N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.

The prevalence of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) and carbapenemases is increasing worldwide. Therapeutic options for infections with these bacteria are limited not only by the production of ESBLs and carbapenemases, which confer resistance to cephalosporins and carbapenems, but also by frequent co-resistance to other antibiotics. The overall aim of this thesis was to obtain a better understanding of multidrug-resistant E. coli and K. pneumoniae in relation to epidemiology, selection and susceptibility to antibiotic therapy. In a prospective study ESBL-producing E. coli was found to spread easily through international travel. Twenty-four of 100 Swedes travelling outside Northern Europe acquired ESBL-producing E. coli in the intestinal flora. The risk was highest for travelers visiting India and those suffering from gastroenteritis during travel. To minimize selection of ESBL-producing K. pneumoniae during a hospital outbreak with these bacteria, an educational antibiotic intervention was performed at Uppsala University Hospital in 2006. The primary aim of the intervention was to reduce the consumption of parenteral cephalosporins. An immediate and radical reduction of cephalosporins was demonstrated with interrupted time series analysis. The outbreak declined during 2007 and no increased resistance to replacement antibiotics was detected. The impact of ESBL production on the antibacterial activity of ertapenem was studied in time-kill experiments. It was shown that porin-deficient subpopulations with reduced susceptibility to ertapenem frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Further, the antibacterial effects of antibiotic combinations against four strains of K. pneumoniae producing carbapenemases of the metallo-beta-lactamase type were studied in time-kill experiments. Double and triple combinations of aztreonam, fosfomycin, meropenem, rifampin and colistin at clinically relevant static concentrations were effective despite that the bacteria were frequently resistant to the individual drugs. These results indicate that there is a largely unexplored potential of antibiotic combination therapy for multidrug-resistant K. pneumoniae.

It has been reported by the American Burn Association that 4,000 people die every year due to burn injury. After survival of the initial trauma, the next major obstacle that must be overcome is combating bacterial infection, the primary cause of mortality for burn victims (Chapter 1). The polyacrylate nanoparticle drug delivery system was created to provide a water-based solution for delivery of highly lipophilic antimicrobials; such as N-thiolated β-lactams, however, with the success of this system for these antimicrobials, it was extended towards other, commercially-available water-soluble antimicrobials through acrylation of the drug monomers, including those with observed bacterial resistance (Chapter 2). Various antibiotics were incorporated into this polyacrylate nanoparticle delivery system by either encapsulation or covalent attachment, and the antibacterial activity was determined in vitro (Chapter 3). Since current treatment of burn wound infections calls for numerous antimicrobials in order to combat the vast array of microbes that may be present in the wound, a multi-drug conjugated nanoparticle system was constructed and analyzed for antibacterial activity against many pathogens commonly found in burn wounds (Chapter 4). In vitro antibacterial assays suggest that the nanoparticle delivery system rejuvenated the activity of penicillin-based antibiotics against formerly resistant microbes, such as methicillin-resistant Staphylococcus aureus. The multi-drug conjugated nanoparticle emulsion had the added benefit of forming a drug-conjugated polyacrylate polymer film through air-drying and polymer coalescence. Upon topical application to a skin abrasion in a mouse model, a protective barrier was created over the wound. This film exhibits mechanical properties similar to elastin, a pliant biological material, giving it the elasticity and flexibility required to move and interact with the wound in the same fashion as intact skin (Chapter 5). This film also permits diffusion of essential nutrients and small molecules (such as oxygen and water) required for wound healing. The emulsion was able to be combined with other biological materials, such as collage, to form a biocomposite material expressing the most optimal properties from each constituent (Chapter 6). In vitro cytotoxicity analysis (Chapter 7) and in vivo toxicity studies (Chapter 8) produced positive results indicating that the multi-drug conjugated nanoparticle emulsion is a promising new treatment for the burn wound and other topical skin and soft tissue infections.

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.

Work in the Turos group over the past five years has focused on the development of N-alkylthio beta-lactams, which show antibacterial activity against Staphylococcus (including MRSA), Bacillus, and others. These compounds do not function in the manner of the traditional beta-lactam antibiotics, but were thought to undergo an intracellular thiol-transfer to coenzyme A. In expanding the SAR of these novel compounds, it was found that N-alkylthio-2-oxazolidinones also exhibit antibacterial activity. Although CoA acts as the thiol-redox buffer in the genera most susceptible to the N-alkylthio beta-lactams, studies on Coenzyme A disulfide reductase (CoADR) show that the redox buffer is not affected by these compounds. However, the recent finding that fatty acid synthesis is affected by the N-alkylthio beta-lactams led to the discovery that these compounds act as prodrugs, and that the asymmetric CoA disulfides produced by in vivo thiol transfer are potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) through a novel thiol-disulfide exchange with the active site cysteine. Lactams 2a and 2g were also found to be potent inhibitors of this enzyme. In an effort to produce a CoA mixed-disulfide mimic which could cross the cell membrane, a series of simple aryl-alkyl disulfides were synthesized and tested against E. coli, S. aureus, and B. subtilis. Several of these compounds were found to be very potent antibacterials both in vitro and in vivo, with MICs less than 0.125 micrograms/mL. Comparison of the activities of these disulfides with those of acyl-CoA analogs and CoA mixed disulfides support the assertion that FabH is indeed the cellular target of these potent new compounds.

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.