Andrew R. Russell

Antibiotic resistance continues to challenge the medical field worldwide, and the need for alternative methods to combat antibiotic resistance is imperative. Plant lectins have shown promising results as a natural anti-biofilm agent by disrupting the biofilm’s ability to adhere by binding to specific monosaccharides in the extracellular polymeric substance (EPS). This study aimed to use the plant lectins, Con A and WGA, to agglutinate to the EPS and inhibit biofilm formation in antibiotic-resistant pathogens like Pseudomonas aeruginosa, Staphylococcus epidermidis, and Stenotrophomonas maltophilia. Initially, minimum inhibitory concentration (MIC) assays were performed, and the final concentration determined was based on a study investigating the impact of plant lectins on biofilm formation for Streptococcus mutans (0.4 mg/mL). 96-well biofilm assays were statistically significant for P. aeruginosa with Con A treatment and S. mutans with WGA treatment. These results indicate that P. aeruginosa treated with Con A reduces the biofilm’s ability to adhere, because of the high volume of mannose-specific monosaccharides in its EPS. Additionally, results for S. mutans indicate that WGA can inhibit the biofilm’s adhesion, because of the presence of N-acetylglucosamine monosaccharides in the EPS. Future research is needed to confirm if plant lectins are a natural anti-biofilm agent.

Microbial biofilms are an increasing threat to human health as they provide protection from both antibiotics and the immune system; therefore, some labs are turning their attention to discovering compounds that can disrupt their formation or break apart established biofilms. Zingerone, a compound found in ginger, has been shown to disrupt biofouling in several Gram-positive and Gram-negative species. To determine if zingerone may be a candidate for general usage, the way in which it disrupts biofouling should be elucidated. Recent results suggest that one of the ways zingerone impacts biofilm formation is by disrupting quorum sensing. Yet many quorum sensing systems exist and it is still unclear which of these are affected by zingerone. This study aims to determine if zingerone disrupts the biofouling of bacteria that use AHL, DSF, and Agr quorum sensing signaling systems. Minimum inhibitory concentration assays were used to determine the maximum concentration of zingerone that the bacteria tolerate, and then biofilm assays were used to determine whether zingerone affected biofilm formation. While this study did not see a statistically significant decrease in biofilm formation in the presence of zingerone, visible differences between the control and treatment wells of Agrobacterium tumefaciens in the qualitative results suggest that technical error may have diminished any statistical significance in the quantitative data.

Dental caries is the most prevalent childhood disease, affecting nearly 30% of children worldwide. Caries occur when oral bacteria form biofilms on tooth surfaces and produce acidic by-products from residual metabolites. While regular brushing and mouth washes are effective measures for some, additional preventative methods are needed to decrease caries in individuals colonized by aggressive biofilm-forming species. Previous research has demonstrated that zingerone, a component of ginger, can decrease biofilm formation in other bacterial species, perhaps by preventing communication and coordination of gene expression. This project investigated if zingerone reduces biofilm formation in the oral bacteria Streptococcus mutans and Lactobacillus gasseri. Each bacterial species was grown and placed in a Drip Flow Biofilm Reactor and treated with a diluted media and zingerone solution. Biofilm formation was analyzed using a confocal microscope and Live/Dead stain. Although biofilm formation of both strains decreased with zingerone treatment, there was not a significant difference between experimental and control groups for S. mutans. Only two trials of L. gasseri were completed in this study; therefore, statistical analysis was not yet possible to determine if the reduction of biofilm in this species was significant. Nevertheless, this research models the dynamic environment of medical treatments and can be used as a bridge between traditional static biofilm assays and clinical trials. Additional trials with increased sample size may reveal zingerone’s ability to significantly decrease biofilm formation in a simulated mouth

environment.

As of 2015, nearly 80% of the world’s virgin plastic ended up in a landfill or the natural environment, a majority accredited to the packaging industry. Due to the composition of the durable plastic, polyethylene terephthalate (PET), the primary polymer found in single‐use packaging, is not naturally biodegradable and thus accumulates. Recently, several microbes were discovered that could degrade the plastic compounds within their gut with no signs of toxicity. Ideonella sakaiensis is a novel bacterium capable of depolymerizing PET using a hydrolase termed PETase. An additional enzyme, MHETase, aids in converting dimers for the complete recovery of monomers within I. sakaiensis . Ultimately, efficiency in degrading PET of varying crystallinity occurs with a preference of low crystallinity PET, in opposition to other species currently known. During degradation, I. sakaiensis forms a thin biofilm over the plastic. It is known that the cells contain special appendages to adhere to the plastic surface, however, little research has been conducted on how the biofilm formation affects the degradation process. Multi‐species biofilms have been shown to create complex communities for enhanced PET degradation in marine plastic debris using other bacterial strains. This research aims to study the biofilm formation as well as its effects on its degradation properties of I. sakaiensis as an isolate, as well as a dual species biofilm with known biofilm forming bacteria: Stenotrophomonas maltophilia, Staphylococcus aureus , and Pseudomonas aeruginosa . The first half of the project characterized the effects of introducing biofilm forming bacteria on I. sakaiensis’ biofilm growth. The second half of the project analyzes the biofilm growth along with a degradation assay to determine the effects of these bacterial strains on the degradation process of I. sakaiensis. These effects were then evaluated to determine the role of biofilm formation in the degradation process. This study provides a foundation that can be used by the biotech, environmental, and molecular communities to lead to a commercialization of plastic degradation products, ultimately creating a circular and stable economy within the consumer world.

Beer draught lines are frequently contaminated with biofilm-forming microorganisms, which forces retailers to spend considerable time and money cleaning and replacing lines. In light of this financial burden, draught tubing composition was examined for its role in the prevention of biofouling in beer lines. Three types of draught tubing - vinyl, polyethylene, and nylon barrier - were inoculated with a combination of biofilm-forming microorganisms (Hafnia paralvei, Raoultella planticola, Pediococcus damnosus and Saccharomyces cerevisiae) and used to simulate a bar environment for sixteen weeks. Following simulation, the degree of biofouling in each draught line was determined by spectrophotometry and microscopy. Absorption values and fluorescence images showed that nylon barrier tubing was superior to the other lines at resisting biofilm maturation.These results suggest that tubing composition plays a significant role in the prevention of biofilm formation in beer draught lines and supports the adoption of nylon barrier tubing as an effective strategy against biofouling in a variety of applications.

Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is harmless to healthy individuals. However, it has become an emerging pathogen in immunocompromised individuals because it readily forms biofilms on both hospital devices and contact lenses. Furthermore, treatment of resulting infections is often difficult, since this bacterium is naturally resistant to many antibiotics. The goal of this study is to prevent S. maltophilia from forming biofilms on contact lenses in the first place, which should reduce the incidence of eye diseases such as keratitis in immunocompromised individuals. Previous studies have shown that S. maltophilia is capable of cell-to-cell communication through quorum sensing, providing a possible target for biofilm inhibition. We tested several quorum sensing blockers for their ability to prevent S. maltophilia biofilms on contact lenses. One of these compounds, zingerone, reduced both surface biofilms, as well as, growth on coverslips and contact lenses. These results are intriguing, since zingerone is a naturally-occurring compound in ginger. Thus, it could easily be added to contact lens solutions as an extra measure of protection against bacteria that cause eye disease in immunocompromised individuals.

The Pseudomonas syringae effector AvrB triggers a hypersensitive resistance response in Arabidopsis and soybean plants expressing the disease resistance (R) proteins RPM1 and Rpg1b, respectively. In Arabidopsis, AvrB induces RPM1-interacting protein kinase (RIPK) to phosphorylate a disease regulator known as RIN4, which subsequently activates RPM1-mediated defenses. Here, we show that AvrPphB can suppress activation of RPM1 by AvrB and this suppression is correlated with the cleavage of RIPK by AvrPphB. Significantly, AvrPphB does not suppress activation of RPM1 by AvrRpm1, suggesting that RIPK is not required for AvrRpm1-induced modification of RIN4. This observation indicates that AvrB and AvrRpm1 recognition is mediated by different mechanisms in Arabidopsis, despite their recognition being determined by a single R protein. Moreover, AvrB recognition but not AvrRpm1 recognition is suppressed by AvrPphB in soybean, suggesting that AvrB recognition requires a similar molecular mechanism in soybean and Arabidopsis. In support of this, we found that phosphodeficient mutations in the soybean GmRIN4a and GmRIN4b proteins are sufficient to block Rpg1b-mediated hypersensitive response in transient assays in Nicotiana glutinosa. Taken together, our results indicate that AvrB and AvrPphB target a conserved defense signaling pathway in Arabidopsis and soybean that includes RIPK and RIN4.

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog.