Indiana University-Purdue University Indianapolis (IUPUI) / For regenerative endodontic procedures (REPs) to be successful an elimination of bacteria from the root canal system must be accomplished. Many different medicaments with antibacterial properties have been used to obtain complete disinfection. Double antibiotic paste (DAP) containing a mixture of ciprofloxacin and metronidazole has been shown to be a promising intracanal medicament. The addition of a radiopaque filler such as zirconium oxide to DAP may affect the antibacterial properties of DAP as well as allow precise placement and radiographic visualization of its position in the canal system. The aim of the proposed study was to evaluate the direct antibacterial properties of zirconium oxide radiopacifier combined with DAP (RoDAP) against a multispecies biofilm from a bacterial isolate from an infected immature tooth with a necrotic pulp and a known single species biofilm. 4x4 mm radicular dentin specimens (n = 112) obtained from human extracted teeth were used prepared and sterilized prior to use. A multispecies clinical bacterial isolate from an immature tooth with a necrotic pulp and a single species Enterococcus faecalis isolate were obtained. These bacterial isolates were used to inoculate dentin slabs and grown for 3 weeks. The dentin slabs were treated for 1 week with 1.0-mg/mL and 10- mg/mL RoDAP, 1.0-mg/mL DAP, and two placebo pastes consisting of methyl cellulose (MC) and methyl cellulose combined with zirconium oxide (RoMC), respectively, as well as two no-treatment controls. Following treatment, the grown biofilm was detached and spiral plated. The plated biofilm cells were cultured for 24 hours and each group examined using a colony counter to determine bacterial numbers (CFUs/mL). Data analysis, using a 5.0-percent significance level was conducted using one-way ANOVA followed by pair-wise group comparisons. Both 1.0-mg/mL and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from an immature tooth with a necrotic pulp as well as an E. faecalis isolate. The precise application of RoDAP confirmed radiographically with its direct antibacterial properties may be beneficial for intracanal disinfection during REPs.

Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed. / 2021-08-21

Listeria monocytogenes response to oxidative stress by sublethal sodium hypochlorite was investigated in this study. Continuous exposure of sublethal chlorine influenced biofilm formation and stress adaptation (homologous and heterologous) in L. monocytogenes. The biofilm forming ability of oxidative stress adapted and control cells were investigated on polystyrene surface at 22°C and 37°C. The oxidative stress adapted cells were found to form less biofilm in the presence of chlorine (p < 0.10) when compared to non-treated control cells at both the temperatures. In addition, the biofilm forming ability of L. monocytogenes was reduced significantly at higher sublethal chlorine concentrations (p < 0.10). In conclusion, oxidative stress adapted L. monocytogenes has developed tolerance to chlorine and some of the antibiotics. However, oxidative stress those cells did demonstrate an antibiofilm effect. This demonstrates that oxidative stress reduces L. monocytogenes biofilm formation but can also increase antibiotic resistance.

Plants rely on innate immunity to perceive and respond to pathogenic microbes. Pathogenic microbes suppress and evade plant immune responses to obtain nutrients and multiply resulting in plant diseases and death. One battleground for the arms race between plants and microbial invaders is located in the leaf intercellular space, specifically between Pseudomonas bacteria and Arabidopsis. This thesis seeks to understand the virulence mechanisms that allow Pseudomonas bacteria to grow within the leaves of Arabidopsis and how the plant immune response reduces pathogen growth and reproduction. Some plant pathogens produce specific extracellular polysaccharides to potentially enhance pathogenicity during infection of plants. The objective of this thesis is to understand the importance of biofilms for Pseudomonas success and determine if Arabidopsis suppresses biofilm formation as part of the plant immune response. It was hypothesized that biofilm formation contributes to Pseudomonas success in planta and Arabidopsis suppresses biofilm formation during PAMP-Triggered Immunity (PTI) to reduce bacterial growth. Wild-type plants and defense mutants were infiltrated with flg22 or mock (water) treatments to induce or mock-induce PTI in plants, followed by observing GFP-expressing Pseudomonas via florescence microscopy to determine if biofilm-like aggregate formation was occurring. In vivo studies in this thesis indicate that biofilm-like aggregate formation contributes to bacterial success during Arabidopsis infection. Additionally, the phytohormone, salicylic acid (SA), accumulates in leaf intercellular spaces of resistant plants during PTI that suppresses biofilm formation, suggesting that SA acts as an anti-microbial and anti-biofilm agent that contributes to the suppression of pathogen growth during plant defense. / Thesis / Master of Science (MSc)

The increasing prevalence of bacterial infections has made it necessary to find novel methods of combatting the resistance of bacteria to conventional antibiotics. Mycobacterium abscessus is an increasingly prevalent pathogen that is intrinsically drug resistant, therefore difficult to treat. The use of phytochemicals as a source of alternate antibiotics has been explored, however, the poor solubility of phytochemicals in water makes it difficult to effectively deliver them to bacterial biofilms. In this study, I investigated the efficacy of nanosponge-emulsified phytochemicals in killing M. abscessus biofilms. The nanosponge technology was used to improve the solubility and stability of the phytochemicals, allowing for improved bioavailability. Results showed that the nanosponge-emulsified phytochemicals effectively reduced the viability of M. abscessus biofilms, compared to non-emulsified phytochemicals. The findings of this study contribute to a development of new strategies for the treatment of bacterial infections and demonstrate the potential of nanosponge-emulsified phytochemicals as a promising alternative to conventional antibiotics.

Biofouling represents the main problem in membrane filtration systems. Biofouling arises when the biomass growth negatively impacts the membrane performance parameters (i.e. flux decrease and feed channel pressure drop). Most of the available techniques for characterization of biofouling involve membrane autopsies, providing information ex-situ destructively at the end of the process. OCT, is non-invasive imaging technique, able to acquire scans in-situ and non-destructively. The objective of this study was to evaluate the suitability of OCT as in-situ and non-destructive tool to gain a better understanding of biofouling behavior in membrane filtration systems. The OCT was employed to study the fouling behavior in two different membrane configurations: (i) submerged flat sheet membrane and (ii) spacer filled channel. Through the on-line acquisition of OCT scans and the study of the biomass morphology, it was possible to relate the impact of the fouling on the membrane performance. The on-line monitoring of biofilm formation on a flat sheet membrane was conducted in a gravity-driven submerged membrane bioreactor (SMBR) for 43 d. Four different phases were observed linking the variations in permeate flux with changes in biofilm morphology. Furthermore, the biofilm morphology was used in computational fluid dynamics (CFD) simulation to better understand the role of biofilm structure on the filtration mechanisms. The time-resolved OCT analysis was employed to study the biofouling development at the early stage. Membrane coverage and average biofouling layer thickness were found to be linearly correlated with the permeate flux pattern. An integrated characterization methodology was employed to characterize the fouling on a flat sheet membrane, involving the use of OCT as first step followed by membrane autopsies, revealing the presence of a homogeneous layer on the surface. In a spacer filled channel a 3D OCT time series analysis of biomass development under representative conditions for a spiral-wound membrane element was performed. Biomass accumulation was stronger on the feed spacer during the early stage, impacting the feed channel pressure drop more than the permeate flux. OCT biofilm thickness map was presented as new tool to evaluate the biofouling development in membrane filtration systems through the use of a false color scale.

The prevalence and mode of persistence of Salmonella on commercial poultry processing equipment was examined in this study. The equipment surfaces of six commercial processing plants were sampled over three-time periods designated as A (after processing), B (after cleaning), and C (after sanitization) and each plant was visited three times. Salmonella prevalence was greater (p<0.0001) at time A than times B and C, while there were no differences (p=0.386) between times B and C. A total of twentyive Salmonella isolates from five distinct serovars were recovered from the processing plants. Further examination of the recovered isolates showed that tolerance to sanitizing agents (chlorine and quaternary ammonium compounds, QAC) and/or biofilm formation was the possible mode of persistence. In addition, all the isolates were resistant to multiple antibiotics in different classes of drugs and more than two genes that are responsible for ⅰ). biofilm formation, ⅱ). resistance to QAC, ⅲ). oxidative stress response, and ⅳ). resistance to antibiotics were detected. Three selected isolates were further characterized based on virulence factors and antimicrobial resistance using whole genome sequencing (WGS) to possibly predict phenotypic characteristics. The WGS data correlates with phenotypic characteristics that were previously observed in the isolates including the ability to produce biofilms and resistance to antibiotics including β-lactams, aminoglycosides, and cephalosporins. WGS predicted the isolates carried resistance genes for antibiotic drug classes that were not observed phenotypically. These include macrolides and fluoroquinolone, which is a concern due to its use in treating foodborne infection. Furthermore, the genome of the three selected isolates were predicted to have over sixty virulence genes that allow Salmonella to invade, attach, and colonize the host cells. The results from this study suggest that the antimicrobials used for sanitization may be insufficient to inactivate Salmonella because of the ability to produce biofilms on processing surfaces. Most importantly, the results suggest the ability of the isolates to cause infection in humans meaning if contamination were to occur, the right antibiotic treatment could be a challenge. Further research is imperative to determine the effective antimicrobial for Salmonella biofilms.

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