Three experiments were conducted to evaluate the impacts of removing antibiotic growth promoters (AGP) on broiler performance and yield and to evaluate alternative products as potential replacements. In experiment one, approximately 552,000 broilers were reared in four solid-wall, tunnel ventilated houses that were divided into two paired-house facilities, each assigned one of two dietary treatments. The treated group received basal diets containing salinomycin (SAL), roxarsone (ROX) and AGP, while the control group received the same diets without ROX and AGP. Removal of ROX and AGP had no affect on average body weight and feed efficiency, while livability was significantly affected negatively by the removal of ROX and AGP. Tender, wing, drum and percentage of total white meat showed significant improvements in yield during the study, while all other parts were not affected by removal of ROX and AGP. In experiment two, an investigation was conducted to evaluate the effects on performance from feeding Bacillus subtilis spores (Gallipro®, Chr Hansen A/S, Denmark), as a direct-fed microbial additive, to commercial broiler chickens. Birds were divided among two paired-house facilities. The treatment group received basal diets supplemented with B. subtilis spores, while the control group was fed the same basal diets containing an AGP. Feed conversion ratio was significantly lower for the treatment group, while average body weight, coccidiosis lesion scores, and footpad scores were not affected by the treatments. In experiment three, 6,000 broiler chickens were equally divided among four treatment groups and reared to 49 d to determine the effectiveness mannan oligosaccharides (MOS, Bio-Mos®, Alltech, Nicholasville, Kentucky, USA) as an alternative for an AGP program and MOS plus Natustat™ (NAT, Alltech, Nicholasville, Kentucky, USA) as an alternative to an enteric health program (AGP+anticoccidial drug). Average body weight for the control (CON) and antibiotic (ANT) groups was significantly different from the MOS+NAT group, but not the MOS group. Carcass front half, carcass hind half, frame and skin yields were improved for all treatments when compared to the MOS+NAT group. Conversely, percent total white meat yield was improved with the inclusion of MOS when compared to the ANT group. The findings of this research suggest that the removal of AGP from the diets of commercial broiler chickens does not affect the performance and yield of the birds over a one year production period. Furthermore, B. subtilis spores and mannan oligosaccharides provide acceptable alternatives to an AGP program.

In the present work, in order to evaluate the seriousness of environmental pollution caused by antibiotics abuse, and by industrial and agricultural pollutants, different strains of Pseudomonas aeruginosa were collected from industrial area, abandoned metal hardware factory, fishery pool, vegetable garden and fruit farm, river mud, and different origins of water bodies in southern Taiwan. The organisms were analyzed for their drug resistance against a variety of antibiotics and agricultural pesticides. They were also analyzed for endurance toward heavy metal ions including mercury, cadmium, arsenic and chromium ions. As the data indicated that, in terms of their resistance to clinical frequently used antibiotics, about 40% of the Pseudomonas aeruginosa isolates of the environment have developed resistance against Cefoperazone, about 20% showed resistance against Tobramycin, and only about 4% revealed resistance against Imipenem and Ceftazidime. As to heavy metal ion resistance, about 27% of the environmental Pseudomonas aeruginosa strains demonstrated resistance against mercury ion, and about 10% exhibited resistance against arsenic ions, whereas no resistance was observed toward chromium ions. In terms of resistance to agricultural pesticides, about 36% environmental isolates demonstrated resistance against Paraquat, but none of the tested Pseudomonas aeruginosa show resistance toward Cartap and Methomyl. To sum up the findings, so far only minor portion of Pseudomonas aeruginosa acquired drug resistance, therefore, immediate measure is required to prevent the spreading of drug-resistant Pseudomonas aeruginosa . It is suggested that all the physicians and pharmacists to prescribe antibiotics should be more careful and responsible manner. Meanwhile, it would also call on the restrained usage of pesticides and antibiotics in the livestock and aquatic product industry, and strengthening pollution control in the industrial sector.

The production of new drugs as potential pharmaceutical targets is arguably one of the most important avenues of medicine, as existing diseases not only require treatment, but it is also certain that new diseases will appear in the future which will need treatment. Indeed, existing medicines such as antibiotics and immunosuppressants maintain their current activities in their respective realms, yet the molecular and stereochemical complexity of these compounds cause a burden on organic synthetic chemists that may prohibit the high yields required to manufacture a drug. The enzyme systems that naturally manufacture these compounds are incredibly efficient in doing so, and also do not use environmentally harmful solvents, chiral auxiliaries, or metals that are utilized in the current syntheses of these compounds; therefore utilizing these enzymes' machinery for the biocatalysis of new medicinally-relevant compounds, as researchers have in the past, is undeniably a rewarding endeavor. In order to harness these systems' biocatalytic potential, we must understand the processes which they operate. This work focuses on ketoreductase domains, since they are responsible for setting most of the stereocenters found within these complex secondary metabolites. We have supplied a library of substrates to multiple ketoreductases to test their limits of stereospecificity and found that, for the most part, they maintain their natural product stereospecificity seen in nature. We were even able to convert a previously nonstereospecific ketoreductase to a stereospecific catalyst. We have also developed a new technique to follow ketoreductase catalysis in real-time, which can also differentiate between which diastereomeric product is being produced. Finally, we have elucidated the structure of a ketoreductase that reduces non-canonically at the [alpha]- and [beta]- position, and functionally characterized its activities on shortened substrate analogs. With the knowledge gained from this dissertation we hope that the use of ketoreductases as biocatalysts in the biosynthesis of new natural product-based medicines is a much nearer reality than before. / text

The Staphylococci are gram-positive bacteria that cause infections in humans and can produce severe morbidity and mortality. Methicillin resistant S. aureus (MRSA) isolates are resistant to all beta-lactam antibiotics, such as methicillin, and cephalosporins making treatment of these infections more difficult. MRSA has become prevalent throughout the United States, spreading in the health care setting and the community.The purpose of this study is to examine methicillin resistance among S. aureus isolates in an outpatient population in the state of Florida and asses possible associations between methicillin resistance and age group, gender, and geographic area. It is important to define methicillin resistance in a population so that clinical practice can adjust to the prevalence of resistance.The dataset used for this analysis is a record of all the S. aureus isolates tested by a large lab company in the state of Florida from January 1, 2003 to December 31, 2005. This is the first study to asses methicillin resistance with a population based dataset and not patients from hospitals. The percent of isolates that were methicillin resistant increased as year increased. This increase in the number of methicillin resistant isolates was significant for both the crude and adjusted analysis. When treated as a continuous variable and adjusted for age category, gender, and county of residence the odds ratio for year is 1.446, 95% CI: 1.410- 1.484. In 2005, 49.7% of the isolates were methicillin resistant. Methicillin resistance also varied by age category, gender, county, and region. For age group and gender the differences were not large and may not be clinically significant. However, there was substantial variation in methicillin resistance by region and county of residence.With nearly half of the S. aureus isolates being methicillin resistant, the beta-lactam antibiotics may no longer be an ideal choice for treating S. aureus infections in Florida. The percentage of MRSA isolates that were resistant to trimethoprim-sulfamethoxazole, tetracycline, gentamycin, and rifampin was low. These antibiotics may be feasible alternatives to treat outpatient S. aureus infections in Florida.

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

<p>The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens. </p><p>In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the &#61538;a-&#61538;b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections. </p><p>In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.</p> / Dissertation

On the verge of entering the post-antibiotic era, numerous efforts are in place to regain the losing potential of antibiotics which are proving ineffective against common bacterial infections. Engineered nanomaterials, especially gold nanoparticles (GNPs) capped with antibacterial agents are proving to be an effective and novel strategy against multi-drug resistant (MDR) bacteria. In this study, we report a one-step synthesis of kanamycin-capped GNPs (20 ± 5 nm) utilizing the combined reducing and capping ability of the aminoglycoside antibiotic, kanamycin. Antibacterial assays showed dosedependent broad spectrum activity of Kan-GNPs against Gram-positive (Staphylococcus epidermidis and Enterococcus durans), Gram-negative (Escherichia coli and Enterobacter aerogenes) and Kan-resistant and MDR bacterial strains. A significant reduction in the minimum inhibitory concentration (MIC) of Kan-GNPs was observed as compared to free kanamycin against all the sensitive and resistant bacterial strains tested. Mechanistic studies using TEM and fluorescence microscopy showed that Kan- GNPs exerted their bactericidal action through disrupting the cellular membrane resulting in leakage of cytoplasmic content and death of bacterial cells. Results of this study provide a novel method in the development of antibiotic capped GNPs as potent next-generation antibacterial agents.

The shikimate pathway is responsible for the biosynthesis of the aromatic amino acids and other aromatic metabolites in plants, micro-organisms and apicomplexan parasites. The shikimate pathway is essential in bacteria and plants, but absent from mammals, which has led to interest in the enzymes of the pathway as targets for the design of antimicrobial and herbicidal agents. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first commit¬ted step of the shikimate pathway, the condensation of phosphoenolpyruvate and erythrose 4-phosphate to yield 3-deoxy-D-arabino-heptulosonate 7-phosphate. The subject of this thesis is the investigation of inhibition and allosteric regulation of the DAH7PS enzyme from Myco¬bacterium tuberculosis (MtuDAH7PS), the pathogen that causes tuberculosis. Tuberculosis remains a major health threat to the global community, and the emergence of multi-drug resistant strains highlights the need for new tuberculosis treatments. Inhibitors of MtuDAH7PS have the potential to be developed into new anti-tuberculosis drugs. Chapter 2 describes the design, synthesis and evaluation of active site inhibitors based on intermediate mimic scaffolds. The intermediate mimics synthesised represent the first reported example of inhibitors targeting the active site of MtuDAH7PS. The most active compounds tested displayed inhibition constants in the sub-micromolar range, making them the most potent inhibitors of any DAH7PS enzyme reported to date. MtuDAH7PS displays a complex and subtle mechanism of synergistic regulation: The enzyme is inhibited by binary combinations of the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr). Three allosteric binding sites were identified using X-ray crystallo¬graphic analysis of MtuDAH7PS in complex with Trp and Phe. While these crystal structures led to the identification of an allosteric binding site which preferentially binds Trp, the role and selectivity of the other two sites with respect to Phe and Tyr remained unclear. The results described in Chapter 3 provide structural and biochemical evidence for the hypothesis that each of the three allosteric binding sites has a preference for binding one of the aromatic amino acids Trp, Phe and Tyr, respectively. The results furthermore show that the ternary combination of Trp, Phe and Tyr synergistically regulates MtuDAH7PS, leading to almost complete loss of enzymatic activity in the presence of all three allosteric ligands. In Chapter 4, the interaction of MtuDAH7PS with the naturally less common D-enantiomers of the aromatic amino acids is described. It was found that the D-enantiomers of the aromatic amino acids have no effect on enzymatic activity of MtuDAH7PS, suggesting an efficient mechanism by which the enzyme can discriminate between allosteric ligands of opposite configuration. Studies of the binding affinity of the D-amino acids to MtuDAH7PS as well as structural characterisation of MtuDAH7PS-D-amino acid complexes by X-ray crystallographic analysis suggest that D-Trp and D-Phe can still bind to their respective sites. The lack of inhibition is attributed to subtle differences in the binding mode of the D-enantiomers of the ligands compared to the L-enantiomers. Chapter 5 details the discovery of alternative ligands and inhibitors targeting the allosteric sites of MtuDAH7PS using virtual screening. Libraries of potential alternative ligands were docked into the allosteric sites of MtuDAH7PS and the predicted docking poses were used to guide the selection of compounds for physical screening. Using this approach, a number of ligands and inhibitors of MtuDAH7PS were discovered and their interaction with the enzyme structurally characterised. Comparison of the crystallographically observed binding modes of new ligands with the docking poses predicted by computational docking highlighted potential improvements to the virtual screening method. The analysis of the correlation between ligand binding modes and inhibition of enzymatic activity provided further insight into which interactions between the allosteric ligand and the binding site are crucial in order to achieve inhibition. The crystal structures of MtuDAH7PS in complex with the new alternative ligands can serve as a starting point for the design of ligands with increased affinity and potency.

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.