Biofilm formation on medical devices is responsible for a substantial portion of healthcare associated infections with approximately 99,000 deaths and estimated financial burden of $28-$45 billion annually. Given the long-standing challenges of biofilm eradication, physical and chemical surface modifications to prevent biofilm formation from the early adhesion stage, continue to gain momentum. Nanoscale structural features, ubiquitous in both natural and synthetic surfaces, are increasingly recognized to have wide-ranging effects on microorganism adhesion and biofilm development. In this thesis, bio-inspired nanofiber-coated polystyrene surfaces were developed to systematically investigate how highly ordered surface nanostructures (200nm-2000nm in size) impact adhesion and proliferation of model fungal pathogen, Candida albicans. A theoretical model for cell-textured surface interaction was also developed using thermodynamic principles to demonstrate that single cell adhesion to surface can be used to describe the population behavior. The trend for adhesion density of C. albicans on nanofiber-textured surfaces of varying diameters correlates with our theoretical finding of adherent single-cell energetic state. Findings from this thesis can be used for enhanced ab initio design of antifouling surfaces for medical applications and beyond. We demonstrate a successful prototypical example of reduction in biofilm formation by optimally designed nanofiber coating of urinary catheters. / Master of Science

The heat resistance was determined for Salmonella typhimurium and Listeria monocytogenes Scott A suspended in 2% UHT processed milk and in a biofilm matrix. Pure cultures at an initial concentration of 10⁵ / ml were used. Heat resistance was determined by two methods. One method was sealed borosilicate glass TDT tubes that were completely submerged in the heating menstrum. Biofilms were grown on Buna-n rubber o-rings (4.46 mm O.D. x 1.41 mm]. D.) for 36 hours. All other cultures used were in stationary phase of growth. The three treatments tested were: inoculated milk, sterile milk and a biofilm on an o-ring, and inoculated milk with a sterile o-ring. At the three temperatures tested (60, 63, 67°C), there was no significant difference (p>0.05) in D-values between treatments. There was a significant difference (p<0.001) between the D-values for Salmonella and Listeria. The second method used a laboratory scale HTST pasteurizer to determine the difference in heat resistance of the same organisms suspended in 2% milk vs. sloughed off pieces of biofilm in milk. Pure cultures of the organisms at an initial inoculum of 10⁵ / ml were used. Flow rates of the pasteurizer were adjusted to achieve two different F-values for each organism at a reference temperature of 71.7°C. Neither S. typhimurium nor L. monocytogenes Scott A was recovered from pasteurized samples of either treatment. The heating involved in come up and cool down of the transit lines was considered in determining F-value. Under commercial HTST processing, concentrations of 10⁵ / ml of S. typhimurium and L. monocytogenes Scott A would not survive pasteurization. The results also show that if pieces of biofilms (3.8 x 10⁻⁴ mm²- 8.8 x 10⁻³ mm²) were sloughed off gaskets in the processing lines they would not survive pasteurization. The heating characteristics of these two systems were so dissimilar they could not be compared. It should however be noted that in the TDT tubes it was necessary to obtain a slightly higher F-value before no growth was seen as compared with the pasteurizer. In the pasteurizer the laminar flow properties would contribute to a more uniform heating. The TDT tube experiences convection heating which can produce cold spots in the tubes and could explain the need for an increased F-value. / Master of Science

Microbial fuel cells (MiFCs) have been suggested as a means to harness energy that is otherwise unutilized during the wastewater treatment process. MiFCs have the unique ability to treat influent waste streams while simultaneously generating power which can offset energy associated with the biological treatment of wastewater. During the oxidation of organic and inorganic wastes, microorganisms known as exoelectrogens have the ability to move electrons extracellularly. MiFCs generate electricity by facilitating the microbial transfer of these electrons from soluble electron donors in feedstocks to a solid-state anode. While MiFCs are a promising renewable energy technology, current systems suffer from low power densities which hinder their practical applicability. In this study, a novel anode design using flame-deposited carbon nanostructures (CNSs) on stainless steel mesh is developed to improve the electron transfer efficiency of electrons from microorganisms to the anode and thus the power densities achievable by MiFCs. These new anodes appear to allow for increased biomass accumulation on the anode and may aid in the direct transfer of electrons to the anode in mediatorless MiFC systems. Experiments were conducted using anaerobic biomass in single-chamber MiFCs with CNS-enhanced and untreated stainless steel anodes. Fuel cells utilizing CNS-enhanced anodes generated currents up to two orders of magnitude greater than cells with untreated metal anodes, with the highest power density achieved being 510 mW m-2. / Master of Science

The biomass from a pilot-scale two-stage (carbon oxidation first stage, ammonia oxidation second stage) fixed-film biological aerated filter (BAF) was divided in three fractions depending on their attachment strength to the media: detached, easily detachable and strongly attached. VSS measurement showed that the detached and easily detachable fractions accounted for 25 to 40% of the biomass in the bed and are present even after backwash. Protein was the major constituent of all fractions of the biomass. The ratio of carbohydrate to protein differed between fractions and between type of biofilms, with a larger value for detached and detachable fractions and a lower value for a largely heterotrophic biofilm, implying a difference in the composition of the biomass matrix that could be related to the attachment state of the biomass. The biomass did not appear to be substrate-limited anywhere in the system, although the specific activity of the biomass was dependent upon the position in the column. Activity of the strongly attached biomass was less than 70% of the total activity, even after backwash. A mass balance on VSS showed that the backwash flushed a mass equivalent to less than 35% to 45% of the detached and detachable fractions, which was less than 15% of the total biomass present in the system. Data also suggested that during backwash, part of the strongly attached biomass was sheared off the media and regenerated the mass of biomass in the detached phase. In conclusion, it can be stated that a non-negligible part of the biomass in a BAF is in a detached state. Actual mechanistic BAF models based solely on biofilm modeling may be overlooking the role of that biomass, especially in the performance recovery of BAF systems after backwash. / Master of Science

Salmonella contamination of spices and other low water activity foods is a growing concern for the food industry due to increased frequency of salmonellosis outbreaks and detection-based product recalls. The impact of inoculation preparation on the survival of a Salmonella enterica and its proposed surrogate, Enterococcus faecium NRRL B-2385, on the whole black peppercorns and cumin seeds was examined. Three liquid inoculation methods (biofilm-inclusion, agar-grown, broth-grown) for Salmonella enterica and surrogate Enterococcus faecium and one dry transfer method for Salmonella enterica were developed then applied to whole peppercorn and cumin seeds. Spices were returned to original water activity (aw 0.3) and stored for 28 days with periodic sampling (0, 1, 7, 14, 21, 28 days) and surviving bacteria enumerated. Average log reductions (LR) over time were statistically analyzed to determine differences in stability during storage. Inoculation preparation was associated with significant differences in recovered Salmonella and Enterococcus from both peppercorn and cumin over the storage period. At 28 days, the most stable inoculations of Salmonella resulted from the biofilm-inclusion (-0.04 CFU/g LR) and agar grown (-0.75 CFU/g LR) methods on peppercorn and the biofilm inclusion method (-0.28 CFU/g LR) on cumin. Log reductions of Enterococcus faecium (-0.02 CFU/g LR biofilm-inclusion-peppercorn, -0.19 CFU/g LR agar-grown-peppercorn, -0.61 CFU/g LR biofilm-inclusion-cumin) were comparable to Salmonella after 28d desiccated storage. These results will guide the inoculation strategies for validating inactivation processes for reducing Salmonella on whole spices, and for comparisons of inactivation of Salmonella and its proposed surrogate Enterococcus faecium. / Master of Science in Life Sciences

Campylobacter jejuni is microaerophilic pathogen and is one of the leading causes of acute diarrhea in the United States. Despite being a microaerophilic pathogen, C. jejuni continues to endure within the domain of food production, especially in poultry processing. In this study, we evaluated the aerotolerance, biofilm forming abilities, and genetic diversity of C. jejuni isolates previously obtained from commercial broiler processing plants. Out of 40 isolates, 25 (62.5%) were aero–sensitive (AS), 10 (25%) were intermediately aerotolerant (IAT), and 5 (12.5%) were hyper aerotolerant (HAT). The isolates belonged to four clonal complexes (CCs) and six sequence types, with the majority of isolates assigned to the CC–353 clonal complexes. Furthermore, the biofilm forming abilities of 12 field C. jejuni isolate on stainless-steel coupons were measured using a crystal violet assay by measuring the optical density (OD600) and viable cell count was enumerated using direct plating. A notable interaction between aerotolerance categories and temperature (P < 0.039) impacting the number of biofilm-attached C. jejuni cells on stainless steel coupons. All isolates had greater counts when incubated at 42ºC compared to room temperature, regardless of oxygen level (P < 0.001). Furthermore, stronger biofilm density was observed at 42°C compared to room temperature, regardless of oxygen level. Eight C. jejuni strains including 3 AS, 3 IAT, and 2 HAT were used to understand the genomic characterization that underlies aerotolerance and biofilm formation in C. jejuni using whole-genome sequencing (WGS). Genes associated with aerotolerance, and biofilms were present in all eight isolates despite the phenotypic differences. The virulence genes associated with Type VI secretion system (T6SS) and VAS effector proteins were unique in aerotolerant isolates. Antimicrobial resistance markers related to antibiotic efflux pumps, beta-lactams, fluoroquinolones, tetracycline, aminoglycosides, and streptothricin were identified. In conclusion, this study elucidates the diverse aerotolerance profiles and genetic characteristics of C. jejuni isolates from poultry processing plants, shedding light on their ability to persist despite environmental stresses. Additionally, the biofilm forming ability at different temperatures emphasizes the importance of targeted interventions to mitigate its impact on food safety.

Biofilms have the potential to form on various abiotic surfaces and have been found to be more resistant to environmental stressors. Therefore, there is a need to investigate the biofilm forming ability and characteristics of Salmonella strains isolated from poultry meat. These studies characterized the biofilm forming capabilities of 5 Salmonella strains on abiotic surfaces, and investigated changes in gene expression that are associated with Salmonella biofilm formation in 3 Salmonella strains. Salmonella biofilms were cultivated on stainless steel, concrete, rubber, and polyethylene under static and shear stress conditions. Biofilm matrix density was determined using a modified crystal violet assay, and attached cells were enumerated by direct plating on tryptic soy agar plates. Additionally, biofilm development was verified using scanning electron microscopy. Extracellular matrix density was affected by a surface-incubation condition interaction, in which the OD600 was higher on stainless steel under shear stress, as compared to static incubation (P < 0.001). On polyethylene, the OD600 was higher under static incubation (P < 0.001). The number of attached cells was highest on polyethylene under shear stress, irrespective of strain (6.4 log/coupon; P < 0.001). The study tracked changes in the expression of the biofilm-associated genes csgD, bapA, bcsA, adrA, and luxS. The gene expression levels in 24 h planktonic cells, 4-day old biofilms, and 5-day old biofilms were compared to those in 12 h planktonic cells and normalized to 16S RNA. Three different Salmonella serovars were used in this study: Salmonella Typhimurium, Reading, and Kentucky. Upregulation of csgD, bcsA, adrA, and luxS at 24 h was observed exclusively in Salmonella Reading (P = 0.028). In 4-day old biofilms, downregulation of all 5 genes was detected (P < 0.001). However, in 5-day old biofilms, the expression of bapA, bcsA, adrA, and luxS increased across all tested Salmonella serovars. These findings highlight the variations in gene expression across different Salmonella serovars, emphasizing the need to remove and prevent Salmonella attachment on food contact surfaces. Through further investigation, it may be possible to establish environmental stress thresholds for biofilm formation and optimize intervention strategies to mitigate Salmonella attachment and persistence on industrial materials.

In this examination of microbial colonization of wood and factors affecting the structure and function of wood biofilms in streams, the first two chapters summarize research conducted in New Zealand and compare the composition of biofilm colonizing wood to that colonizing rocks and leaves. Similar biofilms developed on wood veneer, natural twigs, and beech leaves, but fungi did not colonize stones where diatoms were the predominant colonizer. Comparing wood incubated on the streambed surface to wood buried beneath the streambed, fungal hyphae dominated the biofilm but actinomycetes and bacteria were also present. An assay of microbial activity (¹⁴C glucose uptake) indicated that surface biofilms were more active than biofilms on buried wood. There was no relationship between ¹⁴C glucose uptake and stream pH indicating that acidity did not affect wood biofilm activity in these streams. Biofilm activity on wood buried at 3-9 cm in the streambed was not significantly different than that buried at 19-25 cm. Chapters 3, 4, and 5 examine the processes governing wood biofilms in the presence and absence of leaf litter in 2 small mountain streams at Coweeta Hydrologic Laboratory in the southern Appalachians. Microbial respiration, fungal biomass, extracellular enzyme activity, and the effect of nutrient addition were used as descriptors of wood biofilms. Exclusion of leaf litter from a headwater stream enhanced extracellular enzyme activity, and fungal biomass was 7 times higher than that in the reference stream. Relative activities of selected extracellular enzyme activities suggested that the biofilm in the reference stream was nutrient limited. Also, nutrient releasing substrates placed beneath wood veneers indicated co-limitation of nitrogen and phosphorus on biofilms in the reference stream. Competition for nutrients by microbial biofilms may play a regulatory role in detrital processing in these streams. Laboratory feeding studies using Tallaperla sp. were conducted to explore the suitability of wood biofilms as a food resource for shredders. There were no differences in Jallaperla growth rates on wood and leaves, and Tallaperla grew equally well on wood incubated for 1 or 2 months. At the end of each study, fungal biomass on wood in the feeding chambers was not different from fungal biomass at the beginning indicating that Tallaperla nymphs were not food limited and fungal production was able to compensate for invertebrate grazing. In the absence of leaf litter, stream shredders such as Tallaperla can survive and grow on the microbial biofilm on wood. The carbon stored in wood in streams is transferred to higher trophic levels via microbial biofilms. / Ph. D.

Pasteurella multocida is an important multi-host animal and zoonotic pathogen that is capable of causing respiratory and multi-systemic diseases, bacteremia, and infections resulting from bite wounds. The glycosaminoglycan capsule (CPS) of P. multocida is an essential virulence factor, protecting the bacterium from host defenses. However, chronic infections such as bovine respiratory disease (BRD) and avian cholera may be associated with biofilm formation. Biofilm formation was inversely related to capsule production (determined by uronic acid and N-acetylglucosamine assays), and was confirmed with capsule-deficient mutants of mucoid strains. Capsule-deficient mutants formed biofilms with a larger biomass that was much thicker and smoother than encapsulated strains. Gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion demonstrated that the matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS). Therefore, CPS may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix to encase large numbers of bacterial cells. Chemical mutagenesis was performed on P. multocida strain P1059, resulting in isolation of an acapsular mutant designated as P1059-R8. A uridyltransferase encoded by gene P1059_01979 was mutated in such a way that a polar amino acid was changed to a non-polar amino acid near the active site. The protein product of P1059_01979 is important for the biosynthesis of the CPS subunit N-acetylglucosamine. CPS quantification revealed that the subunit glucuronic acid was produced in equal concentrations to the parent, but the CPS subunit N-acetylglucosamine was not detected in the chemical mutant. Biofilm formation in the chemical mutant was significantly higher than in WT P1059 and the capsule-deficient mutant. We hypothesize that P1059_01979 is essential for CPS production in P. multocida serogroup A. Histophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Following respiratory infection of calves with H. somni, P. multocida is also often isolated from the lower respiratory tract. Because H. somni normally forms a biofilm during BRD, we suspected that P. multocida may co-exist with H. somni in a polymicrobial biofilm. Interactions between the two species in the biofilm were characterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined by fluorescently-tagged lectins (FTL), confocal scanning laser microscopy of in vitro biofilms and bovine pulmonary tissue following dual H. somni and P. multocida infection. FISH and FTL were used to show that P. multocida and H. somni were evenly distributed in the in vitro biofilm, and both species contributed to the polymicrobial biofilm matrix. COMSTAT z-stack image analysis revealed that the average biomass and biofilm thickness of the individual and polymicrobial biofilms were greatest when both species were present. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a polymicrobial biofilm with H. somni, but only the EPS of H. somni could be detected by FTL staining of bovine tissues from which both species were isolated. Bacteria within a biofilm are more quiescent than during planktonic growth and induce less of an inflammatory response, indicating encapsulated P. multocida may take advantage of the H. somni biofilm to persist in the host during less severe, but more chronic, BRD. These results may have important implications for the management of BRD. Acute avian cholera is associated with encapsulated P. multocida, while chronic and asymptomatic cases of avian cholera are associated with acapsular P. multocida isolates. We hypothesize that biofilm formation is present and an important factor for chronic and asymptomatic avian cholera. Experimental infections of chickens with biofilm deficient P. multocida strain WT X73, proficient biofilm forming P. multocida strain X73ΔhyaD, and proficient biofilm forming clinical isolates 775 and 756 showed that virulence inversely correlated with biofilm formation. Histopathological analysis showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver, while biofilm deficient isolates induced greater inflammation. Biofilm material was located in pulmonary tissues of chickens diagnosed with chronic avian cholera using FTL staining.. Quantitative real-time PCR for expression of cytokine genes in the spleens of infected chickens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. Chickens that succumbed to acute avian cholera after experimental challenge with WT X73 had high levels of INF-ƴ, IL-1β, IL-6, IL-12A, IL-22, IL-17A, and IL-17RA expression in the spleen compared to all other experimental groups. Antibody titers were low, indicating that antibodies may be less important in managing and clearing P. multocida infections. / Ph. D. / Pasteurella multocida is a zoonotic pathogen, which means it can be transferred from animals to humans as part of the normal flora of many animals including household pets such as cats and dogs, and agriculture species such as cattle. P. multocida is responsible for infected animal bites, especially those resulting from household and large cats. Additionally, P. multocida is responsible for several diseases of veterinary importance, including avian cholera and bovine respiratory disease (BRD). Capsule, composed of capsular polysaccharide (CPS), is an essential virulence factor for P. multocida. Virulence factors are genetically encoded attributes that aid the bacteria in causing an infection. Capsule covers the surface of bacterial cells, which allows P. multocida to survive within the host and avoid detection by the immune system. The P. multocida capsular serogroup A is composed of hyaluronic acid. Biofilms are communities of bacteria that survive within a hydrated matrix composed of polysaccharides, proteins, enzymes, antimicrobial compounds, extracellular DNA, and other bacterial and host components. Biofilms can be compared to multicellular organs of eukaryotes. While less complex, biofilms similarly regulate nutrients, water, composition, remove waste, and perform other processes such as DNA transfer. Biofilms protect bacterial communities by shielding them from the host immune response. Bacteria living in biofilms also grow slowly, and as a result are protected from many antibiotic treatments. While biofilm formation has been suggested for P. multocida, the biofilm has not yet been characterized. The work reported here characterizes biofilm formation by P. multocida isolates of capsular serogroup A. Biofilms formed by P. multocida were stained with fluorescently-tagged lectins, DNA stain, and other fluorescent dyes, as well as crystal violet stain. Biofilms were imaged using several microscopy techniques. Biofilm formation was prominent for serogroup A strains of P. multocida that were acapsular. However, in the presence of CPS, biofilm formation was inhibited. H. somni forms a biofilm during BRD that allows the bacterium to survive within the heart and lungs of the bovine host. BRD is often caused by several different bacterial, viral, and even parasitic microbes – resulting in a polymicrobial disease. Polymicrobial diseases are more difficult to diagnose and treat, which is a challenge when trying to control this economically important disease. Experimental infections of bovines with H. somni have resulted in polymicrobial infections with P. multocida. We hypothesize that these two bacterial species may form a mutualistic or commensalistic interaction together during BRD to improve the survival of one or both species within the host. The polymicrobial biofilm was observed using fluorescent microscopy techniques. We confirmed that H. somni and P. multocida form a polymicrobial biofilm. Avian cholera can be an acute, chronic, or asymptomatic disease that affects poultry farms and migratory flocks around the world. The spread of P. multocida and avian cholera is thought to occur through infected water, infected insects, and through other infected animals surrounding water supplies such as deer, raccoon, and even fish. We hypothesize that P. multocida can produce a biofilm and survive within the respiratory tract of birds for extended periods of time, that biofilm formation is important for the establishment of chronic and asymptomatic avian cholera, and that a biofilm assists in the spread of disease between flocks of birds. Chickens were challenged in the respiratory tract with a highly encapsulated, poor biofilm forming strain, or a prominent biofilm forming strain. After 7, 14, and 28 days chicken lungs were examined to identify bacteria, biofilm material, and inflammation. Biofilm-forming P. multocida strains were less virulent and caused less inflammation than non-biofilm forming P. multocida strains. Biofilms were visible in the airways of pulmonary tissue by scanning electron microscopy. Biofilm formation by P. multocida was observed within the pulmonary tissue of chickens with chronic and acute avian cholera.

No / Streptococcus agalactiae is a major neonatal pathogen that is able to colonise various host environments and is associated with both gastrointestinal and vaginal maternal carriage. Maternal vaginal carriage represents the major source for transmission of S. agalactiae to the foetus/neonate and thus is a significant risk factor for neonatal disease. In order to understand factors influencing maternal carriage we have investigated growth and long term survival of S. agalactiae under conditions of low pH and nutrient stress in vitro. Surprisingly, given that vaginal pH is normally <4.5, S. agalactiae was found to survive poorly at low pH and failed to grow at pH 4.3. However, biofilm growth, although also reduced at low pH, was shown to enhance survival of S. agalactiae. Proteomic analysis identified 26 proteins that were more abundant under nutrient stress conditions (extended stationary phase), including a RelE family protein, a universal stress protein family member and four proteins that belong to the Gls24 (PF03780) stress protein family. Cumulatively, these data indicate that novel mechanisms are likely to operate that allow S. agalactiae survival at low pH and under nutrient stress during maternal vaginal colonisation and/or that the bacteria may access a more favourable microenvironment at the vaginal mucosa. As current in vitro models for S. agalactiae growth appear unsatisfactory, novel methods need to be developed to study streptococcal colonisation under physiologically-relevant conditions.