Actinomyces species play an important role in the pathogenesis of oral diseases and infections. Susceptibility testing is not always routinely performed, and one may oversee a shift in resistance patterns. The aim of the study was to analyze the antimicrobial susceptibility of 100 well-identified clinical oral isolates of Actinomyces spp. against eight selected antimicrobial agents using the agar dilution (AD) and E-Test (ET) methods. We observed no to low resistance against penicillin, ampicillin-sulbactam, meropenem, clindamycin, linezolid and tigecycline (0–2% ET, 0% AD) but high levels of resistance to moxifloxacin (93% ET, 87% AD) and daptomycin (83% ET, 95% AD). The essential agreement of the two methods was very good for benzylpenicillin (EA 95%) and meropenem (EA 92%). The ET method was reliable for correctly categorizing susceptibility, in comparison with the reference method agar dilution, except for daptomycin (categorical agreement 87%). Penicillin is still the first-choice antibiotic for therapy of diseases caused by Actinomyces spp.

Bacteria-based cancer therapy (BBCT) holds immense promise in addressing the limitations in treatment of solid tumors. Bacterial strains used for BBCT are engineered to express therapeutics, facilitate precise navigation within the tumor microenvironment by enhancing bacteria's motility, chemotaxis (movement toward or away from specific chemicals), or other mechanisms that aid in reaching and infiltrating the tumor tissue effectively, and complementing traditional chemotherapy and immunotherapies while minimizing side effects. Bacterial motility not only influences the ability of bacteria to navigate within the tumor but also plays a pivotal role in optimizing drug delivery, treatment efficacy, and minimizing potential obstacles associated with the complex microenvironment of human tissues. However, the current understanding of bacterial motility remains limited. In this thesis, we use a reductionist approach and study bacteria motile behavior within human tissue phantoms (collagen and agar) and the bacteria-hydrogel interactions. Apart from motility, it is important to analyze the mechanical properties of the hydrogels the bacteria interact with as they play a vital role in overall behavior and physics of bacteria movement. To that extent, there exists a gap in our understanding of the viscoelastic properties of hydrogels. Lastly, systematic and comprehensive investigation of bacteria behavior in hydrogels requires tracking of thousands of individual cells. Thus, there is an unmet need to develop new automated techniques to reduce the labor-intensive manual tracking of bacteria in low-contrast hydrogel environments, with feature sizes comparable to that of bacteria. To address these gaps, this thesis proposes a trident approach towards mechanistic understanding of bacteria motility in time-invariant agar and temporally evolving collagen hydrogels to bridge critical gaps in understanding bacterial motile behavior in these media, non-destructive microelastography-based mechanical characterization of hydrogels with less than 4.7% error compared with rheology, and the development of deep learning-enabled automated bacteria tracking tools with 77% precision. / Master of Science / There exists a huge scope for improvement in cancer therapies. The gold standard chemotherapy and immunotherapies are responsible for a lot of side effects. Over a century ago, bacteria-based cancer therapy started to develop and over the period, it was discovered that they can be effective when used with traditional therapies improving precision and reducing side effects. The motility of bacteria is shown to improve bacterial distribution in solid tumors. However, the physical underpinnings of bacteria motility in the tumor environment remains understudied. This thesis proposes a trident approach, investigating bacteria motility in tissue-like environments (hydrogels), characterizing the mechanical properties of hydrogels using acoustic waves to capture bacteria-induced alterations, and developing deep-learning-enabled automated bacterial tracking approach for high throughput analysis of experimental data. We report bacteria behavior and motility patterns in hydrogels, the mechanics of these hydrogels with less than 4.7% error compared with standard characterization methods, and automated bacteria tracking with 77% precision to inform the development and advancement of bacteria-based drug delivery systems. In summary, these tools can help improve our understanding of bacteria-hydrogel interactions, allowing us to develop innovative bacteria-based cancer therapies in the long term.

No / The isotopic effect of exchanging deuterium with hydrogen on the mechanical and surface properties of agar gel is examined. The elastic modulus of the D2O gels obtained by AFM nanoindentation is significantly higher (factor of 1.5¿2) than the modulus found in H2O agar gels. Furthermore, the modulus is independent of loading rate. Surface imaging reveals that the surface roughness gets progressively smaller with increasing agar concentration. All these data suggest that the isotopic replacement of deuterium enhances the mechanical properties of the agar gel, with significant advantages in its use as a biphasic scaffold. / MRC

Cytopathology plays a significant role in cancer diagnostics. One of the ancillary methods used for prognostication is immunocytochemistry (ICC). Formalin pre-fixed tissue blocks are often used as validated immunohistochemical controls for cytology samples. However, those are not treated as clinical cytology specimen, which are furthermore commonly fixed in alcohol. As cell structure might be affected by added fixatives and block materials, the aim of this project was to compare the effects of fixation in formalin and PreservCyt®, a methanol-based fixative, and the two cell block materials, plasma-thrombin and agar, on the ICC results. The material used in the study consisted of several cell lines provided by the Technological University Dublin, which were chosen to be representative of the clinical cytology samples. The cells were fixed and put into cell blocks for each cell type or as a mixture of different cell types – a composite cell block. All cell blocks were then stained with suitable antibodies. Composite cell blocks could be tested as simultaneously positive and negative ICC control material. The results showed that in general, PreservCyt®-fixed cell blocks preserved sharper cellular morphology compared to those fixed in formalin. However, PreservCyt® also gave unexpected negative results for some antibodies, including some nuclear markers. Plasma-thrombin cell block method showed to have more advantages than the agar method.This study suggests, that with further optimization and validation of both fixative methods and cell blocks, composite cell block has good potential to be used as source of control blocks for cytology samples.

Master of Science / Food Science Institute- Animal Sciences and Industry / Kelly J. K. Getty / The Food and Drug Administration’s Bacteriological Analytical Manual recommends two enumeration methods for Bacillus spp.: 1) standard plating method using mannitol-egg yolk-polymyxin (MYP) agar and 2) most probable number (MPN) method with tryptic soy broth supplemented with 0.1% polymyxin sulfate. Preliminary research evaluated three inoculum preparation methods using EZ-Spore™ B. cereus pellets. Two methods involved EZ-Spore™ B. cereus pellets that were dissolved in deionized (DI) water, grown in brain heart infusion broth with manganese sulfate, and then heated to produce spores. The third inoculum preparation method of dissolving EZ-Spore™ pellets only in DI water was the most efficient due to 100% spores being present in the inoculum. Preliminary research also determined that MPN method recovered greater (p<0.05) B. cereus populations than MYP method in inoculated ultra-high temperature pasteurized skim and 2% milk. The objective of the main study was to compare the MYP and MPN method for detection and enumeration of B. cereus in raw and high-temperature-short-time pasteurized skim, 2%, and whole milk at 4 °C for 96 h. Milk samples were inoculated with B. cereus EZ-Spores™ dissolved in DI water and sampled at 0, 48, and 96 h after inoculation. No differences (p>0.05) were observed among sampling times so data was pooled for overall mean values for each treatment. The overall B. cereus population mean of pooled sampling times for MPN method (2.59 log CFU/mL) was greater (p<0.05) than MYP plating method (1.89 log CFU/mL). B. cereus populations ranged from 3.40 log CFU/mL to 2.40 log CFU/mL for inoculated milk treatments for MYP and MPN methods, which is well below the necessary level for toxin production. Even though MPN method enumerated more B. cereus, the MYP method should be used by industry for enumeration of B. cereus due to its ease of use and rapid turnover time (2 d compared to 5 d with MPN). However, MPN method should be used for validation research due to its greater populations recovered. EZ-Spore™ B. cereus pellets were found to be an acceptable spore inoculum for validation research because the inoculum consists of 100% spores and does not contain vegetative cells.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:09Z No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:21Z (GMT) No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-27T17:03:21Z (GMT). No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-07-17 / Biomphalaria glabrata snails are aquatic and inhabit often ephemeral basins. They have high reproductive capacity, and egg masses are laid on submerged substrates on the banks of breeding sites exposed to desiccation and pathogens like fungi. Virtually nothing is known about the viability of egg masses in breeding and transient sites and their pathogens. This information is important to control this intermediate host of Schistosoma mansoni, which causes schistosomiasis. This study describes the feasibility of egg masses in water films and susceptibility infection with to Beauveria bassiana and Metarhizium ansiopliae. Egg masses were exposed to water film formed in two different devices, one with a stone clay in a cup with water and another one with water agar at different concentrations of agar (0.3% - 3%) in Petri dishes in different relative humidity (RH > 98% and 75%). Conidia or blastospores of B. bassiana (ARSEF 9588) or M. anisopliae (IP 46) (2 x 107 conidia or blastospores/egg mass) were inoculated in egg masses exposed in the water film in stone clay or submerged (2 x 107 conidia/ml or blastospores/ml). Egg masses exposed in the water film in stone clay or in water agar (agar < 1%) at RH > 98% were viable. The eclosion and the displacement of juveniles in the films were observed regardless of the tested device (RH > 98%, agar < 1%), except at 75% RH and glass without water film. Mycelium and conidia developed on egg masses treated with conidia or blastospores of M. anisopliae in the water film in stone clay (RH > 98%), and there was no eclosion of juveniles from egg masses treated with blastospores of M. anisopliae. In egg masses submerged treated with blastospores of M. anisopliae or B. bassiana no eclosion of juveniles and egg masses treated with conidia accumulated average onset was < 52.5%. The feasibility of egg masses and the displacement of juveniles in both devices on RH > 98% was due to the formation of stable water film. Blastospores may have released toxic metabolites that inhibited the eclosion of juvenile B. glabrata. Both devices are promising for testing egg masses in the water film. The feasibility of egg masses in water under laboratory conditions film should be considered to control B. glabrata. Blastospores of M. anisopliae and B. bassiana are promising against B. glabrata. / Biomphalaria glabrata são caramujos aquáticos e habitam coleções hídricas frequentemente efêmeras. Possuem alta capacidade reprodutiva, e massas ovígeras são postas em substratos submersos nas margens dos criadouros expostas ao ressecamento e a patógenos como fungos. Praticamente nada é sabido sobre a viabilidade de massas ovígeras em criadouros transitórios e a sua susceptibilidade a fungos. Estas informações são importantes para o controle desse caramujo hospedeiro intermediário de Schistosoma mansoni, causador de esquistossomose. O presente trabalho relata a viabilidade de massas ovígeras em filme de água e a susceptibilidade a Metarhizium ansiopliae e Beauveria bassiana. Massas ovígeras foram expostas em filme de água formado em dois dispositivos diferentes confeccionados, um com pedra de argila em copo com água e outro com meio ágar água com diferentes concentrações de ágar (0,3% - 3%) em placas de Petri em diferentes umidades relativas (UR > 98% e 75%). Conídios ou blastosporos de B. bassiana (ARSEF 9588) ou M. anisopliae (IP 46) (2 x 107 conídios ou blastosporos/massa ovígera) foram inoculados em massas ovígeras expostas em filme de água em pedra de argila ou submersas (2 x 107 conídios/ml ou blastosporos/ml). As massas ovígeras expostas em filme de água em pedra de argila ou em meio ágar água (ágar < 1%) em UR > 98% foram viáveis. A eclosão e o deslocamento de juvenis nos filmes foram observados independentemente do dispositivo testado (UR > 98%, ágar < 1%), exceto em UR 75% e em vidro sem filme de água. Micélio e conídios desenvolveram sobre massas ovígeras tratadas com conídios ou blastosporos de M. anisopliae em filme de água em pedra de argila (UR > 98%), e não houve eclosão de juvenis a partir de massas ovígeras tratadas com blastosporos de M. anisopliae. Em massas ovígeras submersas tratadas com blastosporos de M. anisopliae ou B. bassiana não houve eclosão de juvenis e em massas ovígeras tratadas com conídios a eclosão média acumulada foi < 52,5%. A viabilidade de massas ovígeras e o deslocamento de juvenis em ambos os dispositivos em UR > 98% foi devido à formação de filme de água estável. Blastosporos podem ter liberado metabólitos tóxicos que inibiram a eclosão de juvenis de B. glabrata. Ambos os dispositivos são promissores para testes com massas ovígeras em filme de água. A viabilidade de massas ovígeras em filme de água em condições laboratoriais deve ser considerada no controle de B. glabrata. Blastosporos de M. anisopliae e B. bassiana são promissores contra B. glabrata.

Nas últimas décadas as espécies de Campylobacter spp. são reconhecidas como importantes agentes de gastroenterites de origem alimentar em humanos em diversos países, tendo como principal veículo de transmissão a carne de frango. Devido ao aumento da frequência com que é isolada a partir de humanos, animais, alimentos e água, esta bactéria tem sido foco crescente de atenção nos últimos 30 anos. No Brasil, ainda são limitadas as informações sobre esta bactéria na cadeia de produção de aves, não existindo legislação que contemple o controle de Campylobacter. A alta incidência na avicultura, a presença natural deste patógeno nos animais e os graves problemas de saúde pública gerados tornam essa bactéria alvo de esforços para prevenção e controle. O objetivo do presente estudo foi avaliar se a metodologia de contagem direta proposta pela normativa MLG 41.02 teria eficiência na monitoria em diferentes amostras avícolas e paralelamente comparar o desempenho de dois ágares (mCCDA e Campy-Cefex) na contagem de Campylobacter spp. Foram realizadas quatro tomadas de amostras em um frigorífico da região Sul do país, durante um mês, sendo a amostragem composta por suabes de cloaca, carcaças pré-chiller, carcaças pós-chiller, água pré-chiller, água do chiller, e amostras de água de abastecimento. O ágar Campy-Cefex obteve maior frequência de isolamento de Campylobacter spp. em diferentes amostras avícolas quando comparado com o ágar mCCDA. Houve redução significativa de contaminação ao longo da linha de abate, com níveis de 9,8 x 102 UFC/mL em carcaças préchiller e 1,5x102 UFC/mL em carcaças pós-chiller. Do total de amostras em que foram realizadas a PCR, 72% foram positivas para Campylobacter jejuni e 38% positivas para Campylobacter coli. A metodologia mostrou-se eficiente e possível de ser aplicada na indústria avícola, em diferentes materiais, para monitoria de Campylobacter. / In recent decades the Campylobacter spp. species are recognized as important agents of foodborne gastroenteritis in humans in several countries and the broiler meat as are pointed as the main vehicle of transmission. Due the increase in frequency isolation from humans, animals, food and water, this bacteria has received great attention in the last 30 years. The information about this bacteria are still limited in Brazil and in the poultry production chain, there are no laws to the Campylobacter’s control. The high incidence in poultry industry, the natural presence of this pathogen in animals and the serious concern in public health, lead this bacteria as target to prevention and control efforts. The aim of this study was to evaluate whether the direct counting methodology proposed by MLG 41.02 rules would be efficient on monitoring different poultry samples, as well as to compare two agars plate (mCCDA and Campy-Cefex) performance’s to Campylobacter cell count. We carried out four samples taken in a slaughterhouse located at southern region of Brazil, during one month. The samples were composed by cloacal swabs, pre-chiller carcasses, post-chiller carcasses, pre-chiller water, chiller water, and water supply samples. The Campy-Cefex agar showed higher Campylobacter spp isolation frequency of among different poultry samples than mCCDA agar. There was a significant reduction in contamination along the slaughter line with levels of 9.8 x 102 CFU/mL in pre- chiller carcasses and 1.5 x102 CFU/mL in post-chiller carcasses. The samples typified by PCR, showed 72% of the samples as Campylobacter jejuni and 38% as Campylobacter coli. The methodology was efficient and also possible to be used in the poultry’s industry for different samples to Campylobacter monitoring program.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 139 pages. Includes vita. Includes bibliographical references.