Methicillin-resistant Staphylococcus aureus (MRSA) is among the most important pathogens affecting the human race in our time. In spite of recent medical advances, our therapeutic choices for MRSA infections remain limited due to the propensity of this organism to develop resistance to antimicrobials. Therefore, there is a continuing need to develop newer methods of treating MRSA infections. This dissertation examines the effects of bacteriophages 88 and 92 on ten clinical isolates of MRSA from the central Florida area. . For the majority of the MRSA isolates, bacteriophages 88 and 92 were unable to induce lysis. However, bacteriophage 88 was found to lyse MRSA Sample 94. Reduced cytotoxicity and apoptosis due to MRSA Sample 94 was also observed. This protective effect was most notable in the 1:10-6 concentration of MRSA 94 and bacteriophage 88. In addition, this effect was observable with both immediate inoculation of the cell culture with the MRSA concurrent with the bacteriophage and with bacteriophage applied one hour after initial inoculation of the MRSA. This effect was likely due to the increased replication of the bacteriophage in the actively growing bacteria found in the 1:10-6 samples. The bacteria in the 1:10-6 concentration were likely more able to replicate in comparison to the higher bacterial concentrations because of less competition between the bacteria for the limited nutrients in the 1:10-6 concentration. The long-term goal of this study is the development of a bacteriophage-containing ointment for the control of MRSA nasal carriage. In addition, the concept of bacteriophage therapy may open a new horizon in controlling infections such as those caused by MRSA. Finally, as for future studies, it would be informative to be able compare these results with other MRSA isolates and bacteriophages samples to examine the effects across a wider sample of bacteria and bacteriophages. In addition, it would be interesting to examine the possibility of being able to modify the bacteriophage in order to allow lysis of the previously resistant bacterial strains.

Streptococcus pneumoniae is a major bacterial pathogen which causes pneumoniae, meningitis, otitis media, and bacteremia. Currently there are two vaccines available, Pneumococcal Polysaccharide Vaccine (PPV) for adults and the Pneumococcal Conjugate Vaccine (PCV) for children. The PCV vaccine was developed in 2000 specifically for children and infants due to the ineffectiveness of the PPV vaccine in children. This is a cross sectional study of invasive S. pneumoniae in Florida during 2003. This study is designed to determine the population characteristics, clinically relevant antibiotic resistance patterns and specific risk factors for development of antibiotic resistance of invasive S. pneumoniae. Participants for the study of antimicrobial resistance will be selected if they are positive for invasive S. pneumoniae, and have been reported to the Florida Department of Health, Bureau of Epidemiology with a laboratory specimen collection date in 2003. A total of 1056 cases were reported. The incidence of invasive S. pneumoniae was calculated. Logistic regression was used to find an association between each risk factor and invasive S. pneumoniae. 95% Confidence Intervals were calculated to determine statistical significance. The incidence of invasive pneumococcal disease was calculated to be 6.61 per 100,000 persons (95% CI 6.21 -- 7.01). The incidence of drug resistant S. pneumoniae was calculated to be 3.3 per 100,000 persons (95% CI 3.03 -- 3.59).The incidence of penicillin resistant S. pneumoniae (PRSP) was estimated to be 2.6 per 100,000 persons (95% CI 2.37 -- 2.87). Fifty percent of the cases qualified as Drug Resistant S. pneumoniae (DRSP), being non-susceptible to one or more antibiotics as defined by the National Committee for Clinical Laboratory Standards (NCCLS). Age, race, gender, county and month of occurrence were evaluated as risk factors for DRSP. Only month of occurrence was determined to be a risk factor. Compared to current studies and previous results for Florida, it appears that Florida has a decreasing incidence of antibiotic resistant Streptococcus pneumoniae. I believe that this is due to the use of the PCV vaccine.

Infectious diseases resulting from bacterial pathogens are the most common causes of patient morbidity and mortality worldwide. The rapid identification of the pathogens and their antibiotic resistances is crucial for proper clinical management. However, the standard culture-based diagnostic approach requires a minimum of two days from the initial specimen collection to result reporting. As a consequence, broad-spectrum antibiotics are often prescribed under the worst-case assumption without knowledge of the pathogens or their resistances. The current clinical practice results in improper treatment of the patient and causes the rapid emergence of multi-drug resistant pathogens. A rapid diagnostics system has therefore been developed which performs hybrid electrokinetic sample preparation and volume reduction, for single-cell antimicrobial susceptibility testing (AST). The system combines multiple electrokinetic forces for sample preparation, which reduces the sample volume for over 3 orders of magnitude and minimizes the matrix effects of physiological samples for enhanced sensitivity. The device is integrated with a single-cell AST system with microfluidic confinement and electrokinetic loading to phenotypically determine the bacterial antibiotic resistance at the single-cell level. The applicability of the system has been demonstrated for performing direct AST with urine and blood samples within one hour, enabling rapid infectious disease diagnostics in non-traditional healthcare settings.

Antibiotic resistance (AR) is reducing options for effective treatment of bacterial infections for clinically ill patients throughout the world. Our investigation examined the impact solids retention time (SRT) may have on the degradation of antibiotic compounds, proliferation of AR bacteria, and the persistence of antibiotic resistance genes (ARGs) during the wastewater treatment process. Results indicate the presence of ARGs related to clinically relevant antibiotics. Data analysis showed that antibiotic classes exhibit different removal efficiencies in the biological treatment processes (i.e. SRT); therefore, operating conditions at each WWTP can be optimized for highest efficiency removal. SRT of 19 days and higher indicated a high removal efficiency for all six ARGs of concern. Ultimately, identifying a critical value to optimize SRTs, where antibiotic degradation is maximized and AR is minimized, will provide information to WWTP operators, allowing treatment optimization to decrease harmful loading of Trace Organic Contaminants (TOrCs) in the environment.

The study evaluated the Mutant Prevention Concentration (MPC) of Pradofloxacin on three Escherichia coli (E.coli) strains, 2 wildtypes and one first-step gyrA resistant mutant. We also measured the value of AUC (Under the Concentration)/MPC that prevents growth of resistant mutants. It is of importance to reach a concentration above MPC that prevent E.coli from developing resistance against the antibiotic. We used an in vitro kinetic model where we added bacteria? and antibiotic. The culture flask was attached to a pump with an adjustable pump-speed. This made it possible to dilute the antibiotics in a satisfying elimination half-life (t1/2= 7 hours) pace. Samples were removed with a syringe at different times in the study. The samples where then cultured on agar- plates to enable counting of the viable colonies after incubation. The optimal concentration to completely eradicate both E.coli wildtypes Nu14 and MG1655 with Pradofloxacin was Cmax ≥8 times MPC and AUC/MPC then became73. Additional experiments needs to be done on the resistant mutant LM378 before we can determine the optimal concentration. But results so far indicate that the concentration of Cmax would be about 8-12 timesMPC to completely eradicate that mutant.

Objective: To explore views of medical doctors, veterinarians and drug sellers on use of antibiotics on humans and nonhumans and on factors that influences the development of resistance to antibacterial agents. Further, to look at the bi-directional relationship between antibiotic use and environment. Methods: The study was a qualitative explorative interview study, analysed using conventional content analysis. It was conducted in Orissa, India. Data were collected by face to face semi structured interview. The interviews were tape recorded and transcribed into Oriya, then translated in to English. Each paragraph or sentence was coded. Similar codes were clustered together and collapsed into sub categories and categories. The main themes were allowed to emerge, based on the relationship between categories. Findings: The main finding of the study was mishandling and abuse of antibiotics in patients as well as at professional level due to weak implementation of legislation, which appears to be the major cause of antibacterial agent resistance. Incomplete course or dose due to poverty in rural area and self medication in urban area are more common. The study also showed that climatic factors, pollution and population density are the major ecological factors which influence antibiotic prescriptions. Another major finding of this study was that, due to improper disposal system of pharmaceuticals; antibiotics are contaminating air, water and terrains which can cause major risk to aquatic and grazing animals. Conclusion: This study emphasises the need for comprehensive actions including information, training, legislation and education at all levels of drug delivery system to rationalize antibiotic use by improving prescribing pattern and creating awareness among consumers. Proper disposal of pharmaceutical wastes is required to prevent the contamination of environment from pharmaceutical pollutants. Further study is essential concerning environmental impact of antibiotics. Key words: Antibiotic use; antibiotic resistance; environment; qualitative; conventional; content analysis; veterinarians; medical doctors; drug sellers; Orissa; India.

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.

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.

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.