With the studies performed in this theses the local drug delivery technique FibMat developed by the biotech company AddBIO, was shown to be applicable to other plasma proteins and drugs than the fibrinogen-bisphosphonate combination that is today being commercialized. Hence the potential for a broader field of application was demonstrated. The application targeted today is as a surface modification giving improved strength to bone around screws used in bone implants. The effect of changing protein and manufacturing conditions was studied with null ellipsometry. It was demonstrated that with changes in incubation temperature, pH and salinity the fibrinogen could be successfully exchanged for the plasma proteins human serum albumin and immunoglobulin G. With liquid scintillation counting it was shown that the developed protein multilayers were able to absorb and release the bone strengthening drug alendronic acid in levels comparable to that of the fibrinogen based ditto. Disk susceptibility tests with the bacteria S. Aureus showed a potential for antibacterial functionalization with gentamicin. The release was, in the case of the fibrinogen multilayer, detectable up to 48 hours. Similar test revealed an inability of silver nanoparticle incorporated protein multilayers to achieve inhibitory levels.

The life of microorganisms is characterized by two main tasks, rapid growth under conditions permitting growth and survival under stressful conditions. The environments, in which microorganisms dwell, vary in space and time. The microorganisms innovate diverse strategies to readily adapt to the regularly fluctuating environments. Phenotypic heterogeneity is one such strategy, where an isogenic population splits into subpopulations that respond differently under identical environments. Bacterial persistence is a prime example of such phenotypic heterogeneity, whereby a population survives under an antibiotic attack, by keeping a fraction of population in a drug tolerant state, the persister state. Specifically, persister cells grow more slowly than normal cells under growth conditions, but survive longer under stress conditions such as the antibiotic administrations. Bacterial persistence is identified experimentally by examining the population survival upon an antibiotic treatment and the population resuscitation in a growth medium. The underlying population dynamics is explained with a two state model for reversible phenotype switching in a cell within the population. We study this existing model with a new theoretical approach and present analytical expressions for the time scale observed in population growth and resuscitation, that can be easily used to extract underlying model parameters of bacterial persistence. In addition, we recapitulate previously known results on the evolution of such structured population under periodically fluctuating environment using our simple approximation method. Using our analysis, we determine model parameters for Staphylococcus aureus population under several antibiotics and interpret the outcome of cross-drug treatment. Next, we consider the expansion of a population exhibiting phenotype switching in a spatially structured environment consisting of two growth permitting patches separated by an antibiotic patch. The dynamic interplay of growth, death and migration of cells in different patches leads to distinct regimes in population propagation speed as a function of migration rate. We map out the region in parameter space of phenotype switching and migration rate to observe the condition under which persistence is beneficial. Furthermore, we present an extended model that allows mutation from the two phenotypic states to a resistant state. We find that the presence of persister cells may enhance the probability of resistant mutation in a population. Using this model, we explain the experimental results showing the emergence of antibiotic resistance in a Staphylococcus aureus population upon tobramycin treatment. In summary, we identify several roles of bacterial persistence, such as help in spatial expansion, development of multidrug tolerance and emergence of antibiotic resistance. Our study provides a theoretical perspective on the dynamics of bacterial persistence in different environmental conditions. These results can be utilized to design further experiments, and to develop novel strategies to eradicate persistent infections. / Das Leben von Mikroorganismen kann in zwei charakteristische Phasen unterteilt werde, schnelles Wachstum unter Wachstumsbedingungen und Überleben unter schwierigen Bedingungen. Die Bedingungen, in denen sich die Mikroorganismen aufhalten, verändern sich in Raum und Zeit. Um sich schnell an die ständig wechselnden Bedingungen anzupassen entwickeln die Mikroorganismen diverse Strategien. Phänotypische Heterogenität ist eine solche Strategie, bei der sich eine isogene Popolation in Untergruppen aufteilt, die unter identischen Bedingungen verschieden reagieren. Bakterielle Persistenz ist ein Paradebeispiel einer solchen phänotypischen Heterogenität. Hierbei überlebt eine Popolation die Behandlung mit einem Antibiotikum, indem sie einen Teil der Bevölkerung in einem, dem Antibiotikum gegenüber tolerant Zustand lässt, der sogenannte "persister Zustand". Persister-Zellen wachsen unter Wachstumsbedingungen langsamer als normale Zellen, jedoch überleben sie länger in Stress-Bedingungen, wie bei Antibiotikaapplikation. Bakterielle Persistenz wird experimentell erkannt indem man überprüft ob die Population eine Behandlung mit Antibiotika überlebt und sich in einem Wachstumsmedium reaktiviert. Die zugrunde liegende Popolationsdynamik kann mit einem Zwei-Zustands-Modell für reversibles Wechseln des Phänotyps einer Zelle in der Bevölkerung erklärt werden. Wir untersuchen das bestehende Modell mit einem neuen theoretischen Ansatz und präsentieren analytische Ausdrücke für die Zeitskalen die für das Bevölkerungswachstums und die Reaktivierung beobachtet werden. Diese können dann einfach benutzt werden um die Parameter des zugrunde liegenden bakteriellen Persistenz-Modells zu bestimmen. Darüber hinaus rekapitulieren wir bisher bekannten Ergebnisse über die Entwicklung solch strukturierter Bevölkerungen unter periodisch schwankenden Bedingungen mithilfe unseres einfachen Näherungsverfahrens. Mit unserer Analysemethode bestimmen wir Modellparameter für eine Staphylococcus aureus-Popolation unter dem Einfluss mehrerer Antibiotika und interpretieren die Ergebnisse der Behandlung mit zwei Antibiotika in Folge. Als nächstes betrachten wir die Ausbreitung einer Popolation mit Phänotypen-Wechsel in einer räumlich strukturierten Umgebung. Diese besteht aus zwei Bereichen, in denen Wachstum möglich ist und einem Bereich mit Antibiotikum der die beiden trennt. Das dynamische Zusammenspiel von Wachstum, Tod und Migration von Zellen in den verschiedenen Bereichen führt zu unterschiedlichen Regimen der Populationsausbreitungsgeschwindigkeit als Funktion der Migrationsrate. Wir bestimmen die Region im Parameterraum der Phänotyp Schalt-und Migrationsraten, in der die Bedingungen Persistenz begünstigen. Darüber hinaus präsentieren wir ein erweitertes Modell, das Mutation aus den beiden phänotypischen Zuständen zu einem resistenten Zustand erlaubt. Wir stellen fest, dass die Anwesenheit persistenter Zellen die Wahrscheinlichkeit von resistenten Mutationen in einer Population erhöht. Mit diesem Modell, erklären wir die experimentell beobachtete Entstehung von Antibiotika- Resistenz in einer Staphylococcus aureus Popolation infolge einer Tobramycin Behandlung. Wir finden also verschiedene Funktionen bakterieller Persistenz. Sie unterstützt die räumliche Ausbreitung der Bakterien, die Entwicklung von Toleranz gegenüber mehreren Medikamenten und Entwicklung von Resistenz gegenüber Antibiotika. Unsere Beschreibung liefert eine theoretische Betrachtungsweise der Dynamik bakterieller Persistenz bei verschiedenen Bedingungen. Die Resultate könnten als Grundlage neuer Experimente und der Entwicklung neuer Strategien zur Ausmerzung persistenter Infekte dienen.

The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-­lactams, (2) expression of PBPs with low-affinity for β-­lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-­lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance. The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-­lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-­lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-­lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction. The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.

BACKGROUND: Trachoma is a leading cause of avoidable blindness. Currently, trachoma is endemic in 57 countries, infects approximately 84 million people globally, and continues to threaten over 10 % of the world’s population with the risk of blindness. Caused by the bacteria Chlamydia trachomatis, blindness due to trachoma is caused by repeated eye infection resulting in the inflammation of the upper eyelid eventually leading the upper lid to pull inward scratching and tearing the cornea causing it to become opaque resulting in loss of vision. The World Health Organization recommends eliminating trachoma as a public health problem using the SAFE strategy: Surgery, Antibiotic, Face washing and Environmental control. OBJECTIVES: This review examined the benefits of the added value of water, sanitation, and hygiene education interventions on preventive mass drug administration for trachoma. METHODS: Trials were identified from MEDLINE, PubMed, and LISTA EBSCO databases using a series of search terms. No restrictions were put on study date, location, design, or language of publication. The abstracts were examined from each of the searches, and any abstract describing risk factors, survey results of mass drug administration (MDA), or providing a general overview of trachoma were automatically discarded. Full text of papers including the combined use of key words including SAFE, WASH, intervention, impact, added value, MDA, azithromycin/ Zithromax® were obtained for review. Twelve full texts articles were retrieved all relevant information were placed in a standardized data extraction form. MAIN RESULTS: Three studies met the complete criteria for inclusion. All studies found a significant change in reduction of active trachoma prevalence. One study focused on the added benefit of antibiotic and environmental components on hygiene education delivered by radio. Another trial compared two villages; the control community performed MDA and the surgery while the intervention village added the F and E components. The final study as well focused the added benefit of ‘F’ and ‘E’ on ‘A’. Two of the three studies found this reduction was from the added benefit of face washing ‘F’ and environmental control ‘E’ to antibiotic use. CONCLUSIONS: In order to eliminate blinding trachoma as a public health problem, recurrence of the active form of the disease must be interrupted before repeated scarring leads to trichiasis. The antibiotic component of the SAFE strategy is a quick fix to the immediate problem. The ‘F’ and ‘E’ components are the more sustainable interventions, yet little research has been done on the actual amount of added value the individual ‘A’‘F’&’E’ components have to one another. After thorough review of the articles, articles were found which documented the ‘F’ and ‘E’ components provide significant value to the overall decrease of prevalence of active. However, the limited results of the search suggest more research can better elucidate the ability of the ‘F’ and ‘E’ components to reduce trachoma prevalence and ultimately impact blinding.

Neracionalus antimikrobinių vaistų (AMV) vartojimas didina mikroorganizmų atsparumą, gydymo kaštus ir hospitalizacijos trukmę. Didėjantis atsparumas AMV yra svarbi visuomenės sveikatos problema. Magistro darbo tikslas - kokybiškai ir kiekybiškai įvertinti AMV skyrimo KMU klinikose paplitimą bei jo pokyčius ir atitikimo racionalaus AMV vartojimo rekomendacijoms ypatumus ir dėsningumus. Tyrimas atliktas naudojant „skersinio pjūvio“ metodiką. 2007 m. kovo mėnesį vienmomentinio apžvalginio tyrimo metu (2 savaičių laikotarpyje) atrinktos ir išanalizuotos pacientų, kurie gauna AMV, ligos istorijos ir paskyrimai 32-iuose KMU klinikų skyriuose. AMV vartojimas yra racionalus, jei jų skyrimas atitinka racionalaus AMV vartojimo rekomendacijoms. Peržiūrėta 1213 paskyrimų, AMV buvo skirti 265 pacientams. Rekomendacijų neatitiko pusė (54 proc.) visų AMV paskyrimų. Daugiau kaip pusė (58 proc.) gydymo tikslu skirtų empirinių AMV paskyrimų vertinti kaip neatitinkantys racionalaus AMV rekomendacijų. Vertinant AMV skyrimo atitikimą rekomendacijoms atskiruose skirtingo profilio skyriuose daugiausia neatitikusių paskyrimų nustatyta chirurginio (74 proc.) ir terapinio (55 proc.) profilio skyriuose, atitikusių – pediatrinio (71proc.) ir intensyvios terapijos (60 proc.) profilių skyriuose. Ryšio tarp skyriaus profilio ir atitikimo rekomendacijoms nenustatyta. (r = 0,5387 , p = 0,4613, Spearmano koreliacija). Didžiąją dalį neatitinkančių rekomendacijų paskyrimų atvejų (56,86 proc.) sudarė... [toliau žr. visą tekstą] / Irrational and excessive use of antibiotics increases resistance to these preparations. Antimicrobial resistance is a serious public health problem worldwide. The main goal of our study was to evaluate and to compare the non-adherence (NA) to guidelines (NfG) on rational antibiotic therapy and prophylaxis (ABT/P) in tertiary hospital setting among two years (2006 and 2007). A cross-sectional study was performed in order to collect the data for patients receiving prescriptions for antimicrobial agents in March, 2007. Descriptive and comparative data were processed and evaluated using Graph Pad Prism 4 statistics program. Rationality of antibiotic therapy was evaluated according for adherence to published guidelines. A total of 265 (21%) patients recieved antibiotic therapy. 54% of all events were considered as non-adherent.The majority of NA cases was determined in the departments of therapeutic profile (55%) and in surgical profile (74%). The adherent events - in paediatric profile(71%) and in the intensive therapy profile (60%). No dependence between the type of profile and adherence to guidelines was determined (r = 0,5387, p = 0,4613, Spearman correlation). The main reasons of NA were inapropriate dosage (57%) and choice of drug (43% ). NA case analysis revealed too high dose prescribed in 40/87 (46%) cases, too low dose 47/87 (54%)cases; too broad spectrum in 28/61 (46%) cases, too narrow - in 9/61(15%), 2/61 ( 3 %) case as unsafe and 22/61 (36%) cases... [to full text]

Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.

Caseinolytic protease (ClpP) is a cylindrical protease forming the core of protein degradation machinery in eubacteria. ClpP is tightly regulated and is non-functional without a member of the Clp-ATPases. A new class of antibiotics, termed ADEPs, bind to ClpP and allow for activation without the Clp-ATPases; leading to cell death. A more efficient synthetic route to the ADEPs utilizing solid-phase peptide synthesis was investigated. A linear peptide was synthesized, however attempts to close the depsipeptidic macrocycle via macrolactonization failed. Further attempts of assembling a branched depsipeptide for ring closure via a macrolactamization resulted in products that were not stable to cleavage conditions. A group of molecules termed Activators of Self-Compartmentalizing Proteases (ACP) were identified through a screen for activity towards ClpP. Compound ACP1 was synthesized along with twelve analogs and their activity towards ClpP evaluated. The project resulted in a compound with a higher activity than its natural product counterpart.

Background: Considerable amount of inappropriate antibiotic utilization has been observed in both acute and non-acute care hospitals. Literature has shown that strategies such as an order entry (OE) and computer decision support system (DSS) have improved prescribing practices in acute care settings. However, there is limited research on the effect of OE on antibiotic utilization in non-acute care settings. Objective: To determine the relationship between OE with DSS and antibiotic utilization in a complex continuing care and rehabilitation hospital. Methods: A retrospective analysis of OE and Pharmacy dispensing data, prospectively collected between July 1, 1999 and December 31, 2005, was conducted. Dispensing events for oral and intravenous antibiotics were merged with corresponding OE’s (when present) to form a final database of 4,739 dispensing events with 2,397 OE’s. The presence of OE and the proportion of OE to dispensing events formed the exposure variable while antibiotic utilization in defined daily dose (DDD) was calculated using dose and number of doses of an antibiotic. Antibiotic utilization was examined at the hospital and individual service in-patient unit levels (complex continuing care/CCC and rehabilitation medicine/REH). Statistical analysis consisting of multiple regression modeling was conducted to determine the association between use of OE and antibiotic utilization. Results: A best-fit model using multiple regression analysis at hospital level indicated a significant positive relationship between the presence of OE and antibiotic utilization when service, patient age, gender and antibiotic classes were accounted for. This model explained 11% of the variation in antibiotic utilization. No significant associations were found in the CCC in-patient unit while in the REH in-patient unit a significant positive relationship between the presence of OE and antibiotic utilization was observed. Similarly, antibiotic utilization increased significantly with increase in the proportion of OE to dispensing events at the hospital and REH in-patient unit levels but not in the CCC in-patient unit. Conclusion: The results of this study demonstrate that antibiotic utilization increased over the years following the inception of the OE system with DSS at the study hospital. Further research is required to examine the effect of OE with a rudimentary DSS on antibiotic utilization management in non-acute care. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2007-07-13 10:47:38.035

<p><font face="Tahoma"> <p align="left">The purpose of this study is to evaluate the need for prophylactic antibiotic treatment in third molar surgery and to establish specific guidelines for antibiotic prophylaxis in the department of Maxillo-Facial and Oral Surgery (MFOS) at Tygerberg Academic, Groote Schuur and Mitchells Plain Hospitals.</p> </font></p>

Environmental bacteria and especially soil bacteria are active producers of antibiotic molecules and most drugs used nowadays are isolated from saprophytic soil bacteria and these microorganisms have also evolved numerous resistance pathways leading to an arsenal of Antibiotic Resistance Genes Determinants (ARGD) known as the environmental resistome. A survey of ARGD prevalence is required in order to characterize this natural phenomenon with critical implications in our current infectious diseases management. In order to perform such analysis we compiled a set of 71 metagenomic datasets from various environmental origins: soils, oceans, lakes, human feces, indoor air, etc., and compared their sequences with a database of known antibiotic resistance gene determinants (ARGD). ARGD-annotated reads are found in every environment analyzed confirming their ubiquity. Soil is found to be the richest and shares a large part of ARGD with the human gut microbiome, indicating ARGD transfers between these environments. Experiments using qPCR and metagenomic DNA sequencing on soil samples from two sites with known and distinct antibiotic pollution history were conducted to understand how ARGD abundance and diversity in soil are affected when impacted by antibiotic molecules. The first site is a reference soil from a long-term experiment without history of antibiotic pollution (Rothamsted Park Grass, UK). Soil microcosms are setup with addition of either antibiotic-containg animal manure or pure molecules and incubated for 6 months to monitor changes in ARGD concentration following these perturbations. Our second study-site is a very remote settlement in French Guiana where antibiotics are available since recently and may have impacted the local soil microbial community. Soil samples are taken following a line-transect going from the village (antibiotic source) to 3km deep in the forest in a gradient of human-impact. Our results all confirm prevalence of ARGD in soil at significant abundance but also that ARGD distribution is more correlated to environmental factors such as soil type, microbial taxonomy composition or microcosms incubation conditions than antibiotic molecules exposure in both sites. Pathogens ARGD diversity is far lower than ARGD diversity found in the environment and not all the soil resistome is readily accessible for transfer. In order to characterize the soil mobile gene pool, a strategy is proposed to isolate specifically mobile DNA directly from the environment for sequencing purposes. Better knowledge on the microbial ecology factors limiting ARGD transfers to pathogens may greatly help us reduce the current threat on our limited medical antibiotic molecules resource.