FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e as doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas em todo o mundo, fato que impulsionou a realizaÃÃo de estudos de prospecÃÃo por novas drogas antifÃngicas. A histoplasmose à uma micose sistÃmica, causada pelo fungo Histoplasma capsulatum var. capsulatum que, em pacientes hÃgidos, pode mimetizar a tuberculose quanto aos aspectos clÃnicos e radiolÃgicos. Alguns casos de histoplasmose refratÃria ao tratamento com drogas antifÃngicas convencionais vÃm sendo descritos. O objetivo deste trabalho foi determinar o efeito inibitÃrio, in vitro, das drogas antituberculose: isoniazida (INH), pirazinamida (PZA) e etambutol (EMB); antifÃngicas: anfotericina B (AMB), fluconazol (FLC), itraconazol (ITC) e voriconazol (VRZ) e de anÃlogos quÃmicos da isoniazida frente a cepas de H. capsulatum (n=30), assim como avaliar o emprego de diferentes meios de cultura para a realizaÃÃo dos testes de sensibilidade. Para isso, primeiramente, foram repicadas 18 cepas de H. capsulatum em Ãgar BHI e utilizadas na realizaÃÃo dos testes de sensibilidade frente aos agentes antituberculose citados e anÃlogos quÃmicos da isoniazida, isolados e em combinaÃÃo com os antifÃngicos FLC, ITC e VRZ por meio da tÃcnica de macrodiluiÃÃo em caldo. Cada uma das 12 cepas restantes foi repicada em Ãgar batata dextrose, Ãgar BHI, Ãgar malte a 2% e Ãgar lactrimel e, analisadas ao microscÃpio Ãptico quanto a presenÃa de macroconÃdios tuberculados, sendo quantificados de acordo com os parÃmetros: (0-10); (10-50); (>50) macroconÃdios/campo. As culturas foram empregadas na determinaÃÃo dos testes de sensibilidade frente aos agentes antifÃngicos AMB, FLC, ITC e VRZ, utilizando a tÃcnica de microdiluiÃÃo em caldo. As drogas antituberculose inibiram o crescimento das cepas in vitro com valores de CIM de 0,04 a 0,30 mg/mL para INH, 0,55 a 3,13 mg/mL para PZA e 1,56 a 6,25 mg/mL para EMB. No tocante Ãs drogas antifÃngicas, todas as cepas foram sensÃveis apresentando valores de CIM que variaram de 0,0625 a 0,25 Âg/mL para AMB; 15,62 a 62,5 Âg/mL para FLC; 0,0039 a 0,0312 Âg/mL para ITC e 0,00156 a 0,25 Âg/mL para VRZ. Quanto Ãs combinaÃÃes entre os fÃrmacos antituberculose e os derivados azÃlicos, todas foram capazes de inibir o crescimento in vitro das cepas de H. capsulatum, sendo detectado sinergismo nas nove combinaÃÃes. Os anÃlogos da isoniazida apresentaram valores de CIM 2, 4, 8 e 15 vezes superior a atividade da droga antituberculose padrÃo. A partir da anÃlise micromorfolÃgica do fungo repicados nos quatro meios de cultura foi identificado a menor quantificaÃÃo (0-10 macroconÃdios/campo) para Ãgar batata, Ãgar BHI, Ãgar malte e Ãgar lactrimel, perfazendo um total de 11, 10, 6 e 7 cepas, respectivamente. O meio de cultura Ãgar malte foi o mais adequado para produÃÃo de macroconÃdios (10-50) e (>50), norteando um total de 6 cepas, seguido do meio lactrimel, 5 cepas. Em relaÃÃo a determinaÃÃo da CIM e o meio de cultura utilizado para o procedimento, observou-se que quando o inÃculo era proveniente de cepas em Ãgar malte e Ãgar BHI foi possÃvel a visualizaÃÃo da CIM em 11 cepas. Enquanto repiques feitos em Ãgar batata e lactrimel nÃo foi possÃvel determinar os valores de CIM para 8 e 5 cepas, respectivamente. Os resultados deste estudo fornecem dados adicionais sobre o potencial antifÃngico das drogas antituberculose e suas interaÃÃes com os derivados azÃlicos. Entretanto, novos estudos se fazem necessÃrio, visando determinar os mecanismos de aÃÃo desses compostos no metabolismo celular dos fungos. / In the past years, the improvement of mycological diagnosis methods and immunosuppressive diseases have caused a great impact in the incidence of opportunistic and deep mycoses all around the world, which motivated the performance of new antifungal drugs prospective studies. Histoplasmosis is a systemic mycosis caused by the fungus Histoplasma capsulatum var. capsulatum, which may mimic tuberculosis, in healthy individuals, concerning clinical and radiological aspects.ome cases of histoplasmosis that are refractory to the treatment with conventional antifungal drugs have been described. The aim of this study was to determine the in vitro inhibitory effect of the antituberculosis drugs: isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB); antifungal drugs: amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC) and voriconazole (VRZ) and chemical analogs of isoniazid against strains of H. capsulatum (n=30), as well as to evaluate the use of different culture media for the performance of the susceptibility tests. For such, first, the antituberculosis agents INH, PZA and EMB and the analogs of isoniazid were tested isolatedely, and then, in association with the antifungal drugs FLC, ITC and VRZ, against 18 strains of H. capsulatum, previously grown onto BHI agar, through broth macrodilution technique. Each of the 12 remaining strains grown onto potato agar, BHI agar, 2% malt extract agar and lactritmel agar were microscopically analyzed, concerning the presence of tuberculate macroconidia, which were quantified as follows: 0-10, 10-50 and >50 macroconidia/field. Fungal cultures were used to determine the susceptibility of H. capsulatum to the antifungal agents AMB, FLC, ITC and VRZ, through broth microdilution methodology. The antituberculosis drugs inhibited the in vitro growth of the fungal strains, with MICs ranging from 0.04 to 0.30 mg/mL for INH; 0.55 to 3.13 mg/mL for PZA and 1.56 to 6.25 mg/mL for EMB. Concerning antifungal drugs, all the strains were susceptible, with MIC values ranging from 0.0625 to 0.25 Âg/mL for AMB; 15.62 to 62.5 Âg/mL for FLC; 0.0039 to 0.0312 Âg/mL for ITC, and 0.00156 to 0.25 Âg/mL for VRZ. When associating antituberculosis drugs with azole derivatives, all associations inhibited the in vitro growth of H. capsulatum strains, and synergy was observed for the nine combinations tested. Analogs of isoniazide presented MICs of 2, 4, 8 and 15-fold better than the standard antituberculosis drug. Basing on micromorphological analysis, the lowest quantification of macroconidia/field (0-10) was observed for 11, 10, 6 and 7 strains previously grown onto potato agar, BHI agar, malt agar and lactritmel agar, respectively. Malt agar was the most adequate medium for the production of macroconidia, 10-50 and >50/field, with a total of six strains; followed by lactritmel agar, with 5 strains. Concerning the relationship between MIC and culture medium used during the test, it was observed that inoculum from strains grown onto malt agar and BHI agar allowed the detection of the MIC for 11 strains. On the other hand, for those inocula grown onto potato agar and lactritmel agar, the MIC values were not detected for 8 and 5 strains, respectively. The results of this study provide additional data on the antifungal potential of antituberculosis drugs and their interactions with azole derivatives. However, new studies are necessary in order to determine the mechanism of action of these compounds on fungal cellular metabolism.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In recent years, advances in computer architecture and lipid force field parameters have made Molecular Dynamics (MD) a powerful tool for gaining atomistic resolution of biological membranes on timescales that other tools simply cannot explore. With many key biological processes involving membranes occurring on the nanosecond timescale, MD allows us to probe the dynamics and energetics of these interactions in molecular detail. Specifically, we can observe the interactions taking place as a peptide or protein comes into contact with a lipid bilayer, and how this may shape or alter the bilayer either locally (changes in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane curvature). The resolution achieved through atomistic MD can be directly compared with other tools such as NMR and EPR to gain a full perspective of how these biological systems behave over different timescales. As my background is in computational physics, this thesis not only looks into broadening our understanding of various interactions with biological membranes, but also into the development of construction and analytical software to assist in my research and benefit others in the field. One aspect of biological membranes that could vastly benefit from MD simulations is that of antimicrobial peptides (AMPs). These peptides primarily target and destroy microbes by permeabilising the cell membrane through a variety of proposed mechanisms, where each mechanism relies on the AMP to adopt specific conformations upon contact with bacterial membranes. In this thesis, I present an investigation into the interactions between a synthetic AMP and an inhibitor peptide designed to regulate antimicrobial activity through the formation of a coiled coil structure, which restricts the AMP from adopting new conformations. Simulations captured the spontaneous formation of coiled coils between these peptides, and specific residues in their sequences were identified that promote unfolding. This knowledge may lead to better design of coiled coil forming peptides. Another aspect of biological membranes that can be explored with MD is the interactions between model bacterial membranes and amphipathic helices, such as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix is responsible for anchoring the MinD protein to the inner membrane of Bacillus subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit sensitivity to transmembrane potentials (TMVs), whereby its localisation and binding affinity to bacterial membranes are disrupted upon removal of the TMV. Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup region of a model bacterial membrane. Analytical software was constructed to measure the membrane properties of the lipids surrounding inserted MinDMTS peptides, which revealed splayed lipid tails and suggests the MinD-MTS may be capable of inducing membrane curvature. Additional simulations were conducted to investigate the influence of a TMV on model bacterial membranes, where software was constructed to measure changes in membrane properties. An analysis of these simulations suggests that a TMV is capable of lowering the transition temperature of a model bacterial membrane by a few degrees, yielding increased fluidity in the lipids and increased perturbations on the membrane surface. Finally, another aspect of biological membranes that can be explored through MD is that of electroporation. This induction of transient water pores in cell membrane provides an exciting aspect for drug delivery applications into cells, whereby electric fields are applied to cells to increase the uptake of therapeutic drugs. Simulations of membranes with high voltage TMVs were conducted that sought to investigate the implications of electroporation across a variety of bilayer compositions at different temperatures. Software was constructed to measure changes in membrane and system properties, which revealed that pore formation occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL), implying that bilayers with bulkier lipids or those transitioning from gel to fluid will experience smaller TMVs and fewer pore formations. These simulations also revealed lipid flip-flopping through pores, where charged lipids tended to translocate in the direction of the electric field to produce an asymmetrically charged bilayer. Finally, simulations utilising charged peptides with membranes yielded electroporation effects, whereby the charged peptides generate an identical TMV to those produced by an ion imbalance of equal magnitude. This suggests that charged peptides, such as AMPs, may be capable of permeabilising cell membranes through electroporation mechanisms.

Class of 2011 Abstract / OBJECTIVES: To evaluate the appropriateness of discharge antimicrobial medications for UTI in an adult population based on therapy prescribed. METHODS: In this retrospective chart review study the appropriateness of discharge antimicrobial therapy for patients admitted to an academic medical center during 3 weeks in 2010 was assessed based on culture results, estimated renal function, reported drug allergies, route of administration, and change in UTI from in-house to discharge prescribed therapy. RESULTS: A total of 35 patients with discharge UTI antimicrobials within the study period met inclusion criteria and were evaluated. According to available urinary culture and susceptibility data, 22 of 35 (62.8%) of received an appropriate antimicrobial therapy. Based on reported gastrointestinal function, all 35 patients could take oral medications but two patients with an appropriate oral therapy option received intravenous therapy. All patients were discharged with antimicrobials that were appropriate according to patients’ reported drug allergies and only one patient received an antimicrobial agent that was inappropriately adjusted based on the patient’s estimated renal function. UTI antimicrobial therapies were the same at 24 hours prior to discharge and as the discharge antimicrobial in 100% of patient cases evaluated CONCLUSION: The antimicrobial UTI discharge therapy was evaluated for appropriateness based on urine culture results, patients’ allergies, and patients’ estimated renal function. Overall, antimicrobial therapy was only appropriate in 22 of 35 (62.8%) of patients based on the available culture results.

Sorghum grains contain phenolic compounds that have been shown to have many favourable effects. In this study the levels of phenolic compounds in condensed tannin and condensed tannin-free sorghums have been determined and the antimicrobial activity of phenolic extracts from bran fractions of the respective sorghums has been further evaluated against pathogenic bacteria Bacillus cereus ATCC 1178, Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 7644. Defatted bran fractions prepared from a condensed tannin sorghum variety (red) and a condensed tannin-free sorghum variety (white) were analysed for their content of total phenols and condensed tannins. Total phenols were determined using the Folin-Ciocalteu method and condensed tannins with the vanillin-HCL method. Total phenols and condensed tannins of the bran fractions were extracted with aqueous acetone (75 % v/v) and acidified methanol (1% HCL v/v in methanol) respectively, using a bran-to-solvent ratio of 1:4 (w/v). Red sorghum bran contained a higher amount of total phenols and condensed tannins (33.18 mg tannic acid equivalent/g and 117.98 mg catechin equivalent/g of the bran fractions, respectively) than white sorghum bran (6.81 mg tannic acid equivalent/g and 8.52 mg catechin equivalent/g of the bran fractions, respectively). Freeze-dried sorghum crude phenolic extracts (CPE) obtained from defatted bran fractions of condensed tannin and condensed tannin-free sorghum varieties were evaluated for their antimicrobial activities against Bacillus cereus ATCC 1178, Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 7644 pathogenic bacteria. The extracts were tested at 1, 2, 4 and 20 % concentrations (w/v) in methanol using the paper disc diffusion method and absolute methanol was used as a control. The condensed tannin-free sorghum CPE at concentrations 1, 2 and 4 % had no inhibitory effects on the bacteria tested but was effective against Gram-positive bacteria, B. cereus ATCC 1178 and L. monocytogenes ATCC 7644 at a concentration of 20 %. The condensed tannin sorghum CPE was effective against B. cereus ATCC 1178 and L. monocytogenes ATCC 7644 at concentrations 1 , 2 , 4 and 20 %. None of the tested sorghum extracts inhibited the Gram-negative bacteria, E. coli ATCC 25922. Phenolic extracts from condensed tannin sorghum may be used as antimicrobial agents to prevent the growth of Gram-positive bacteria, B. cereus ATCC 1178 and L. monocytogenes ATCC 7644. / Dissertation (MSc (Food Science and Technology))--University of Pretoria, 2007. / Food Science / unrestricted

Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate. / Land and Food Systems, Faculty of / Graduate

This dissertation documents the development of an environmental framework for monitoring antimicrobial resistance gene (ARG) dissemination in the aquatic environment. The work opens with a review of the relevant literature and outlines the importance of an environmental framework for monitoring ARG dissemination as part of antimicrobial resistance risk assessments. The ability to interrogate sequencing data quickly and easily for the presence of ARGs is crucial in order to facilitate their monitoring in the environment. As current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria in the environment were limited in their effectiveness and scope, the dissertation begins by describing the design and implementation of a Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally-acquired ARGs in raw sequencing data. The suitability of metagenomic methods for monitoring the ARG content of effluents from faecal sources was then assessed via a pilot study of a river catchment. Novel metagenomes generated from effluents entering the catchment were interrogated for ARGs. The relative abundance of ARGs in effluents were determined to be higher relative to the background environment, as were sequences relating to human and animal pathogens and mobile genetic elements. Thus, effluents were implicated in the dissemination of ARGs throughout the aquatic environment. To determine if ARGs were potentially in use in the environment, the expression of ARGs within effluents was then evaluated across a series of longitudinal samples through the use of metatranscriptomics, and the presence of potential environmental antimicrobial selection pressures was examined. This demonstrated that the abundance of ARGs, as well as antimicrobial usage at the effluent source, was correlated with the transcription of ARGs in aquatic environments. The work described in this dissertation has also found that horizontally transmitted ARGs were present in pathogenic endospore-forming bacteria commonly found across the aquatic environment, potentially providing a mechanism for ARG persistence in the environment. Finally, these findings were integrated into a universal framework for monitoring ARG dissemination in aquatic environments and used to highlight the developments required to incorporate this framework into future environmental ARG research and to facilitate antimicrobial resistance risk assessments.

Peptides represent a source of novel therapeutics for recalcitrant human diseases, but screening for bioactivity from natural or synthetic sources can be uneconomic. In contrast, in vivo expression of peptides from DNA libraries in a heterologous host such as Escherichia coli may combine production with screening. This dissertation aimed to use such an approach to discover novel bioactive peptides in a high throughput and cost-effective manner, with a focus on antimicrobials and antiaggregants as proof-of-principle. Antimicrobial peptides (AMPs) are innate defence effectors that may combat antibiotic-resistant pathogens. An inducible, autocleaving fusion tag was utilised to produce the model murine cathelicidin K2C18, along with a number of variants, which exhibited varying degrees of antimicrobial activity against a panel of microbes. Importantly, K2C18 also exhibited a bacteriostatic effect in vivo when secreted to the periplasm. This allowed for the implementation of an in vivo whole cell screen for novel AMPs, using genomic DNA libraries as an input. One putative hit, the peptide S-H4, showed similar in vivo behaviour to K2C18 and was active when added exogenously to microbial cultures. A second in vivo screen was constructed to search for inhibitors of Aβ42 aggregation, a process implicated in Alzheimer’s disease. The aggregation state of Aβ42 was coupled to the fluorescence of a chromophore fusion partner, and used to screen co-expressed peptides from a random DNA library for putative antiaggregants. Additionally, the system incorporated an internal fluorescent reference to allow ratiometric comparison between samples. Several hits were identified and further validated using flow cytometry, with work ongoing to assess their activity in vitro. Proof-of-principle of these two screens was achieved, indicating that such in vivo approaches to bioactive peptide discovery could lead to the development of new and useful therapeutics.

Antimicrobial resistance is a serious and imminent threat to human health, though its rise may be controlled with improved stewardship strategies that limit the emergence and spread of resistant strains. Motivated by theoretical models from population genetics and ecology, my M.Sc. experimentally evaluates how varying drug availability in either time or space impacts the prevalence of resistance in a population. By experimentally evolving Pseudomonas aeruginosa under different antibiotic selection regimes in vitro, I show that spatial, but not temporal, drug free refuges delay the fixation of resistance by promoting the coexistence of sensitive and resistant genotypes. Second, I establish that this polymorphism is underlain by a trade-off between resistance and growth rate in the absence of antibiotic that underpins the maintenance of diversity through negative frequency dependent selection. Third, I demonstrate that spatially varied drug selection cannot prevent the fixation of resistance because continued selection leads to the evolution of resistant types that pay smaller costs of resistance and gradually displace sensitive strains. These results provide insight into the fate of diversity under long-term selection and highlight the value of incorporating the principles of evolutionary ecology into antimicrobial resistance stewardship.

This study aims to evaluate the removal efficiency of microbial contaminants from two hospitals on-site Wastewater Treatment Plants (WWTPs) in Saudi Arabia. Hospital wastewaters often go untreated in Saudi Arabia as in many devolving countries, where no specific regulations are imposed regarding hospital wastewater treatment. The current guidelines are placed to ensure a safe treated wastewater quality, however, they do not regulate for pathogenic bacteria and emerging contaminants. Results from this study have detected pathogenic bacterial genera and antibiotic resistant bacteria in the sampled hospitals wastewater. And although the treatment process of one of the hospitals was able to meet current quality guidelines, the other hospital treatment process failed to meet these guidelines and disgorge of its wastewater might be cause for concern. In order to estimate the risk to the public health and the impact of discharging the treated effluent to the public sewage, a comprehensive investigation is needed that will facilitate and guide suggestions for more detailed guidelines and monitoring.