Orientadores: Francisco Carlos Groppo, Maria das Graças Machado Freire / Dissertação (mestrado profissional) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T09:31:23Z (GMT). No. of bitstreams: 1 Oliveira_MaraRubeaTinocoRodriguesde_M.pdf: 229602 bytes, checksum: 5369ca1218480e5687d9ee3827b671ce (MD5) Previous issue date: 2005 / Resumo: A medicina natural e complementar, especialmente a fitoterapia, suprem as necessidades em saúde de grande parte da população, particularmente nos países em desenvolvimento. Dentre os fitoterápicos, as lectinas podem ter valia como agentes antiplaca, uma vez que podem estar intimamente relacionadas com a aderência de microrganismos. O objetivo deste estudo foi testar in vitro a capacidade de inibição das lectinas isoladas (TEL - derivada da semente de Talisia esculenta e LABRAMIN - purificada da planta Labramia bojeri), na adesão e crescimento de microrganismos orais (Streptococcus sanguinis, S. mitis, S. oralis, S. mutans, S. sobrinus). A atividade antimicrobiana das duas lectinas foi determinada pelo teste convencional da macrodiluição de caldo, sendo testada as concentrações de 400, 200, 100, 50, 25 µg/mL contra 105 CFU/mL dos microrganismos em estudo. Os tubos foram incubados (10% CO2, 37oC, 18h) e submetidos à leitura de densidade óptica (OD a 600nm). A MBC foi determinada pela adição das amostras de cada tubo em placas de petri contendo agar BHI (10% CO2, 37oC, 18h). Para avaliação de aderência foi feito um ensaio semiquantitativo de aderência em placas de microtitulação de poliestireno, onde foi adicionado 100 µL de saliva clarificada e incubada por 2h a 37°. Após lavagem com PBS, foram adicionadas às placas em triplicata 100 µL de lectinas (6.25, 12.5, 25, 50 e 100 µg/mL) e incubados durante 1h. A seguir foi adicionada 100 µL de suspensão bacteriana (106 UFC/mL) e incubado a 37ºC em uma atmosfera de 10% de CO2. A aderência foi revelada e quantificada por tintura com cristal violeta. A absorção do cristal violeta foi determinada por um leitor de placa (575nm). Nem a TEL nem a LABRAMIN foram capazes de inibir ou matar os microorganismos estudados. No teste de inibição de aderência, a LABRAMIN reduziu significativamente a aderência de S. mutans e S. sobrinus, na concentração de 100µg (p< 0,05). A TEL não inibiu a aderência dos estreptococos e ainda favoreceu a aderência do S. mitis. Concluiu-se que embora nenhuma das lectinas estudadas tenham atividade antimicrobiana, a LABRAMIN foi capaz de inibir a aderência de estreptococos cariogênicos / Abstract: Natural and complementary medicine, with special regard to phytotherapy, provide care for health needs of a great part of the world¿s population, particularly in developing countries. Among phytotherapic compounds, lectins could be of value as anti-plaque agents, once they can be closely related to microorganisms¿ adherence. Our study intended to test, in vitro, the capacity of two isolated lectins ¿ TEL (derived from Talisia esculenta seeds) and LABOL (purified from the plant Labramia bojeri ¿ for inhibiting the adherence and growth of oral microorganisms (Streptococcus sanguinis, S. mitis, S. oralis, S. mutans, S. sobrinus). Both lectins¿ antimicrobial activities were determined by a conventional macrodilution broth test. Study¿s microorganisms concentrations were tested for 400, 200, 100, 50, 25 µg/mL against 105 CFU/mL. Tubes were incubated (10% CO2, 37oC, 18h) and submitted to optical density reading (OD at 600nm). MBC was determined by the addition of samples from each tube to petri dishes containing agar BHI (10% CO2, 37oC, 18h). A semiquantitative assay was performed for adherence assessment in polystyren microtiter plates. A hundred microliter (100µL) of clarified saliva were added and incubated for 2h at 37°. Following PBS wash, 100 µL lectins (6.25, 12.5, 25, 50 e 100 mg/mL) were added to the triple plates and incubated for 1h. Then, 100 µL of bacterial suspension (106 UFC/mL) were added and incubated at 37ºC in a 10% CO2 atmosphere. Adherence was revealed and quantified by crystal violet staining. A plate reader (575nm) determined crystal violet absorbance. Nor TEL nor LABOL were able to inhibit or kill the studied microorganisms. Regarding adherence inhibition, 100µg concentration of LABOL showed statistically significant (p < 0.05) on S.mutans and S. sobrinus. TEL did not inhibt streptococci adherence; yet, it favored S. mitis adherence. It was concluded that, although none of the studied lectins had presented antimicrobial activity, LABOL was capable of inhibiting cariogenic streptococci adherence. Further studies will be necessary to assess such lectins¿ potential over other microorganisms and over oral biofilm / Mestrado / Mestre em Odontologia em Saúde Coletiva

Streptococcus sanguis 12 adhesins mediating attachment to saliva-coated hydroxyapatite beads (S-HA) were isolated and characterized. Cell surface fibrils were released from this organism by a method of freeze-thawing followed by brief homogenization. Fibrils in the homogenate were precipitated by ultracentrifugation or ammonium sulphate precipitation. This precipitate was shown to contain fibrils by electron microscopy. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of fibrils showed a single band which stained with Coomassie blue and periodate-Schiff. The molecule had a Mr in excess of 300,000. This protein has been given the name long-fibril protein (LFP). Antibody raised against the LFP reacted with long fibrils of S. sanguis 12. LFP was degraded by subtilisin, pronase, papain, and trypsin, but not by chymotrypsin and muramidases. Fibrils were hydrolyzed by subtilisin into discrete lower Mr protein bands which reacted with both anti-fibril and anti-LFP serum. F(ab')₂ prepared from anti-fibril IgG inhibited adhesion of S. sanguis 12 to pH modified S-HA, indicating that fibrils were acting as an adhesin mediating attachment via the neuraminidase-sensitive receptor on S-HA. Five recombinant clones expressing surface antigens of S. sanguis 12 were isolated by ligating a partial digest of S. sanguis 12 chromosomal DNA with the plasmid vector pUC 18, and transforming into Escherichia coli JM83. Recombinant clones were screened by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or with anti-fibril serum. Positive clones were then analyzed by SDS-PAGE, Western blotting and restriction endonuclease digestion of recombinant plasmids. One recombinant plasmid, pSA2 expressed two proteins of Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated as SsaB (S. sanguis adhesin B). Both proteins were purified to homogeneity by gel filtration and ion exchange chromatography. Anti-SsaB serum was used in an immunogold bead labelling experiment to demonstrate that this protein was present on the surfaces of S. sanguis 12 and in the non-saliva-aggregating variant 12na, but not on the non-adhering non-aggregating hydrophilic variant 12L. Western blot analysis with anti-SsaB and anti-20 kd sera showed that both SsaB and the 20 kd proteins were present in cell extracts of S. sanguis 12 and its variants. SsaB inhibited adhesion of S. sanguis 12na to S-HA, indicating that it was the adhesin which mediates the binding to the pH-sensitive receptor. SsaB was found to be present on all S. sanguis strains tested, but not on other oral streptococci. Chemical cross-linking studies of SsaB on S. sanguis 12 cell surface suggested that this protein may be present in a higher Mr complex. This study provides direct evidence that binding of S. sanguis 12 to S-HA involves at least two adhesin-receptor interactions. The adhesin mediating binding to the neuraminidase-sensitive receptor on S-HA involves the long fibrils and the adhesin binding to the acid labile receptor is a 36,000 Mr protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Microbes in nature live collaboratively in adherent communities, known as biofilms. Biofilms can be contextually beneficial or detrimental. In medical implants, biofilms cause infections leading to additional healthcare costs of billions of dollars. Studies have found that micro/nanoscale surface topography can significantly alter (i.e., promote or hinder) the process of biofilm formation. The formation of biofilm starts with planktonic microbes attach to the surface. To further understand the biophysical underpinning of this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. To this end, aligned nanofiber coating with controlled fiber diameter and edge-to-edge spacing were manufactured using the Spinneret-based Tunable Engineered Parameters (STEP) techniques. The effect of surface topography on bacterial near-surface motility was studied. The experimental results showed that the bacterial attachment and near-surface motion can be greatly impacted by surface topography. Furthermore, the finding was applied to ureteral stents. The results showed that the aligned nanofiber can significantly reduce the biofilm formation process on ureteral stents. / Master of Science / Many microbes in nature live in adherent communities called biofilm. Biofilms contain individual microbes inside polymeric matrix which protect them from environmental stressors such as antibiotics. Biofilms are a significant contributor to the infection of implantable medical devices, which leads to additional healthcare costs of billions of dollars annually in the U.S. alone. Studies have found that sub-micron scale surface topography can significantly promote or hinder biofilm formation; however, the exact mechanism remains poorly understood. To further understand this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. The formation of microbial biofilm starts with swimming bacteria sensing the liquid-solid interface and attaching to the surface. Microbes are more likely to settle on a surface if a surface is favorable to attach. However, the decision-making process has not been fully understood. Our experimental results showed that the bacterial attachment and near-surface motion can be greatly influenced by surface topography. Furthermore, the finding was applied to ureteral stents, which is a type of medical implants used to maintain the flow of urine in the urinary tract. Ureteral stents serve great for medical purposes, but as foreign bodies, they also lead to urinary tract infection. The results showed that some types of aligned fiber coating increased microbial attachment density, while other types of aligned fiber coating reduced the bacterial surface coverage by up to 80%, which provides directions for future studies.

Bacteria in nature predominantly grow as biofilms on living and non-living surfaces. The development of biofilms on non-living surfaces is significantly affected by the surface micro/nano topography. The main goal of this dissertation is to study the interaction between microorganisms and nanopatterned surfaces. In order to engineer the surface with well-defined and repeatable nanoscale structures, a new, versatile and scalable nanofabrication method, termed Spun-Wrapped Aligned Nanofiber lithography (SWAN lithography) was developed. This technique enables high throughput fabrication of micro/nano-scale structures on planar and highly non-planar 3D objects with lateral feature size ranging from sub-50 nm to a few microns, which is difficult to achieve by any other method at present. This nanolithography technique was then utilized to fabricate nanostructured electrode surfaces to investigate the role of surface nanostructure size (i.e. 115 nm and 300 nm high) in current production of microbial fuel cells (MFCs). Through comparing the S. oneidensis attachment density and current density (normalized by surface area), we demonstrated the effect of the surface feature size which is independent of the effect on the surface area. In order to better understand the mechanism of microorganism adhesion on nanostructured surfaces, we developed a biophysical model that calculates the total energy of adhered cells as a function of nanostructure size and spacing. Using this model, we predict the attachment density trend for Candida albicans on nanofiber-textured surfaces. The model can be applied at the population level to design surface nanostructures that reduce cell attachment on medical catheters. The biophysical model was also utilized to study the motion of a single Candida albicans yeast cell and to identify the optimal attachment location on nanofiber coated surfaces, thus leading to a better understanding of the cell-substrate interaction upon attachment. / Ph. D. / Formation of surface associated multicellular communities of microorganisms known as biofilms is of concern in medical settings as well as in industries such as oil refineries and marine engineering. It has been shown that micro/nanoscale surface features can highly regulate the process of biofilm formation and the attached cell activities. In this dissertation, we study the interaction between surface nanoscale structures and bacterial adhesion by experiments and biophysical modelling. We develop the Spun-Wrapped Aligned Nanofiber (SWAN) lithography, a versatile, scalable, and high throughput technique for masterless nanopatterning of hard materials. Using this technique, we demonstrate high fidelity whole surface single step nanopatterning of bulk and thin film surfaces of regularly and irregularly shaped 3D objects. SWAN lithography is used to texturize the electrode surface of microbial fuel cells (MFCs), which are envisioned as an alternative sustainable energy source. Compared to the non-patterned electrodes, the electrodes with 115 nm surface patterns facilitate larger biofilm coverage and 40% higher current production. We also develop a biophysical model to optimally texturize the surface of central venous and uretic medical catheters to prevent biofilm formation by fungal pathogen, Candida albicans. We show that the surface structures that result the highest cell total energy retained the least C. albicans. Furthermore, the adhesion behaviour of a single yeast cell is also experimentally studied in conjunction with the developed model.

Na natureza, os microorganismos podem apresentar forma de vida planctônica ou podem estar aderidos a superfícies formando comunidades conhecidas como biofilmes. A formação de biofilmes na indústria alimentícia é uma constante preocupação visto que os microorganismos aderidos podem causar contaminações persistentes, levando a deterioração do alimento e a transmissão de doenças. Uma alternativa para evitar a adesão bacteriana e a formação de biofilmes é o pré-condicionamento de superfícies com biosurfatantes, que são compostos tensoativos de origem microbiana capazes de alterar as propriedades físico-químicas e conseqüentemente modificar as interações entre a bactéria e a superfície. Os biosurfatantes, surfactina obtida de Bacillus subtilis e ramnolipídeo de Pseudomonas aeruginosa, foram testados quanto a capacidade de evitar a adesão e remover biofilmes de bactérias patogênicas de interesse alimentar. Foram avaliadas culturas individuais e mistas de Staphylococcus aureus, Listeria monocytogenes, e Salmonella Enteritidis utilizando-se como modelo superfícies de poliestireno. O pré-condicionamento da superfície com surfactina na concentração de 0,25% reduziu a adesão de Salmonella Enteritidis e Listeria monocytogenes em 42%, enquanto que o tratamento com ramnolipídeo a 1% reduziu a adesão de Listeria monocytogenes e Staphylococcus aureus ao poliestireno em 57,8% e 67,8% respectivamente. O condicionamento com os biosurfatantes não se mostrou eficiente na redução da adesão das culturas mistas das bactérias se comparado aos resultados obtidos para as culturas individuais. O poliestireno condicionado com os biosurfatantes apresentou redução na hidrofobicidade devido ao caráter aniônico destas moléculas. A repulsão eletrostática e a redução das interações hidrofóbicas promovidas pelo condicionamento do poliestireno com ramnolipídeo foram fatores determinantes na atividade antiadesiva observada para L. monocytogenes e S. aureus, entretanto os resultados obtidos para a superfície tratada com surfactina sugerem que outros parâmetros influenciaram nos resultados observados. Após 2 h de contato a surfactina na concentração de 0,1% promoveu a remoção de 63,7% do biofilme de S. aureus, 95,9% do biofilme de L. monocytogenes, 35,5% do biofilme de S. Enteritidis e 58,5% do biofilme da cultura mista das três bactérias. Já o ramnolipídeo na concentração de 0,25% removeu 58,5% do biofilme de S. aureus, 26,5% do biofilme de L. monocytogenes, 23,0 % do biofilme de S. Enteritidis e 24% do biofilme da cultura mista após 2 h contato. De modo geral, o aumento do tempo de contato e da concentração dos biosurfatantes reduziu a remoção dos biofilmes. A surfactina e o ramnolipídeo demonstraram potencial para uso como agentes anti-adesivos assim como para a remoção de biofilmes de bactérias patogênicas de importância alimentar. / In nature, microrganisms can live as planktonic cells or can be found living in communities attached in surfaces forming biofilms. Biofilm represents a great concern for food industry, since it can be a source of persistent contamination that can lead to food spoilage and the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants that are microbial compounds that can modify the physico-chemical properties of the surfaces changing bacterial interactions and consequently adhesion. The biosurfactants, surfactin obtained from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated as agents to avoid the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes e Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0,25% reduces in 42,0% the adhesion of L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1,0% reduced in 57,8% the adhesion of L. monocytogenes and in 67,8% the adhesion of S. aureus to polystyrene. The conditioning of surface with biosurfactants was less effective to avoid adhesion of mixed cultures of the bacteria when compared with the results obtained for individual cultures. The polystyrene surface conditioned with the biosurfactants showed a reduction in the hydrophobicity due to the anionic character of the molecules. The electrostatic repulsion and the reduction on hydrophobic interactions promoted by the conditioning of surface with rhamnolipids were determinant factors to explain the anti-adhesive activity observed for L. monocytogenes and S. aureus, however, the data obtained with surfactin suggest that other parameters have influenced the results observed. After 2 h contact with surfactin at 0,1% concentration, the pre-formed biofilms of S. aureus were reduced by 63,7%, L. monocytogenes biofilms were reduce by 95,9% , S. Enteritidis biofilms by 35,5% and the mixed culture biofilm by 58,5%. The rhamnolipids at 0,25% concentration removed 58,5% of biofilm of S. aureus, 26,5% of the biofilm of L. monocytogenes, 23,0% the biofilm of S. Enteritidis and 24,0% the biofilm of the mixed culture after 2 h of contact. In general, the increase in concentration of biosurfactants and in the time of contact decreases the biofilm remove percentage. These results demonstrate that surfactin and rhamnolipids present potential to be used as agents to control the attachment and to disrupt biofilms of food-borne pathogens.

Orientador: Lafayette Nogueira Junior / Coorientador: Estevão Tomomitsu Kimpara / Banca: Tarcísio José de Arruda Paes Junior / Banca: João Maurício Ferraz da Silva / Banca: Fabíola Pessôa Pereira Leite / Banca: Júlio César Brigolini de Faria / Resumo: O objetivo deste estudo foi avaliar se há alteração no comportamento mecânico e na aderência microbiológica da cerâmica à base de dissilicato de lítio com a técnica de pigmentação extrínseca aplicada, após ser submetida a diferentes condições experimentais. Foram confeccionadas 160 amostras, divididas em grupos com e sem pigmentação (n=80). Destes, cada grupo foi subdividido em Controle, Desgate, Biodegradação e Desgaste com Biodegradação (n=20).15 amostras de cada subgrupo foram submetidas ao teste de resistência à flexão e 5 para o teste de aderência microbiológica. As amostras passaram anteriormente por testes complementares para caracterização da superfície (rugosidade, perfilometria volumétrica e microscopia eletrônica de varredura (MEV)). Os resultados foram submetidos a análise estatística descritiva (média e desvio padrão) e inferencial, mediante o teste paramétrico de análise de variância (ANOVA) dois fatores e teste de Tukey ( = 0,05). O fator pigmentação extrínseca influenciou negativamente no comportamento mecânico da cerâmica, apresentando significância estatística (p = 0,000), assim como a interação entre o tipo de condição experimental e a pigmentação (p = 0,020). Entretanto, na aderência microbiológica, foi a condição experimental que influenciou negativamente no comportamento microbiológico (p = 0,000), assim como a interação entre a condição experimental e a pigmentação (p = 0,000). Nas análises complementares, observou-se que a interação entre os fatores a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the mechanical and microbiological behavior of the ceramics based on lithium disilicate with extrinsic characterization. For this, 160 discs were made, divided into two large groups, with extrinsec characterization and without, after which each was divided into four groups (n = 20): Control, Wear, Biodegradation and Biodegradation with Wear. Fifteen samples from each group were submitted to the flexural strength test and 5 submitted to the microbiological adherence test. Prior to the destructive test of flexural strength, the representative samples of each group underwent complementary tests for surface characterization. The results were submitted to descriptive statistical analysis (mean and standard deviation) and inferential, using the parametric analysis of variance (ANOVA) two way and Tukey test ( = 0,05). The extrinsec characterization factor influenced the mechanical behavior of the ceramic, presenting statistical significance (p = 0.000), as well as the interaction between the type of experimental condition and the extrinsec characterization (p = 0.020). However, in the microbiological adherence, it was the experimental condition that influenced the microbiological behavior (p = 0.000), as well as the interaction between the experimental condition and the extrinsec characterization (p = 0.000). It was concluded that the makeup influenced the mechanical behavior of the ceramic, and the experimental condition influenced the m... (Complete abstract click electronic access below) / Doutor

"Bacterial infections persist as a public threat due to the ease by which bacteria adapt to commonly used antibiotics. In addition, bacteria on surfaces develop protective communities called biofilms that hinder the ability of antibiotics to completely eliminate the pathogens. The rapid development of bacterial resistance to antibiotics has made pharmaceutical companies reluctant to fund new antibiotics research. Hence, novel approaches to prevent and treat infections are needed. The development of infections can be divided into three steps: adhesion, invasion and multiplication. Antibiotics target at the latter two step and are prone to bacterial resistance as passive strategies. Bacterial adhesion to host cells/implanted medical devices is the first step leading to following invasion and multiplication. However, fundamental understanding of bacterial adhesion process is still lacking. The current studies are aimed to systematically investigate biological interactions between pathogenic bacteria and host cell, proteins and biomaterials with both macro and micro scale approaches. The macro scale methods include bacterial adhesion assay, viability studies, and thermodynamic modeling. The micro scale methods include direct adhesion force measurements, ultra surface visualization via atomic force microscopy (AFM) and surface structure modeling. Our work combines experiments and modeling aimed at understanding the initial steps of the bacterial adhesion process, focusing on two case studies: 1) Mechanisms by which cranberry can prevent urinary tract infections through interfering with bacterial adhesion; and 2) Design of anti-adhesive and antimicrobial coatings for biomaterials. We make direct adhesion force measurements between bacteria and substrates with an atomic force microscope (AFM), and combine such experiments with thermodynamic calculations, in order to develop a set of tools that allows for the prediction of whether bacteria will attach to a given surface. These fundamental investigations of the bacterial adhesion process help elucidate the underlying mechanisms behind bacterial adhesion, thus leading to improved clinical outcomes for a number of biomedical applications. "

"The adhesion of bacteria to surfaces has been analyzed in terms of surface charge, surface energy, and the characteristics of polymers on bacteria, to understand the factors that control bacterial adhesion. Pseudomonas aeruginosa has received a great deal of interest because it is responsible for a variety of chronic bacterial infections such as airway infections in cystic fibrosis patients and ulcerative bacterial keratitis in soft contact lens users. Over the past few years, force measurement techniques such as atomic force microscopy (AFM) have made it possible to examine interactions between colloidal particles and surfaces. In the present study, the AFM was used to study the interactions between each of two Pseudomonas aeruginosa strains with proteins. Topographical images and force cycles of bacterial cells and proteins were analyzed. Bovine serum albumin (BSA) and concanavalin A (Con A) were the model proteins chosen to represent protein molecules that might affect bacterial adhesion. In addition, the role of LPS structure in bacterial adhesion was investigated. The magnitude of adhesive forces for two P. aeruginosa stains was not statistically significant when they interact with silicon. Although it is not clear if the pull-off distances are accurate representatives of the absolute length of bacterial surface molecules, the trend indicates that the surface molecules of strain AK1401 are shorter than those of strain PAO1. The semi-rough strain AK1401 was more hydrophobic than the smooth strain PAO1, according to the water contact angle measurements. However, surface free energy components and zeta potential values were not significantly different for both strains. Zeta potential of bacterial cells decreased when they were suspended in HEPES/DTT buffer instead of ultrapure water. The AFM results demonstrate the importance of nano-scale interactions between proteins and bacterial cells. Our results show that the lipid A and core oligosaccharides are the most important molecules influencing the interactions of P. aeruginosa with protein molecules. The interactions of P. aeruginosa with model proteins in our study were weak. Therefore, the role of protein molecules may be inadequate for the purpose of enhancing subsurface delivery for bioremediation. Our results suggest that the semi-rough mutant, AK1401, can adhere to the protein receptors of the epithelial cells or protein coated implants stronger than the smooth strain, PAO1, and therefore can cause serious infections."

Studying the interactions between bacteria and soil colloidal particles in the environment is important for bioaugmentation purposes. Different factors affect the transport of the bacteria in porous media. For example, the soil type, the ionic strength of the substrate, and biological properties, such as the bacterial cell motility. Since organic materials are present in almost all subsurface media, the presence of natural organic matter (NOM) is considered an important factor influencing bacterial transport in porous media. In this work, a model system was developed to examine the interactions between natural colloidal particles and environmental bacteria using Atomic Force Microscopy (AFM). The natural colloids in the environment were modeled by a surface film of adsorbed NOM onto spherical SiO2 particles. Poly(methacrylic acid) (PMA), a simple linear polyelectrolyte, was used to mimic NOM since both are dominated by carboxylic acid functional groups. Soil Humic Acid (SHA) and Suwannee River Humic Acid (SRHA), two acidic polyelectrolytes, were used in further experiments to represent more complicated NOM. A smooth strain of Pseudomonas aeruginosa (PAO1) that coexpresses A-band and B-band polysaccharides, and its rough mutant (AK1401) that only expresses the A-band polysaccharides, were chosen to represent environmental bacteria. The model system was characterized through analysis of the measured forces between the chemically-modified colloidal probes and the bacterial cells. Interestingly, we found that PMA was not a good model for the more complex NOM substances. Differences were also observed in how each bacterium interacted with the three forms of NOM. For example, P. aeruginosa PAO1 had the highest adhesion with both complex forms of NOM, while P. aeruginosa AK1401 had the lowest adhesion with the complex forms of NOM. Since the lipopolysaccharide (LPS) structure is the only difference between the two strains, we attribute the different interactions to differences in LPS structure. The polymer density on the bacterial surface was found to be the most important factor in controlling the nature of the interaction forces.

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an Atomic Force Microscope (AFM) cantilever, and a biomaterial or biofilm substrate. Distinct adhesive interactions exist between Candida parapsilosis and both unmodified silicone rubber and Pseudomonas aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive interactions with magnitude of 2.3 ± 0.5 nN (SD) in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force increases to 3.5 ± 0.75 nN (SD), and is preceded by a 2.5 nN repulsion at approximately 175 nm from the cell surface. This repulsion may be attributed to steric and electrostatic interactions between the two microbial polymer brushes. Young's moduli for microbes and biofilms were calculated using Hertzian contact models. These produced values of 0.21 ± 0.003 MPa (SD) for the C. parapsilosis-silicone rubber system, and 0.84 ± 0.015 MPa (SD) for the C. parapsilosis-biofilm system. This technique may be extended to calculate the work per unit contact area involved in the attractions in experimental data. For example, the work of adhesion using a spore probe is an order of magnitude greater for unmodified silicone rubber than for a P. aeruginosa biofilm. This indicates a high affinity for silicone rubber, and suggests that this material is vulnerable to infection by C. parapsilosis in vivo. We have also demonstrated that AFM force curve analysis using established qualitative and quantitative models fails to accurately represent the physical interactions taking place between the probe and sample for the case where a polymer brush exists on the substrate, the probe, or both. As such, an approximate method defining the sample surface as the actual surface plus some vertical dimension associated with the maximum compressible thickness of the polymer brush is discussed. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative evaluation of the materials used for medical implantation. It also provides a link between the physicochemical and physicomechanical properties of these materials and the nanoscale interactions leading to microbial colonization and infection. The goal of this research is to study this link and determine how best to exploit it to prevent microbial infections of medical implant materials.