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)

No description available.

Bacterial outbreak publications outline the key factors involved in uncontrolled spread of infection. Such factors include the environments, pathogens, hosts, and antimicrobial resistance (AMR) genes involved. Individually, each paper published in this area gives a glimpse into the devastating impact drug resistant infections have on healthcare, agriculture, and livestock. When examined together, these papers reveal a story across time, from the discovery of new resistance genes to their dissemination to different pathogens, hosts, and environments. My work aims to extract this information from publications by using the biomedical deep-learning language model, BioBERT. BioBERT is pre-trained on all abstracts found in PubMed and has state-of-the-art performance with language tasks using biomedical literature. I trained BioBERT on two tasks: entity recognition to identify AMR-relevant terms (i.e., AMR genes, taxonomy, environments, geographical locations, etc.) and relation extraction to determine which terms identified through entity recognition contextualize AMR genes. Datasets were generated semi-automatically to train BioBERT for these tasks. My work currently collates results from 204,094 antimicrobial resistance publications worldwide and generates interpretable results about the sources where genes are commonly found. Overall, my work takes a large-scale approach to collect antimicrobial resistance data from a commonly overlooked resource, i.e., the systematic examination of the large body of AMR literature. / Thesis / Master of Science (MSc)

It is predicted that by 2050 there will be 10 million deaths annually due to super-resistant bacterial infections. Antimicrobial resistance (AMR) is already responsible for nearly 5 million deaths a year. Ribosomes serve as an ideal drug target being frequently targeted by antibiotics and having a highly conserved structure with few options for resistance. However, computer aided drug design (CADD) using ribosome crystal structures presents several challenges and is underutilized in the field. In this work we establish a successful protocol for antibiotic redocking and docking within the high interest sites of the peptidyl transferase center (PTC). Molecular visualization and interaction mapping were used to atomistically delineate binding patterns in the ribosomal PTC that could be used for CADD. Eleven ribosome crystal structures were validated for computational testing, which revealed derivative binding patterns in the A-site and P-site that can be used to increase antibiotic efficacy. Ribosome overlays revealed high interaction frequency nucleotides that were widely conserved throughout the different species and could be used to inform bidentate design to target two pockets at once. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR. / Master of Science in Life Sciences / Antimicrobial resistance (AMR) to antibiotics by bacteria is a rapidly increasing problem. Current trends predict that there will be more death due to super-resistant bacterial strains than cancer by 2050. Ribosomes are essential cellular machinery for bacteria and make an ideal antibiotic target. Using computational tools to optimize antibiotics with available ribosome crystal structural data presents several challenges and is underutilized throughout the field. In this work we establish a successful protocol for determining and exploiting antibiotic binding patterns within the functional center of the ribosome, the peptidyl transfer center (PTC). Nearly a dozen ribosome crystal structures were validated for computational testing, and binding patterns were revealed within the PTC that allowed antibiotic derivatives with increased efficacy to be developed. Ribosome validation also helped inform new drug class design so that multiple drug sites could be targeted at once, which were docked sharing high frequency nucleotide interactions with both parent antibiotics. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR.

Pathogenic E. coli infections cause approximately one quarter of disease losses in commercial turkey flocks. A small subgroup of E. coli causes most infections. Epidemiologic studies of this disease have been hindered by a lack of reliable markers to discriminate between pathogenic and fecal E. coli and by the diversity of poultry strains. Reliance on antimicrobials to control E. coli infections has caused widespread antimicrobial resistance. One hundred five clinical E. coli were obtained, and 1104 isolates were collected from fecal specimens of 20 flocks in an integrated turkey operation. Biochemical fingerprinting and antimicrobial susceptibility tests were performed on all isolates, and somatic antigen serologic testing and PCR for potential virulence genes were conducted on 299 strains including all clinical isolates and fecal isolates that had similar traits to clinical isolates. Most avian E. coli infections were caused by a few clonal strains that were uncommon in normal fecal flora. The potential virulence genes iss, K1 and tsh were detected more frequently among clinical than fecal isolates; however, the pattern of occurrence did not suggest that these genes were useful markers for identifying pathogenic strains. Syndromes consistent with colibacillosis were the most commonly reported illness and principal rationale for antimicrobial therapy in sampled flocks. Most clinical E. coli isolates were resistant to gentamicin, sulfamethoxazole and tetracycline. Although resistance to fluoroquinolones and β-lactam antibiotics occurred less frequently, the potential for resistance to emerge to these antimicrobials was evident. A Bayesian model to estimate sample size confirmed the diversity of avian fecal E. coli strains. Studies are needed to define risk factors for infection with and identify markers for avian pathogenic E. coli strains. These research priorities are complementary and may lead to the identification of new interventions to prevent this important infectious disease of poultry. / Ph. D.

One of the most concerning characteristics of microbial biofilms is that of increased resistance to antimicrobial agents such as the commonly used biocide chlorhexidine (CHX). This can have huge impact on clinical, household and environmental settings. This is particularly alarming when it involves opportunistic pathogenic environmental organisms such as Delftia acidovorans as routine mitigation practices may fail to be effective. This thesis examines tolerance mechanisms of D. acidovorans biofilms exposed to CHX at inhibitory and sub-inhibitory concentrations. To achieve the study goals and objectives, a CHX-tolerant D. acidovorans strain (WT15), (Minimum Inhibitory Concentration; MIC-15 μg ml-1) was compared to a CHX-sensitive strain (MT51, MIC-1 μg ml-1) that was obtained by mutating the wild type strain using transposon mutagenesis. Specific morphological, structural and chemical compositional differences between the CHX-treated and untreated biofilms of wild type and mutant strains were documented using microscopic techniques including confocal laser scanning microscopy (CLSM), scanning transmission x-ray microscopy (STXM), transmission electron microscopy (TEM) and infrared (IR) spectroscopy. Molecular level changes between biofilms formed by these two strains due to CHX treatment were compared using whole-cell proteomic analysis (determined using differential in-gel electrophoresis, or DIGE) along with fatty acid methyl ester (FAME) analysis. The gene disrupted by transposon insertion that led to increased susceptibility to CHX in the mutant strain was identified as tolQ. CLSM revealed differences in biofilm architecture and thickness between the biofilms formed by strains WT15 and MT51. STXM analyses showed that WT15 biofilms contained two morpho-chemical cell variants; whereas, only one type was detected in MT51 biofilms. STXM and IR spectral analyses revealed that CHX-susceptible MT51 cells accumulated the highest levels of CHX, an observation supported by TEM wherein prominent changes in the cell envelope of CHX-susceptible MT51 cells were observed. DIGE analysis demonstrated that numerous changes in protein abundance occurred in biofilm cells following CHX exposure and that most of these proteins were associated with amino acid and lipid biosynthesis, protein translation, energy metabolism and stress-related functions. Overall, these studies indicate the probable role of the cell membrane and TolQ protein in CHX tolerance in D. acidovorans biofilms, in association with various proteins that are differentially-expressed.

Master of Public Health - MPH / Background: The irrational use of medicines is a global public health challenge, particularly in developing countries like Nigeria. One of the consequences of irrational medicine use is rising antimicrobial resistance, which continues to contribute to the increase in morbidity, mortality, and high cost of care, despite breakthroughs in medicine and new treatment options. Community pharmacists have been identified as contributors to antimicrobial resistance through their antibiotic dispensing practices. However, there is little research on community pharmacists who provide private healthcare in Nigeria. Aim: This study described the antibiotic dispensing practices of community pharmacists and factors associated with such dispensing practices in Jos, Nigeria. Methodology: The study used a cross-sectional descriptive design. Simple random sampling was used to select a sample of 84 community pharmacies out of a total of approximately 107 community pharmacies in Jos, and one community pharmacist from each community pharmacy. A research assistant was trained to administer the questionnaire along with the researcher and collect information on community pharmacists’ demographics, antibiotic dispensing practices, and the factors associated with those dispensing practices. The socio-demographic data was analysed via descriptive analytical tools such as simple percentages and crosstabulations. These tools were used to generate a descriptive picture of the data, patterns and associations using SPSS version 25. Quantitative content analysis was done on responses to scenario-based questions, and recommendations made as to how the dispensing of antibiotics could be improved. Ethical clearance was obtained from the University of the Western Cape Biomedical Research Ethics Committee and Jos University Teaching Hospital, while informed consent was obtained from all community pharmacists before the commencement of the study. Results: The majority of the community pharmacists (87%) indicated that patients could purchase antibiotics without prescription from their pharmacies, and most pharmacists (98%) asked for reasons why antibiotics were demanded for without prescriptions. While 58% indicated that patients could purchase partial quantities of prescribed antibiotics at their pharmacies, 96% investigated the reasons for partial requests, and 94% counselled on the right dosage and frequency of the prescribed antibiotics. Sixty-seven percent of the pharmacists indicated that one of the major reasons for dispensing antibiotics without prescription was self-medication by patients, while most (87%) indicated that financial constraint was a major reason why patients requested for partial quantities of prescribed antibiotics. Conclusion: The dispensing of non-prescribed and part-prescribed antibiotics is a common practice amongst community pharmacists in Jos, Nigeria. Enhancing the financial status of Nigerians, as well as ensuring stricter regulatory measures on antibiotic use, would help promote rational use of antibiotics and reduce rising antimicrobial resistance rates.

Philosophiae Doctor - PhD / The emergence of virulent and drug-resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA) is a global public health burden. The World Health Organization (WHO) has placed MRSA and vancomycin-intermediate-sensitive S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) on a high global priority pathogens list of antibiotic-resistant bacteria to promote the research and development of novel and effective antibiotic therapeutic rationales. Uncomplicated S. aureus bacteraemia (e.g., mild skin infections) may be treatable with the conventional regimens of antibiotics, but resistance strains of the bacteria (e.g., invasive infections), often persist as a high load of bacterial DNA in blood, and has been linked to increased mortality in world populations, irrespective of country or location. Several lines of evidence imply that combinations of vancomycin (a glycopeptide antibiotic that targets cell wall synthesis) and ß-lactam antibiotics that target the penicillin-binding proteins (PBPs) improve clearance of MRSA bloodstream infections (BSIs).

O uso indiscriminado de antimicrobianos na suinocultura nacional e mundial tem sido uma prática comum, visando minimizar possíveis falhas no manejo e no ambiente em que vivem os animais, no entanto, este uso é descrito como potencial fator de risco na seleção de estirpes resistentes à antimicrobianos, entre elas as estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). O presente estudo revelou o uso médio de 358,0 mg diferentes antimicrobianos/ kg de suíno produzido nos 25 sistemas de produção pesquisados, sendo este valor considerado elevado, quando comparado a tendência global de 172,0 mg, bem como período médio de exposição de 66,3% da vida dos animais e exposição à 7 diferentes princípios ativos em média, variando de 2 a 11. A pesquisa de animais carreadores de estirpes MRSA revelou 80,0% dos sistemas de produção positivos, sendo 68,0% positivos para LA-MRSA-ST398, e 60,0% positivos para a presença do gene czrC, codificador de resistência ao óxido de zinco e cádmio. Não foi evidenciada correlação significativa entre o uso de antimicrobianos, nível de biossegurança, produtividade, status dos rebanhos para Mycoplasma hyopneumoniae, tipo de criação em sítio único (ciclo completo) ou dois sítios e positividade para MRSA. Apesar da ausência das correlações significativas ficou evidente no presente estudo a possibilidade de grandes melhorias nos programas de biossegurança, manejo, assim como nos programas de utilização de antimicrobianos. A alta frequência de sistemas de produção positivos para as estirpes MRSA são um importante alerta para o risco de disseminação deste agente para os seres humanos. / The indiscriminate use of antimicrobials in Brazilian and global pig farms has been a common practice in order to minimize possible failures in the management and environment in which animals live, however, this use is described as a potential risk factor in the selection of strains resistant to antimicrobials, including strains of methicillin resistant Staphylococcus aureus (MRSA). The present study revealed the average use of 358.0 mg of different antimicrobials / kg of pig produced in the 25 production systems surveyed, which is considered high when compared to the overall trend of 172.0 mg, as well as the mean exposure period of 66.3% of the animals life and exposure to 7 different active principles on average ranging from 2 to 11. Research on MRSA strains showed 80.0% of the positive production systems, and 68.0% were positive for MRSA strains. LA-MRSA-ST398, and 60.0% positive for the presence of the czrC gene, encoding resistance to zinc oxide and cadmium. There was no significant correlation between antimicrobial use, biosafety level, productivity, herd status for Mycoplasma hyopneumoniae, single site (complete cycle) or two sites and MRSA positivity. Despite the absence of significant correlations, it was evident in the present study the possibility of great improvements in biosafety programs, management, as well as in programs for the use of antimicrobials. The high frequency of positive production systems for MRSA strains is an important warning for the risk of dissemination of this agent to humans.

Staphyloccocus aureus is an opportunisitic pathogen that causes soft skin and tissue infections (SSTI) such as endocarditis, osteomyelitis and meningitis. In recent years, the re-emergence of antibiotic-resistant S. aureus such as MRSA presents a formidable challenge for infection management worldwide. Amidst this global epidemic of antimicrobial resistance, several research efforts have turned their focus towards exploiting the cell-wall biosynthesis pathway for novel anti-bacterial targets. Recently, the lipoteichoic acid (LTA) biosynthesis pathway has emerged as a potential anti-bacterial target. LTA is an anionic polymer found on the cell envelope of Gram-positive bacteria. It comprises of repeating units of glycerol-phosphate (GroP) and is important for bacterial cell physiology and virulence. For example, it is critically involved in regulating ion homeostasis, cell division, host colonization and immune system invasion. Several reports showed that bacteria lacking LTA are unable to grow. At the same time, they suffer from severe cell division defects and also exhibit aberrant cell morphologies. The key protein involved in the LTA biosynthesis pathway is the Lipoteichoic acid synthase (LtaS). LtaS is located on the cell membrane of Gram-positive bacteria and can be divided into two parts: a transmembrane domain and an extra-cellular domain responsible for its enzymatic activity (annotated eLtaS). Given that LtaS is important for bacterial survival and there are no known eLtaS homologues in eukaryotic cells, this protein is an attractive antibacterial target. In 2013, a small molecule eLtaS inhibitor (termed 1771) was discovered. Although 1771 was able to deplete LTA production, the binding mechanism of 1771 to eLtaS remains unknown. Additionally, 1771 could only prolong the survival of infected mice temporarily because of its in vivo instability. Therefore, the need for finding more potent and metabolically stable inhibitors of eLtaS still remains. Computational-aided drug design (CADD) is a cost-effective and useful approach that has been widely integrated into the drug discovery process. The protein eLtaS lends itself to be a good target for CADD since its crystal structure and a known inhibitor (with limited structure-activity data) is available. In this work, I have targeted eLtaS using CADD methodology followed by prospective validation using various biophysical, biochemical and microbiological assays. My project can broadly be sub-divided into three phases: (a) identification of small molecule binding “hot spots”, (b) optimization of existing inhibitor and (c) discovery of new hits. Through a systematic use of different computational approaches, I modelled a plausible 1771-bound eLtaS complex and used the structural insights to generate new inhibitors against eLtaS. To this end, I discovered EN-19, which is a more potent inhibitor of eLtaS. Additionally, by targeting transient cryptic pockets predicted by Molecular Dynamic simulations, I have discovered a new inhibitor chemotype that seems to exhibit a different mode of action against eLtaS. Taken together, my work presents a computational platform for future drug design against eLtaS and reinforces the notion that targeting eLtaS is a viable strategy to combat Gram-positive infections.