Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the need for designing novel bacterial-sensing modalities. Among the most prominent disease-causing bacteria are strains of Escherichia coli, like the E. coli O157:H7 that produces the Shiga-like toxin (Stx). Apart from diarrheagenic E. coli strains, others cause disease varying from hemolytic uremic syndrome and urinary tract infections to septicemia and meningitis. Therefore, the detection of E. coli needs to be performed fast and reliably in diverse environmental and clinical samples. Similarly, Mycobacterium avium spp. paratuberculosis (MAP), a fastidious microorganism that causes Johne's disease in cattle and has been implicated in Crohn's disease (CD) etiology, is found in products from infected animals and clinical samples from CD patients, making MAP an excellent proof-of-principle model. Recently, magnetic relaxation nanosensors (MRnS) provided the first applications of improved diagnostics with high sensitivity and specificity. Nucleic acids, proteins, viruses and enzymatic activity were probed, yet neither large targets (for instance iv bacterial and mammalian cells) nor multiple bacterial disease parameters have been simultaneously monitored, in order to provide thorough information for clinical decision making. Therefore, the goal of this study was to utilize MRnS for the sensitive identification of multiple targets associated with bacterial pathogenesis, while monitoring virulence factors at the microorganism, nucleic acid and virulence factor levels, to facilitate improved diagnosis and optimal treatment regimes. To demonstrate the versatility of MRnS, we used MAP as our model system, as well as several other pathogens and eukaryotic cell lines. In initial studies, we developed MRnS suitable for biomedical applications (Chapter II). The resulting MRnS were composed of an iron oxide core, which was caged within a biodegradable polymeric coating that could be further functionalized for the attachment of molecular probes. We demonstrated that depending on the polymer used the physical and chemical properties of the MRnS can be tailored. Furthermore, we investigated the role of polymer in the enzyme-mimicking activity of MRnS, which may lead to the development of optimized colorimetric in vitro diagnostic systems such as immunoassays and small-molecule-based screening platforms. Additionally, via facile conjugation chemistries, we prepared bacterium-specific MRnS for the detection of nucleic acid signatures (Chapter III). Considering that MAP DNA can be detected in clinical samples and isolates from CD patients via laborious isolation and amplification procedures requiring several days, MRnS detected MAP's IS900 nucleic acid marker up to a single MAP genome copy detection within 30 minutes. Furthermore, these MRnS achieved equally fast IS900 detection even in crude DNA extracts, outperforming the gold standard diagnostic method of nested Polymerase Chain v Reaction (nPCR). Likewise, the MRnS detected IS900 with unprecedented sensitivity and specificity in clinical isolates obtained from blood and biopsies of CD patients, indicating the clinical utility of these nanosensors. Subsequently, we designed MRnS for the detection of MAP via surface-marker recognition in complex matrices (Chapter III). Milk and blood samples containing various concentrations of MAP were screened and quantified without any processing via MRnS, obtaining dynamic concentration-dependent curves within an hour. The MAP MRnS were able not only to identify their target in the presence of interferences from other Gram positive and Gram negative bacteria, but could differentiate MAP among other mycobacteria including Mycobacterium tuberculosis. In addition, detection of MAP was performed in clinical isolates from CD patients and homogenized tissues from Johne's disease cattle, demonstrating for the first time the rapid identification of bacteria in produce, as well as clinical and environmental samples. However, comparing the unique MAP quantification patterns with literatureavailable trends of other targets, we were prompted to elucidate the underlying mechanism of this novel behavior (Chapter IV). We hypothesized that the nanoparticle valency – the amount of probe on the surface of the MRnS – may have modulated the changes in the relaxation times (ΔΤ2) upon MRnS – target association. To address this, we prepared MAP MRnS with high and low anti-MAP antibody levels using the same nanoparticle formulation. Results corroborated our hypothesis, but to further bolster it we investigated if this behavior is target-size-independent. Hence utilizing small-moleculeand antibody-carrying MRnS, we detected cancer cells in blood, observing similar detection patterns that resembled those of the bacterial studies. Notably, a single cancer vi cell was identified via high-valency small-molecule MRnS, having grave importance in cancer diagnostics because a single cancer cell progenitor in circulation can effectively initiate the metastatic process. Apart from cells, we also detected the Cholera Toxin B subunit with valencly-engineered MRnS, observing similar to the cellular targets' diagnostic profiling behavior. Finally, as bacterial drug resistance is of grave healthcare importance, we utilized MRnS for the assessment of bacterial metabolism and drug susceptibility (Chapter V). Contrary to spectophotometric and visual nanosensors, their magnetic counterparts were able to quickly assess bacterial carbohydrate uptake and sensitivity to antibiotics even in blood. Two MRnS-based assay formats were devised relying on either the Concanavalin A (Con A)-induced clustering of polysaccharide-coated nanoparticles or the association between free carbohydrates and Con A-carrying MRnS. Overall, taking together these results, as well as those on pathogen detection and the recent instrumentation advancements, the use of MRnS in the clinic, the lab and the field should be anticipated.

Tesis por compendio / This PhD dissertation thesis has been focus on the development and characterization of antimicrobial packaging films based on the incorporation in the polymer matrix or on the attachment to the film surface of naturally occurring antimicrobial compounds with the purpose of inhibiting the proliferation of microorganisms and extend the microbiological shelf life of packaged food products. The studied active films are based on the use of ethylene vinyl copolymers (EVOH) containing 29% (EVOH29) or 44% (EVOH44) molar percentage of ethylene as polymeric vehicle for the incorporation of several antimicrobial compounds -oregano essential oil (OEO), citral, ethyl lauroyl arginate (LAE), epsilon-polylysine (EPL), green tea extract (GTE) and lysozyme. These antimicrobial agents have been incorporated in the film-forming solution or immobilized to the film surface by covalent bonding. Prior to the preparation of the active films, the antimicrobial activity of the selected compounds against selected microorganism was demonstrated, confirming that they could be good candidates to be used as preservatives for active food packaging applications, and an alternative to synthetic additives. The effect of the incorporation of the antimicrobial agents on relevant functional properties of the developed EVOH films was studied. In general, the polymer properties as materials for food packaging were not relevantly affected. In order to evaluate the potential of EVOH matrices as sustain release systems of active compounds, the release kinetics of the active compounds from the film to different media was evaluated; for that the agent release rate and extend into food simulants was monitored, and it was concluded that the agent concentration, release temperature, type of EVOH, interaction of EVOH with the food simulant, and the solubility of the active compound in the release media were the main controlling factors. EVOH matrices have also shown good properties to be used for the attachment of active molecules. In this regard, lysozyme was successfully immobilized on the film surface of EVOH. Several experiments were conducted to determine the antimicrobial properties of the resulting films in vitro against different microorganisms responsible for foodborne illness and in vivo with real foods –minimally-process salad, infant milk, surimi sticks and chicken stock- to enhance their preservation. All the materials presented a strong in vitro antimicrobial activity. Although the results obtained through in vivo tests showed activity reductions caused by food matrix effects, all materials presented significant microbial inhibition and, therefore, great potential to be used in the design of active food packaging. They can be applied as an inner coating of the packaging structure, releasing the active agent or acting by direct contact, producing a great protection against contamination with a prolongation of the microbiological food shelf life. / Muriel Galet, V. (2015). Novel antimicrobial films based on ethylene-vinyl alcohol copolymers for food packaging application [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48522 / TESIS / Premios Extraordinarios de tesis doctorales / Compendio

The effect of ozone on the inactivation of two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa) and one Gram-positive endospore (Bacillus subtilis) bacteria, often present in water and the cause of some waterborne diseases was investigated as a function of ozone concentration and ozonation duration. Ozone was generated in situ using corona discharge methods where the ozone concentration ranged from 0.906 - 4.724 mg/L and the inactivation of the three microbes followed pseudo-first order kinetics with respect to the microbes. Three microbes were cultured and the influence of temperature and pH of the aqueous systems on the ozone initiated inactivation rate of the three microbes was also investigated. This study reports that molecular ozone is more effective than hydroxyl radicals initiated by the ozone chain reactions. Two suggested mechanisms for the antimicrobial effectiveness of ozone in water systems from the literature is discussed. The study also found that ozonation significantly decreased the Biological Oxygen Demand (BOD) value of natural water. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.

Pathogenic bacteria secrete various virulence factors as their biochemical weapons to gain access to and destroy the target cells. They can directly interact with the outer lipid bilayer membrane of eukaryotic cells, inducing the premature cell death by either apoptosis or necrosis. Such virulence factors account for much of the toxic actions associated with bacterial infection; therefore the detection of such proteins could provide a methodology for sensing/detection of pathogenic bacteria in, for example, food or human tissue. Detection and identification of pathogenic bacteria by conventional methods such as plating and counting in laboratory is expensive and time consuming. With growing concerns over emergence and re-emergence of pathogenic bacteria with high resistant to current antibiotics, there is a potential need for effective detection of pathogenic toxins invitro. On the other hand, artificially prepared lipid bilayer membrane on planar metallic surfaces provides the cell membrane mimics which are extremely useful in exploring the cellular functions and processes at the molecular level. Therefore in this work, an application of planar tethered bilayer lipid membrane (pTBLM) as a biomimetic sensing platform for the detection of clinically important pathogens, Staphylococcus aureus and Pseudomonas aeruginosa via their secreted virulence factors was presented. Planar TBLM was modified by incorporation of cholesterol and detection of bacterial toxins at human body temperature was examined by impedance and surface plasmon resonance methods. The results of pathogenic bacterial toxin detection were compared with those of Escherichia coli (DH5α), the human gut normal flora with non-pathogenic strain, as a control. Additionally pTBLM was transferred onto single nanoporous Si3N4 membrane to enhance the toxin sensitivity and extend the lifetime for the possible realization of future membrane chips for ion channel characterizations and drug screenings. Then the single ion channel measurement was demonstrated with nanopore-suspended TBLM (Nano-psTBLM) using α-toxin of S. aureus. The results presented in this work therefore, may pave the more effective and efficient ways for future pathogenic bacterial detection in which the sensing mechanism was solely based on the nature of interactions as well as modes of action between bacterial toxins and artificial lipid bilayer membranes.

Les bactéries du genre Flavobacterium sont retrouvées dans des types d’habitats très divers. Ce genre contient trois espèces ichtyopathogènes : columnare, branchiophilum et psychrophilum qui est responsable de pertes économiques importantes pour l’élevage des salmonidés. Un projet de séquençage et de comparaison des génomes de plusieurs flavobactéries pathogènes de poissons ainsi qu’isolées de différents environnements a été mis en place pour améliorer les connaissances sur ce genre. Les objectifs étaient l’identification des déterminants de virulence et la caractérisation de différents marqueurs moléculaires des traits phénotypiques associés à leur mode de vie. L’analyse des génomes de F. psychrophilum a permis de mettre en évidence une diversité des structures chromosomiques au sein de l’espèce et d’identifier des cibles moléculaires prometteuses pour le développement de tests de diagnostic ainsi que des cibles vaccinales potentielles. Le génome de F. branchiophilum a permis d’identifier des mécanismes moléculaires de virulence originaux. Les caractéristiques du génome de F. indicum révèlent un mode de vie environnemental : sa petite taille et ses faibles capacités de dégradation des bio-polymères suggèrent que F. indicum est adapté à une niche écologique restreinte. Ces nouvelles données ont permis de caractériser in silico des marqueurs moléculaires de caractères phénotypiques. En particulier, un groupe de gènes (dnd) rare et responsable d’une modification étonnante de la molécule d’ADN a été décrit pour la première fois chez les Flavobacteriaceae. Ce projet a permis d’enrichir les connaissances sur les bactéries du genre Flavobacterium et a contribué au développement d’outils pour la santé animale. / Flavobacterium species occur in a wide range of habitats. This genus includes three fish-pathogenic species, namely F. columnare, F. branchiophilum and F. psychrophilum. The latter is responsible for serious economic losses for salmonids farming in France and worlwide. A comparative genomics project including several fish-pathogenic flavobacteria as well as various environmental species has been set up in order to improve the knowledge on this poorly studied genus. Our aims were the identification of virulence determinants associated with pathogenicity and the characterization of various molecular elements reflecting phenotypes associated with their life-style. Analysis of the genomes of several F. psychrophilum isolates revealed the diversity of chromosomal structures within the species and identified in silico promising molecular targets for the development of diagnostic tests as well as potential vaccines targets. Analysis of the F. branchiophilum genome enabled to identify particular molecular virulence mechanisms. The features of the F. indicum genome reflected its environmental lifestyle : its small size and its limited bio-polymers degrading abilities suggested that F. indicum is adapted to a quite narrow ecological niche. These new data have allowed the in silico identification of many molecular elements reflecting phenotypic traits. In particular, a rare gene cluster (dnd) responsible for an unusual DNA structure modification was described for the first time within members of the family Flavobacteriaceae. This project enriched the knowledge on Flavobacterium species and contributed to the development of tools for animal health.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The human population in sub-Saharan Africa has been increasing due to decreases in mortality rates and increases in average human age; in turn increasing poverty and pressure placed on agriculture and agricultural production. However, livestock production in South Africa, and globally, is declining due to disease and parasite prevalence, lack of feed, poor breeding, marketing management, change in nutrition in both livestock and humans, rapid urbanization, encroachment on wildlife and unfavourable climatic conditions brought about by global change. One unintended consequence has been the emergence and spread of transboundary animal diseases and, more specifically, the resurgence and emergence of zoonotic disease. Zoonotic diseases are sicknesses transmissible from animals to humans, resulting from direct contact or environmental reservoirs. Previous studies have identified small-scale farmers as the group most prevalent to contracting zoonotic diseases, especially those working in a communal dispensation. Therefore, this study focused on the communal farming area of Steinkopf in the semi-arid Namaqualand region of South Africa. Steinkopf is one of the largest Act 9 areas, with communal land tenure and a mixed farming system, sheep and goats, on about 759 ha. Steinkopf is divided into two rainfall regions, the Succulent Karoo (winter rainfall region) and the Nama Karoo (summer rainfall region). This study aims to identify and characterise the bacterial microbial communities found in the topsoil layer and faecal matter (dung) within the winter and summer rainfall regions of Steinkopf communal rangeland using Next-generation sequencing. Further, the aim is to assess whether pathogenic bacteria are present within the rangeland and what their potential impact on the local farming community might be if present. A high-throughput sequencing technique (Next-generation sequencing) was used to amplify 16S rRNA targeting the V3-V4 hypervariable regions. The phylotypes produced were 37 phyla, 353 families and 634 genera of which the most abundant bacterial phyla were Planctomycetes, Firmicutes and Bacteroidetes and the most abundant genera were Gemmata, Akkermansia and Arthrobacter. Alpha diversity indices showed a variation in species diversity, evenness and richness between soil and dung samples, it shows a higher species richness, evenness and unique OTUs detected in summer soil samples and at natural water holes. Through these analysis soil samples were regarded as superior to dung samples within this particular environment and for this particular study. Natural water holes were identified as a safer option when compared to man-made water holes as there are natural systems in place that combat the spread and growth of harmful bacterial microbes. It was found that seasonality has a great impact on the development and growth of environmental bacterial microbiota and that the current randomness of grazing routes and migrations within the Steinkopf communal rangeland is not a detriment but instead acts as a benefits to environmental and livestock health. Furthermore, a total of three pathogenic bacteria were identified however, they occurred at relatively low abundances. It can thus be concluded that this study thoroughly describes the usefulness of using a high-throughput sequencing technique such as Next-generation sequencing when amplifying a small sample size in order to achieve a large volume of information; and that currently the Steinkopf communal rangeland is not subjected to or at risk of a potential zoonotic threat.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Dr. Christian Baron. Includes bibliographical references (leaves 94-101).

Ball State University LibrariesLibrary services and resources for knowledge buildingMasters ThesesThere is no abstract available for this thesis.

The six major drainage basins of Indiana were sampled for Aeromonas hydrophila. Daring the summer of 1979 a statewide mean of 53 colony forming units (W u) per ml with a range of 0-383 was obtained using membrane filter techniques and Rimler-Shotts differential media. Density values were correlated with the following physico-chemical water quality parameters: temperature, dissolved oxygen, and conductivity. Temperature showed the highest significant correlation when regressed with Gru, accounting for 25% of the variation during the statewide survey. Four sites on the White River, Delaware County, Indiana, were sampled biweekly, in the same manner as the statewide surrey, for cne year to determine seasonal variatic± in A:. hydrophila densities. The mean varied from a low of 2 CFC ml-1 in the winter months , a summer high in July of 205 CFU ml-1. No significant correlations could be found between CFU and water quality parameters during summer months. Antibiotic sensitivities were performed on 112 environmental isolates. All were resistant to ampicillin and carbenicillin and all but seven were resistant to cephalothin.Ball State UniversityMuncie, IN 47306

Ball State University LibrariesLibrary services and resources for knowledge buildingMasters ThesesThere is no abstract available for this thesis.