Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.

Bacteria are inarguably the most ubiquitous and adaptive organisms on the planet. The vast, diverse community of microbes residing in soil are mostly studied using sequencing technologies because over 99% of them are currently uncultivable in the laboratory. This lack of diverse bacterial cultivation presents a serious challenge for modern microbiological and medical science where the discovery of novel antibiotic producers and microbial products has been outpaced by the rise in drug resistance. This study designed and tested two new cost-effective culture systems called the “Dynamic Soil Environment” and Soil Extract Systems with the goal of increasing the cultivable communities of diverse bacteria in a soil sample over standard methods. Illumina MiSeq sequencing and DADA2 pipeline protocols were used to analyze community DNA from cultivated samples and source soil metagenomes. Autoclaved soil extract media in the Soil Extract Experiment yielded a statistically significantly greater Shannon’s (p = 0.008) and Simpson’s diversity (p = 0.007) of bacteria over pH modified (6.4) nutrient agar media over 30 days of incubation. Autoclaved soil extract media was also able to cultivate, on average, 33% of species in bulk soil sequences compared to 27% from standard nutrient agar however these differences weren’t statistically significant. The length of incubation had a lesser effect than media type on yield of bacteria over 30 days in batch culture conditions. Species richness and diversity generally decreased over time except in soil extract samples. In the Dynamic Soil Environment experiment, membrane plates placed on a live soil environment produced a slightly higher diversity than autoclaved membrane plates and control plates without soil, however, these differences were not statistically significant except when analyzed with Chao1 diversity (0.041). Cultivated bacterial diversity and communities differed more according to media type than soil environment with statistically significant differences between standard and pH modified nutrient agar. Media with a 5.8 pH buffer produced a significantly higher relative abundance of the well-known antibiotic-producers, Actinobacteria (t(10) = -5.715, p < .000) and also Proteobacteria (t(10) = -10.127, p < .000). This study establishes cost-effective methods of cultivating more diverse bacterial communities for low-funded laboratories. Culture conditions for the reliable cultivation of higher relative abundances of bacterial groups belonging to Actinobacteria and Proteobacteria are also established with the Dynamic Soil Environment Experiment. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

The specific identification of a megalurous cercaria found in the snail Oxytrema plicifera was accomplished by completing the life cycle in the laboratory. This species is compared with the eastern Philophthalmus megalurus and P. gralli with which it was once confused. The eggs, miracidia, and rediae of the Oregon species were found to be similar to those of the above species. The body and organ sizes and sucker ratios for the cercariae and adult stages obviously indicate that the Oregon species is Philophthalmus megalurus.

I have investigated the possibility that specific conalbumin (ovotransferrin) iron saturation levels enable less virulent strains of Salmonella to become more virulent. Iron starved cells of two pathogenic Salmonella strains, S. paratyphi B var. java and S. thompson, were cultured in iron limited media at 3 different iron conalbumin saturation levels. Results indicate that strains differ significantly at both low and high iron saturation conalbumin. These differences depict a growth advantage for S. paratyphi B which correlates with reports by the Centers for Disease Control that S. paratyphi B was 3 times more frequent in blood isolates than S. thompson. The ability to use protein bound iron may account for the higher involvement of S. paratyphi B in bacteremia.

Periodontal diseases (PD) affect 46% of American adults over age 30. These diseases cause symptoms including bleeding and swelling of the gums, bone resorption, and tooth loss, that affect quality of life and have a high economic burden. Periodontal diseases are caused by an imbalance in the oral microbiome, from a healthy state that contains anti-inflammatory commensals like Streptococcus gordonii and mitis, to a diseased state that has pro-inflammatory anaerobic pathogens including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. The latter initiate disease progression in the oral cavity. However, it’s the host immune response that causes a majority of the symptoms. Ideally, treatment for PD would be approached from both sides to reduce the numbers of pro-inflammatory bacterial cells in the oral cavity but also reduce the host immune response. A novel therapeutic, amixicile, has been created, which specifically targets anaerobes through the pyruvate:ferredoxin oxidoreductase (PFOR) system, the mechanism of energy metabolism found in anaerobic organisms. Our studies show that amixicile inhibits in vitro growth of oral anaerobes in monospecies cultures at concentrations as low as 0.5 µg/mL in broth and 1 µg/mL in biofilms, without affecting the Gram-positive commensal species. In multispecies cultures, amixicile specifically inhibited anaerobes, even in biofilms, with the concentration as low as 5 µg/mL in broth and 10 µg/mL in biofilms. By not affecting the commensal bacteria, we think this treatment could restore a healthy oral microbiome. Aside from the bacterial presence, the host response, particularly the innate immune response is not well understood. Using a Drosophila melanogaster infection model, we elucidated the innate immune response to both mono- and multispecies infections. The 7-Species infection included bacteria mentioned above and Aggregatibacter actinomycetemcomitans in order to replicate in vivo-like disease conditions. We determined that both Drosophila Toll and Imd pathways, which mimic TLR/IL-1 and TNF signaling pathways of mammalian innate immunity respectively, respond to the 7-Species challenge. We also verified virulent bacteria in Drosophila, including P. gingivalis and P. intermedia. Future directions include RNA sequencing to determine the full scope of immune gene expression and using human immune cells to further clarify the response.

Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease.

Production of volatile sulfur compounds in cheese is associated with desirable flavors. The direct source of these compounds has been assumed to arise from the metabolism of methionine and cysteine. However, the methionine concentration in cheese rises above the amount found in casein during aging, suggesting that alternative sulfur sources are present in milk. This led us to hypothesize that lactococci may acquire sulfur from the inorganic sulfur pool of milk, in addition to methionine and cysteine, to generate volatile sulfur compounds during cheese ripening. A turbidimetric method to determine total sulfate content in milk samples was developed. The average sulfate content of milk was determined to be ~49 mg/L ± 2.0 mg/L. The limit of detection of the test was ~2.5 mg/L in Tris buffer and ~10 mg/L in milk. Skim milk samples had significantly higher total sulfate content as compared to whole milk samples. Transport of sulfate by three strains of Lactococcus sp. was studied after we determined that milk had small, but measurable amounts of inorganic sulfate. A decrease in the environmental pH increased sulfate transport. The maximum transport occurred during exponential cellular growth phase. All strains tested had the ability to transport much more sulfate than is native in milk. The last phase of study was to determine the metabolic fate of sulfate. Incorporation of radio-labeled sulfate into cellular protein was studied by two-dimensional gel-electrophoresis of crude cellular lysate followed by auto-radiography. Production of volatile sulfur compounds from inorganic sulfur was determined with analysis of the head space gas with gas chromatography and scintillation counting. The incorporation of radio-labeled sulfur from sulfate was not detected in proteins on two-dimensional gels. Detectable volatile sulfur compounds were found only in the case of gas chromatographic analysis of ML3 head space. However, radio-labeled volatile sulfur was detected in the head space of all the three strains with scintillation counting. This study defined that lactococci can fix inorganic sulfur into volatile sulfur compounds in small amounts.

Early studies on the bacterial content of milk were made mainly to satisfy the interst of people who wished to determine the various materials that contained bacteris. Soon the value of bacterial counts, as an indication of the general conditions of production, of handling, and of the keeping qualities of milk, became evident and bacterial counts were used to obtain information concerning these problems. Numbers of bacteria in milk have been used also in the studies of the desirable and undesirable changes in milk. In the last few years our citizenry has been made more importance of bacteria in milk. As the number of milk dealers have increased and our population in the cities has become more conjested, more stringent regulation of our milk supplies has been practiced. Of major importance in this regulatory program is the bacterial count of milk. Large dairy manufacturing plants, which have also recognized the importance of high bacterial counts in influencing the quality of their products, have encouraged production of low count milk even to the extent of giving bonuses to such producers and rejecting milk that did not come within their standards.

Some ill. mounted. Bibliography: leaves 109-129.

Le genre Legionella regroupe des bactéries pouvant causer chez l'homme une pneumonie fatale dans 10% des cas, la légionellose. Elles sont capables de coloniser tous les réseaux d'eau. Le génome de ces bactéries révèle une forte plasticité génomique, aux niveaux fonctionnel et structural. La première partie de cette thèse analyse l'impact des protéines de type histone sur la régulation de la virulence chez L. pneumophila. Ces protéines structurent le chromosome bactérien et influencent l'expression génique. Des mutants des gènes codant les protéines Dps et IHF ont été obtenus chez L. pneumophila et analysés pour leur sensibilité aux stress et leurs propriétés de virulence. Ces deux protéines sont impliquées dans la régulation de la virulence chez Legionella. De plus, Dps permet de diminuer la sensibilité au stress oxydatif et IHF régulerait l'entrée dans l'état VBNC (viable but non-culturable), un état physiologique dans lequel les bactéries sont viables mais ne sont plus cultivables. La seconde partie vise à utiliser la plasticité structurale, notamment celle induite par les éléments génétiques mobiles IS, comme outil épidémiologique. A l'heure actuelle, les méthodes d'identification ne permettent pas de discriminer les isolats de même espèce. Afin d'éviter de nouvelles contaminations, il est impératif d'identifier rapidement et avec précision l'installation contaminée, à partir des prélèvements de patients comme élément comparatif. L'utilisation d'une méthode RFLP-IS a permis de mettre en évidence une IS particulière, ISLpn11, possédant un taux de discrimination de 80% au sein de la souche L. pneumophila Paris, qui est responsable de plus de 10 % des épidémies en Europe.