Philosophiae Doctor - PhD / Pneumonia remains the major cause of death in children and the elderly and several efforts have been intensified to reduce the rate of pneumonia infection. The major breakthrough has been the discovery of certain biomarkers for the diagnosis of pneumonia through immunogenic techniques.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

Microbiological screening of antimicrobial residues in eggs needs special attention because of the high level of naturally occurring inhibitors contained in eggs which often lead to false positive results. However, it was discovered that heating egg samples at 800C for 10 minutes inactivated the inhibitors. The new bacteriological screening test for antimicrobial residues in eggs which was developed during this study, contains viable spores of Geobacillus stearothermophilus which are sensitive to antimicrobial residues including beta-lactams, tetracycline’s and macrolides. The new test method was validated based on the comparison with a reference method, namely the Kundrat micro-screening four-plate test,1 and published literature of another standard reference method, the Premi®Test. A preliminary trial was conducted on 36 hens that were given therapeutic oral doses of overthe- counter antimicrobials daily for seven days with one of eleven antimicrobials based on the manufacturer’s recommendations. Eggs were collected from the hens during and after treatment and tested for the presence or absence of antimicrobial residues. Several performance criteria and minimum detection concentrations were estimated and discussed. Some agreements and differences were found between the new and the reference tests with the new test being more sensitive to beta-lactams, tetracyclines and macrolides than the Kundrat and Premi®Test on the average. The use of florfenicol and norfloxacin in laying hens is banned and therefore there are no maximum residue limits (MRL) or published Premi®Test values. For meat, the MRL is 100mg/kg. It was therefore concluded that the new screening test could be used for routine screening of antimicrobial residues in eggs. A two seasonal survey was also conducted to determine the prevalence of antimicrobial residues in commercial chicken eggs in Tshwane area of Gauteng Province, South Africa. Although the season did not impact statistically on the antibiotic residues found in the eggs; eggs sold at the roadside (informal businesses), certain egg brands, and those sold at lower prices were found to be more likely to contain antibiotic residues than those obtained from formal outlets and at higher prices. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Production Animal Studies / unrestricted

There is an increasing interest in how therapeutic drugs could alter the human gut microbiota composition and function. While some knowledge is accumulating on the antimicrobial impact of some psychotropics on isolated strains or the gut microbiota of animal models, information about other classes of psychotropics and representative species from the human gut is poorly investigated. The antimicrobial effect of psychotropic drugs is usually neglected as a confounding factor when investigating gut microbiome biomarkers, knowing that patients are generally put in long-term medication. The purpose of the present study was to investigate (in vitro and ex-vivo) the antimicrobial activity of some oral commonly prescribed psychotropics from different therapeutic classes on colonic microbiota diversity and metabolism and the potential capacity of probiotics to alleviate related dysbiosis. The findings of this study revealed an important in vitro inhibitory activity of psychotropic drugs, which were also expressed as drastic alterations in gut microbiota composition ex-vivo. Indeed, the relative abundances of Firmicutes and Actinobacteria were lowered while the Proteobacteria population was increased. Families of Lachnospiraceae, Lactobacillaceae, and Erysipelotrichaceae were also declined by psychotropics (aripiprazole) treatment. These microbial changes were translated into a decrease of the major SCFA (butyrate, acetate, and propionate) at the metabolic level. The addition of a probiotic combination (Lactobacillus rhamnosus and Bifidobacterium longum) concomitantly with a psychotropic (aripiprazole) had a protective effect by attenuating the decline of microbiota composition and increasing the concentrations of SCFA. These findings provide evidence that psychotropics, through their antimicrobial effect, have the potential to alter the human gut microbiota composition and metabolism, while probiotics can mitigate the related dysbiosis.

Muscadine seeds from three cultivars (‘Carlos’, ‘Noble’ and ‘Ison’) were processed by 100% v:v methanol, 95% v:v ethanol and 70% v:v acetone. The acetone seed extracts exhibited the highest (p<0.05) total phenolics (21.62~24.84mg/g of dw) and antioxidant activity. A strong correlation (R2=0.891, p<0.01) was found between total phenolics and antioxidant activity. Methanol and ‘Carlos’ seed extracts generally showed the highest antimicrobial capacity against pathogen strains tested, which correlated well with tannic acid, catechin, epicatechin and tartaric acid content. Hot water-soluble muscadine skin extracts from 17 muscadine cultivars (6 bronze and 11 dark) showed effective antioxidant properties. Significant variations (p<0.05) were observed among the 17 cultivars in total phenolics, organic acids and antioxidant activity. ‘Alachua’, ‘Albermarle’, ‘Southland’, ‘Janebell’ and ‘CA9-37’ were the cultivars found to have the highest antioxidant capacity. Skin extracts of dark-skin cultivars showed higher (p<0.05) content than bronze-skin cultivars in phenolics, tartaric acid, tannic acid and ellagic acids.

The presence of wide spectrum antimicrobial agents triclosan (TCS) and triclocarban (TCC) in the environment has become a concern due to the adverse impact on the environment. Montmorillonite, a widely distributed clay mineral in the natural environment, has been used as an obstacle in landfills to avoid contamination of soil and water by contaminants in leachates due to its high surface area, cation exchange capacity, and abundance. The research reported here focuses on understanding the abiotic oxidative transformation of TCS and TCC by Fe(III)-modified montmorillonite. The overall objectives of this work were: 1) to investigate TCS and TCC oxidative transformation kinetics and transformation products in different environmental conditions, and 2) to elucidate their reaction pathways. TCS was reacted with Fe(III)-modified montmorillonite under the following experimental conditions: 1) at 40% relative humidity and room temperature for up to 100 d with and without UV light exposure; and 2) in aqueous environment with different initial TCS concentrations, light exposure, pH levels, and in the presence of natural organic matters. Reaction in the presence of Na- montmorillonite was conducted for comparison with results from TCS reaction in the presence of Fe(III)-modified montmorillonite. In addition, transformation of TCS in the presence of other types of minerals was also investigated. Transformation of TCC on Fe(III) and Na-montmorillonite in an aqueous environment with and without exposure to light was also studied at different initial TCC concentrations. TCS and TCC transformation products were a) characterized using LC/MS, GC/MS, and computational modeling, and b) quantified using HPLC/UV and GC/MS. The main TCS transformation products were 2,4-dichlorophenol, 2,4-dichlorophenol dimer, chlorophenoxy phenols and, TCS dimers and trimers. 2,8-dichlorodibenzo-p-dioxin was identified under UV light and the sun simulator experiments. Formation of 4-chloroaniline and 3,4-dichloroaniline were confirmed as transformation products of TCC. To the best of our knowledge, this is the first time that 4-chloroaniline and 3,4-dichloroaniline were confirmed as abiotic transformation of TCC. This research has generated a better understanding of the abiotic environmental fate of TCS and TCC and demonstrates the feasibility of utilizing Fe(III)-modified montmorillonite as remediation material for TCS, TCC and other related pharmaceutical and personal care products (PPCPs).

Hydrolysis of casein using chymotrypsin results in the formation of polypeptides (CH) with a hydrophobic aromatic amino acid on one end of the chain because the enzyme selectively cleaves the adjacent peptide-bond. Due to resonance of the aromatic micro-domain, thiols become redox-sensitive and actively participate in electron transfer. These types of amphipathic peptides also tend to be membrane-lytic. The two prong approach of this investigation was to, (1) assess antibacterial effect of the CH in beef steak, and (2), to determine its antioxidative efficacy as a constituent of Cheddar whey based edible coating mix. The edible coating prevented coliform growth even at a minute concentration range of 0.15-0.2 % (w/v). Marked antioxidative efficacy of the CH, particularly at a concentration of 0.3% (w/v), was also evident from its remarkable free radical scavenging ability and extended resilience in an abusive model system saturated with peroxyl-radicals generated through controlled pyrolysis.

Antimicrobial resistance (AMR) poses a substantial threat to public health and clinical medicine. By 2050, it’s predicted that AMR will be responsible for a yearly mortality rate of 10 million people, surpassing the mortality of cancer. Despite this daunting future we face, there are many efforts currently employed to combat the growth of AMR. One significant effort involves surveillance and early identification of novel resistant bacteria circulating in high antibiotic exposure environments. The second chapter of this thesis focuses on sampling 25 patients from a hospital environment, rich with antibiotics, to build a collection of AMR bacteria that will be tested and added to surveillance efforts/future study. This chapter allowed for the identification of several worrying AMR bacteria that provide greater insights into circulating AMR in Canadian hospitals and their patients. From the AMR collection created in chapter 2, we are also able to advance our scientific understanding of how antibiotic resistance develops within us and causes issues with treatment. In chapter 3, we looked at the effects of antibiotic administration routes on the level of AMR observed in our patient sample. We saw that current approaches to limit selection for AMR in the gut still resulted in clinically significant and concerning increases in AMR. Furthermore, this chapter allowed greater understanding of contributors to increased AMR in patients. AMR increases are not fully explained by exposure/colonization in hospital settings, but also by evolution of AMR originating from non-resistant bacteria in the gut. Additionally, analysis of these bacteria will inform expected AMR evolutionary trajectories and help us plan against them. During analysis of patient data, we also came across evolution of a less understood resistance phenotype, hetero-resistance, to a very important antibiotic, colistin. We investigated a commonly prescribed antifungal, fluconazole, for its ability to promote this resistance phenotype; however, it appeared that fluconazole did not promote this phenotype. Ultimately, this thesis serves as a valuable reservoir of AMR bacteria for future study and contributes to a greater understanding of AMR development in patients, one day leading to more informed clinical decision making. / Thesis / Master of Science (MSc)

In the early phases of drug design and development, scientists must overcome many challenges involved in identifying potential drug-like or lead-like compounds. This has led to the need of creating large sets of chemical data which will aid in improving the identification of pharmacophores and active compounds. Various scientific fields especially pharmacology, medicinal chemistry and biochemistry have begun to employ the use of computer sciences to aid in the screening for potential leads with more specificity with regards to drug-like compounds' or substances' bioactivity. The emphasis of this project was to create a database containing a collection of pyrazine compounds synthesized overtime in the Faculty of Pharmacy (Charles University, Hradec Kralove) with the aim of having anti-mycobacterium (and possible antibacterial and antifungal) activity, and further utilize this database to predict certain pharmacokinetic and bioavailability properties. This project seeks to demonstrate how certain molecular descriptors can be used as reliable chemoinformation to determine the likeliness or possibility of developing a lead-like or drug-like compound by utilizing computer software. An in-house database of 623 compounds saved in SMILES format was created and used in demonstrating quantitative...

In order to better understand the mechanism of antimicrobial chemokine activity, including binding to and killing of bacteria, random transposon mutagenesis was performed in Yersinia pseudotuberculosis. Resulting mutants were screened for increased binding to chemokine and high binding clones were selected for further study. One mutant, designated mutant 27, was found to have a single insertion mutation in the rfaD gene. The rfaD gene product is involved in heptose biosynthesis, one of the sugars of the inner core oligosaccharide of Gram- negative lipopolysaccharide (LPS). Mutant 27 was found to bind both CCL25 and CCL28, two antimicrobial chemokines, more efficiently than the wild type bacteria. This clone was also found to be more susceptible to CCL28- mediated killing and polymyxin activity. Complementation with a plasmid bearing the full rfaDFC operon restored the wild type phenotype in both regards. These data suggest that normal LPS expression by Y. pseudotuberculosis serves to protect the bacteria from the antimicrobial function of chemokines and other antimicrobial proteins of the mammalian innate immune system.