Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA / Science, Faculty of / Microbiology and Immunology, Department 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.

N. Gonorrhoeae is a major public health concern due to its capacity to develop antibiotic resistance and its potential complications including pelvic inflammatory disease, epididymitis, infertility, and disseminated infection. In order to keep up with resistance trends, the treatment recommendations for gonorrhea have changed frequently. In other areas of medicine, guideline adherence has been shown to be limited, even without frequent guideline changes. In the case of gonorrhea, inappropriate treatment can have serious individual and public health implications, making the quality of and compliance with clinical guidelines critical. This thesis is a three-part mixed methods research project assessing the development and uptake of gonorrhea treatment guidelines. We conducted a systematic review of current gonorrhea treatment guidelines and used the AGREE II tool to assess the quality of guideline development (Chapter 2). We observed that guideline quality varied greatly with many guidelines having weaknesses in their use of existing evidence to develop recommendations, their reporting of potential conflicts of interest and how they were addressed, and their consideration of barriers to the implementation of their recommendations. We then assessed physician adherence to first-line treatment recommendations in Ontario by conducting a segmented time series analysis of Ontario gonorrhea treatment data from iPHIS, the province’s reportable disease database (Chapter 3). Following the introduction of new guidelines that recommended substantial changes from current practice, we found very dramatic drops in guidelines adherence that then improved slowly over time. We then explored the use of process mapping as a tool to look at the local management of cases in the City of Ottawa by following them across the various possible treatment pathways (Chapter 4). Here, we noted differences in practice between the management of cases at Ottawa Public Health’s Sexual Health Clinic and the management of cases elsewhere in the community.

Orientador: Lourenço Correr Sobrinho, Lucas Zago Naves / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T09:40:26Z (GMT). No. of bitstreams: 1 Soares_EvelineFreitas_M.pdf: 1985707 bytes, checksum: 7527104238577fafc96b875141e96321 (MD5) Previous issue date: 2013 / Resumo: O propósito deste estudo in vitro foi a avaliar a ação da radiação na interface de união dente humano-compósito fotoativado, através do ensaio de resistência de união à microtração (?TBS) variando: dois sistemas adesivos, com ou sem aplicação da Doxiciclina, com ou sem radiação e análise do padrão de fratura. A superfície oclusal de sessenta terceiros molares humanos foi desgastada com lixa de carbeto de silício de granulação 600. Os dentes foram divididos em 3 grupos (n=20) de acordo com o sistema adesivo (Adper Scotchbond MP e Clearfil SE Bond), com ou sem aplicação da Doxiciclina e submetidos ou não a radiação com 60 Gy, fracionados em doses diárias de 2Gy durante 5 dias por semana, num período de 6 semanas. Grupo Controle (n=20) não recebeu radioterapia; Grupo IrrRe (n=20) submetido à radioterapia antes de ser restaurado; e, Grupo ReIrr (n=20) submetido à radioterapia após ser restaurado. Um bloco de resina Z250 (3M ESPE) com 5 mm de espessura foi construído sobre a dentina com os sistemas adesivos, preparados de acordo com as recomendações do fabricante e cada camada de 2 mm de espessura foi fotoativada por 40 segundos com o aparelho UltraLume 5 (Ultradent). Após 24 horas à 37º C, as amostras foram seccionadas perpendicularmente à área de união, para obter palitos com área de secção transversal de 1mm2. Em seguida, os palitos foram submetidos ao ensaio de resistência de união à microtração em máquina de ensaio universal (EZ-Test) a velocidade de 0,5 mm/minuto. A análise do modo de falha foi observada em lupa estereoscópica (Leica Mz 9.5). Os dados foram submetidos à Análise de Variância com 3 fatores e ao teste de Tukey (5%) e mostram que nenhuma diferença estatística foi observada entre os sistemas adesivos. O uso do antimicrobiano Doxiciclina reduziu significativamente a resistência de união, quaisquer que fossem as demais condições. A radiação das amostras antes do procedimento da restauração em compósito reduziu significativamente a resistência de união à microtração. O modo de falha coesiva na dentina foi predominante nas amostras submetidas à radiação antes do procedimento restaurador, e mistas nas amostras restauradas e submetidas à radiação. Falhas mistas e adesivas foram observadas no grupo controle / Abstract: The purpose of this in vitro study is to evaluate radiation effect on human interface bonding to dentin- light cured composite, using microtensile bond strength test (?TBS) ranging: two adhesive systems, with or without Doxycycline application; with or without radiation submission, and the analysis of mode of failure. Flat occlusal dentin of sixty third human molars was exposed using wet ground with 600-grit SiC paper. The teeth were distributed into 3 groups (n=20) according to adhesive systems (Adper Scotchbond MP and Clearfil SE Bond), with or without application of Doxycycline and submitted or not to 60 Gy radiation, in 2 Gy daily fractionated doses during 5 days per week in a total of 6 weeks. Control group (n=20) did not go throw radiotherapy; Group IrrRe (n=20), submitted to radiotherapy before the restoration procedure; Group ReIrr (n=20) submitted to (IrrRe; n=20) submitted to radiotherapy after the restoration procedure. A block of 5mm thickness resin composite Z250 (3M ESPE) was constructed on dentin using adhesive systems according to manufacturer's instructions and each 2mm thickness was light cured for 40 seconds using a light curing unit UltraLume 5 (Ultradent). After 24 h storage at 37oC in deionized water, the specimens were sectioned perpendicularly to bond area to obtain beams in a sectional area of 1 mm2. After that, the beams were submitted to microtensile bond strength (?TBS) test in a universal test machine (EZ-Test) at a crosshead speed of 0.5mm/min. The mode of failure analyses was observed under optical microscopy (Leica Mz 9.5). Data were submitted to two-way ANOVA, Tukey's test (p<0.05) and showed that no statistical difference was observed between the adhesive systems. The use of antimicrobial Doxycycline decreased significantly bond strength, whatever other conditions. The specimens submitted to radiation before restoration procedure decreased significantly microtensile bond strength. Dentin cohesive failure mode was predominantly for specimens submitted to radiation before restoration procedure, and mixed failure mode for specimens restored then submitted to radiation. Mixed and adhesive failures were observed in control group / Mestrado / Materiais Dentarios / Mestra em Materiais Dentários

No description available.

Silk is a natural protein produced by members of the class Arachnida (over 30,000 species of spiders) and by several worms. Silk-based materials have been investigated for medical and biotechnological applications for many years. Although silkworm silk has been studied extensively because of ready availability of the protein, lately the advancements in recombinant technology has made production of spider silk proteins increasingly available. Due to the characteristics like biocompatibility, biodegradability and mechanical strength, silk is highly desirable as a biomaterial for medical purpose. Along with this, techniques for functionalization, has further aided in the development of silk into highly sophisticated material for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein Masp2. Two distinct approaches were used, chemical modification and genetic fusion. In the first modification, we created an infection responsive silk nanospheres by chemically grafting a thrombin sensitive peptide to the silk protein encapsulating antibiotic. These particles were then evaluated for in vitro infection responsive drug release and antimicrobial activity. From these assessments, we found that these particles can release the drug effectively in the presence of infection providing the evidence that these particles are enzyme responsive and can be used to formulate targeted drug release. In the second modification, spider silk was genetically modified with a heparin binding peptide to create a fusion protein which can prevent both thrombosis and infection simultaneously. This fusion protein was evaluated for its heparin binding ability and anticoagulant properties in its solution form. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property. After establishing these properties, next this fusion protein was utilized as a coating for hemodialysis catheter. Deposition of coating was evaluated after which anticoagulant and anti-infective properties of the protein as a coating material was investigated. This thesis provides evidence of successful production of a recombinant silk-based biopolymer that can be chemically and genetically embedded with a various functional motif to create a hybrid product for different applications.

<p><i>Mycobacterium abscessus</i> is an emerging infectious pathogen capable of causing pulmonary disease similar to tuberculosis, but has many intrinsic and extrinsic properties making it more drug-resistant than <i>Mycobacterium tuberculosis</i>. Current treatments, including those used for <i>M. tuberculosis</i> infection, have had poor results. Although <i>in vitro</i> studies have shown promise with drug treatment for this microorganism, clinical trials have been mostly unsuccessful. An <i>in vitro </i>model that mimics the physiological stresses encountered within the human body is likely to enable the discovery of mechanisms of antibiotic resistance used by <i>M. abscessus</i> during infection. Therefore, we subjected <i>M. abscessus</i> to a combination of stresses thought to be encountered by mycobacteria inside the human body. We subjected the pathogen to low oxygen, low pH, and nutrient starvation. This is the first report on subjecting <i>M. abscessus</i> to such a combination of stresses. It is also the first to investigate the effect of the combination of stresses on the tolerance of the pathogen to antibiotics, and the effect of efflux pump inhibitors under such conditions. We found that under these conditions, <i>M. abscessus</i> entered a non-replicating state. We investigated whether the multiple-stressed <i>M. abscessus</i> displayed altered tolerance to antibiotics commonly used to treat infection, and whether efflux pump inhibitors affected the antibiotic resistance under such conditions.<i> </i>We found that when subjected to our multiple stress model, <i>M. abscessus</i> in the reactivating phase had higher tolerance to erythromycin in combination with efflux pump inhibitors verapamil and reserpine compared to non-replicating <i>M. abscessus</i>. Reactivating phase cells had a higher tolerance to antibiotic erythromycin than non-replicating cells. Reactivating phase cells also showed antibiotic tolerance in the presence of ATP. This physiologically-relevant experimental model for <i>M. abscessus</i> could potentially be used in discovering the mechanisms of antibiotic resistance in the pathogen.</p>

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

Rhodococcus, a species of bacteria commonly found in the soil, is a under-explored producer of small bioactive compounds including siderophores, pigments and antibiotics.. MTM3W5.2 is a strain of Rhodococcus that was previously discovered to produce an antibiotic-like compound that has inhibitory effects on other Rhodococcus strains, including the veterinary pathogen, R. equi. The biosynthetic gene cluster responsible for production of the antibiotic has been identified, and a small gene, BTZ20_3964 at the start of the operon is believed to be the regulator of the gene cluster. To test this, a deletion construct was created using an overlap-extension PCR method to remove most of gene 3964. The deletion construct was cloned into the plasmid pEX18Km, along with a kanamycin resistance marker. The deletion construct, pEX18Km3964AD was transformed into competent MTM3W5.2 cells for a double crossover recombination event to replace the functional gene with the deletion construct, using the KanR gene to select for the merodiploids, and the SacB gene to select for the deletion mutants after the second recombination event. The deletion mutant’s ability to produce the inhibitory compound will be determined. Production of the compound will be restored by complementing the deletion mutant with the functional gene cloned into the expression plasmid pDD57.

Thesis advisor: Eranthie Weerapana / Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The overuse and misuse of broad-spectrum antibiotics has contributed to the antibiotic resistance crisis. Additionally, treatment of infections with broad-spectrum antibiotics can cause disruption to the host gut microbiome. The development of narrow-spectrum antibiotics would be ideal to avoid unnecessary cultivation of antibiotic resistance and damage to the human microbiota. Bacteria present many mechanisms of resistance, including modulating their cell surface with amine functionalities. In an age where infections are no longer responding to typical antibiotic treatments, novel drugs that target the characteristics of antibiotic resistance would be beneficial to remedy these defiant infections. Herein, we describe the utility of iminoboronate formation to target the amine- presenting surface modifications on bacteria, particularly those that display antibiotic resistance. Specifically, multiple 2-acetylphenylboronic acid warheads were incorporated into a peptide scaffold to develop potent peptide probes of bacterial cells. Further, by engineering a phage display library presenting the 2-acetylphenylboronic acid moieties, we were able to perform peptide library screens against live bacterial cells to develop reversible covalent peptide probes of target strains of bacteria. These peptide probes, which were developed for clinical strains of Staphylococcus aureus and Acinetobacter baumannii which display resistance, can label the target bacterium at submicromolar concentrations in a highly specific manner and in complex biological milieu. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. It is conceivable that this phage display platform is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics. Furthermore, it is intriguing to speculate that even higher potency binding could be accomplished with better designed phage libraries with dynamic covalent warheads. This work is currently underway in our laboratory. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.