteria in the biofilm conformation, to determine if these organisms are metabolically active when they are situated within a biofilm. We found that all of the organisms isolated were capable of forming a biofilm that was metabolically active. Many of these organisms demonstrated the ability to degrade phenanthrene and fluoranthene, but only a few showed the potential for degrading pyrene. These results confirmed that the isolated organisms from Republic site sediment can degrade PAHs and form a biofilm structure, which will be beneficial for their application to sediments in a bioaugmentation strategy.

The final aim of this work was to evaluate the use of an activated-carbon amendment based technology for the delivery of a bacterial consortium to PAH-contaminated sediment. While validated for use as a remediation technology and delivery strategy for organisms capable of degrading polychlorinated biphenyls (PCBs), this approach has not yet been tested for use with sediments contaminated with PAHs.

The focus of this dissertation is the extension of the innate immune response in wound healing and non-wound healing contexts. I am interested in interactions at the interface between macroorganisms and microorganisms from marine/aqueous environments. This dissertation explored two aspects of the interactions: 1) the presence and function of macroorganism secretions and 2) the role of secretions in managing microfouling on macroorganism surfaces. Particularly of interest are how barriers are biochemically reinforced to mitigate microfouling and the potential consequences of a breach in those barriers. The innate immune response, an evolutionary conserved system in vertebrates and invertebrates, provides an evolutionary context for developing the hypotheses.

In this dissertation the biochemical composition and uses of crustacean secretions are explored for barnacles, fiddler crabs and blue crabs. Fluids of interest were secretions released during barnacle settlement and metamorphosis and those collected from living adult barnacles, fluids on fiddler crab sensory appendages including dactyl washings and buccal secretions, and fluids from blue crab egg masses. The biochemical composition was determined using a combination of fluorescent probes and confocal microscopy, proteomics, and enzyme-specific substrates with a spectrophotometer.

I demonstrated that self-wounding is inherent to the critical period of settlement and metamorphosis, in barnacles. Wounding occurs during cuticle expansion and organization and generates proteinaceous secretions, which function as a secondary mode of attachment that facilitates the transition to a sessile juvenile. I showed extensive proteomic evidence for components of all categories of the innate immune response, especially coagulation and pathogen defense during attachment and metamorphosis. This work provides insight into wound healing mechanisms that facilitate coagulation of proteinaceous material and expands the knowledge of potential glue curing mechanisms in barnacles.

In order to test macroorganism secretions in a non-wound healing context, I examined fluids sampled from body parts that macroorganisms must keep free of microorganisms. I showed that two types of decapod crustaceans can physically manage microorganisms on most parts of their body, but certain parts are particularly sensitive or difficult to clean mechanically. I examined sensory regions on the fiddler crab, including dactyls that are important for chemoreception and the buccal cavity that is used to remove microorganisms from sand particles, and blue crab egg mass fluids that protect egg masses from fouling through embryo development.

This dissertation explores organismal interactions across scales in size, space, and time. The findings from the barnacle work inform mechanisms of attachment and glue curing, both central to understanding bioadhesion. The work on fiddler crabs and blue crabs contributes to our understanding of chemoreception of feeding and reproductive behaviors.

The work presented here highlights how biological secretions from macroorganisms serve multifaceted roles. In cases of physical breaches of barriers, or wounding, secretions coagulate to obstruct loss of hemolymph and have antimicrobial capabilities to prevent infection by microorganisms. In non-wounding cases, secretions remove microorganisms from surfaces, whether that is on the body of the macroorganism or in the immediate environment.

Total joint arthroplasty (TJA) is the replacement of damaged or arthritic joints with synthetic components to improve patient mobility and quality of life. It is a highly effective procedure with a low rate of complications. However, due to the sheer volume of TJA operations, which is over a million per year in the U.S., there are tens of thousands of patients who experience severe complications, such as mechanical loosening, periprosthetic fracture, implant failure, and periprosthetic joint infection (PJI). Perhaps the most severe is PJI, which is an infection of the tissue surrounding the implant, generally as a result of a pathogen, primarily Staphylococcus aureus (S. aureus), entering the joint through the open wound during the surgical procedure. Although the immune system can usually eliminate invading pathogens effectively, the presence of a foreign body, in this case a synthetic joint, significantly reduces the concentration of bacteria required to induce infection due to the ability of bacteria to attach and grow on the surface of the implant. Over time this leads to pain, redness, swelling, and weakening of the joint that could necessitate amputation if untreated.The gold standard treatment for PJI is a two-stage exchange. In the first stage, the infected device is removed, and the infected tissue is debrided and irrigated to eliminate as much of the infection as possible. Then, a new device, called a spacer, is implanted into the joint in place of the original prosthetic. A spacer is an articulating device composed of poly(methyl methacrylate) (PMMA), commonly called bone cement, that releases antibiotics long-term to treat any remaining infected tissue. The advantage of PMMA over possible alternative biomaterials is 1) the ability to mix antibiotics into the cement before molding it into a desired shape, 2) a porous structure that enables antibiotics to leach from the matrix, and 3) sufficient mechanical strength to support partial load-bearing activity during the treatment period. Currently, the FDA has only approved pre-formed spacers with gentamicin, which limits them from treating bacteria that are resistant to gentamicin. While there used to be a mold available for surgeons to form PMMA spacers in the operating room with antibiotics of their choosing, that mold has since been recalled by the FDA. The spacer requires 6-12 weeks of antibiotic elution to eliminate the infection. After the surgeon determines the infection is gone, the second stage is the removal of the spacer and replacement with new sterile prosthetic components. The second stage is necessary since PMMA is too weak to support long-term full load-bearing activity, so the spacer must be replaced with stronger metal components to make sure the implant does not break, which could lead to serious consequences for the patient. The weak mechanical properties are a significant limitation of PMMA during the 6-12 week treatment period because the spacer frequently breaks when patients do not use ambulatory assist devices to facilitate mobility. A broken spacer can lead to soft tissue and bone damage and requires an additional surgery to replace the spacer. Several surgeries can be painful for the patient and extremely costly to the healthcare system. An ideal alternative spacer would have greater mechanical strength to reduce mechanical complications and the number of surgeries needed to treat PJI. In addition to poor mechanical properties, a second limitation of PMMA spacers is an inadequate antibiotic release profile to treat PJI. Since PMMA is non-biodegradable, antibiotics mixed throughout the cement cannot easily escape from the matrix to elute into the surrounding tissue. In fact, only antibiotics close to the implant surface are able to escape. This causes a burst release of antibiotics in the first few days of implantation with sub-therapeutic release thereafter. This can not only lead to persistence of the infection but can also induce antibiotic resistance, making it more challenging to treat the infection. Furthermore, PMMA cement sets with an exothermic reaction that is incompatible with many antibiotics. Currently only certain antibiotics can maintain their therapeutic efficacy following the exothermic reaction. Overall, PMMA spacers are an ineffective treatment for PJI due to poor mechanical properties and antibiotic release. An ideal alternative device would have greater mechanical strength and long-term release of therapeutic levels of antibiotics to effectively eliminate an infection. In this dissertation, I propose a novel spacer that can more effectively treat PJI vs. PMMA spacers due to the ability to modulate its antibiotic release to a desired profile, increase its mechanical strength tantamount to primary prosthetic components used in TJA, and minimize the ability of bacteria to attach and grow on its surface. The proposed technology takes advantage of the rapidly growing technology of additive manufacturing, also known as 3D-printing, which enables the manufacture of devices with complex structures. I propose 3D-printing joint devices with an internal reservoir to be loaded with a biodegradable carrier that releases antibiotics through 3D-printed channels that connect the reservoir to the surrounding tissue. While conventional manufacturing methods cannot make an internal reservoir with channels, 3D-printing enables precise control of the reservoir and channel geometry to tune the antibiotic release profile. A major advantage of this design over PMMA is the separation of the biomaterial that provides mechanical strength and the biomaterial that releases the antibiotics. In this way, I can 3D-print the device from a material with greater strength than PMMA, such as cobalt-chrome and titanium alloys, and I can load the reservoir with a biodegradable material that releases therapeutic levels of antibiotics over longer periods of time than PMMA. In chapter 3 of this dissertation, I assess the ability to modulate antibiotic release from 3D-printed reservoirs. I vary the geometry of reservoirs, including channel diameter, length, and quantity, to determine the effect on the in vitro antibiotic release from a carrier, in this case calcium sulfate, which is a biodegradable bioceramic frequently used as a bone void filler. Decreasing channel diameter and increasing channel length extends the release of antibiotics by extending the distance the antibiotics must diffuse through the carrier to reach the surrounding medium. To understand the mechanism of release, I develop a computational model of antibiotic release by simulating the reservoir as a 3D matrix in MATLAB. I model diffusion as a random walk and degradation as a progressive erosion over time. After fitting the diffusion and degradation constants to the data, the model effectively predicts the antibiotic release profile of a large range of reservoir geometries. This simple model can help rapidly prototype devices with a customized antibiotic elution profile to effectively treat PJI. In addition, I tested two carriers, calcium sulfate and tri-calcium phosphate, which degrades more slowly than calcium sulfate. Increasing the ratio of tri-calcium phosphate to calcium sulfate extends the release of antibiotics by decreasing the degradation of the bioceramic. Thus, modifying the properties of the carrier is another method to tuning antibiotic release in addition to reservoir geometry. Lastly, I studied the mechanical properties of 3D-printed reservoirs. Increasing the diameter of the channels decreases the mechanical strength of the reservoirs due to reduced structural support of the 3D-printed material. However, using 3D-printing, I can create lattice structures within the reservoir to provide additional mechanical support while still leaving sufficient volume to load an antibiotic-impregnated carrier. Overall, chapter 3 shows a multitude of methods to flexibly modulate both antibiotic release and mechanical strength to improve the ability to treat PJI vs. PMMA. In chapter 4, I study the ability of bacteria to form biofilm on the surface of different orthopedic materials, including 3D-printed metals that could potentially be used to make reservoirs. Biofilm is a conglomerate of microorganisms, most commonly S. aureus in PJI cases, that forms on the surface of an implant. Biofilm organizes in such a way to provide protection from the host immune system and antibiotics, making biofilm significantly harder to eliminate than planktonic, or free-floating, bacteria. It requires up to a thousand times higher concentration to eliminate bacteria in biofilm vs. planktonic bacteria. Therefore, in addition to high mechanical strength and long-term antibiotic release, another important property of an effective spacer is a reduced affinity for bacteria to form biofilm on the implant surface. I use a CDC biofilm reactor to model the in vivo conditions of PJI to grow bacteria on the surface of numerous relevant biomaterials. Using several quantitative methods, I found that biofilm grows more readily on non-polished 3D-printed metals and plastics, such as PMMA and ultra-high molecular weight polyethylene compared to polished and machined metals. To understand the mechanism, I measured the surface roughness and hydrophobicity of the materials. Materials with greater roughness and hydrophobicity developed more biofilm than smoother, hydrophilic materials. Metals are more hydrophilic and can be polished to a lower surface roughness value than PMMA. The data show that PMMA is not only mechanically weak and has a poor antibiotic release profile but also provides an additional surface for bacteria to readily form biofilm that can potentially worsen the existing infection. Taken together, this dissertation provides a comprehensive study of the advantages of hand-polished 3D-printed metal spacers with antibiotic-eluting reservoirs to effectively treat PJI vs. PMMA spacers due to 1) improved mechanical properties, 2) the ability to modulate antibiotic release to a wide range of release profiles, and 3) a reduced affinity of bacteria to form biofilm on the spacer surface.

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.