The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

Obiettivo di questo lavoro è stato valutare la diffusione di AR in differenti ceppi di S. thermophilus isolati tra il 1947 e il 2004 e provenienti da differenti ambienti, in modo da avere un chiaro andamento del fenomeno; questo è stato possibile analizzando un numero significativo di ceppi isolati in un periodo di tempo che va da prima dell'utilizzo degli antibiotici fino ai giorni nostri. L'espressione fenotipica è stata valutata con tre differenti metodi (microdiluizioni in brodo, E-test e Disk Diffusion), in accordo con gli standard NCCLS, per la determinazione delle MICs (Minimum Inhibitory Concentration, ovvero Concentrazioni Minime Inibenti). Per la valutazione genetica è stata impiegata la tecnica dei microarrays a DNA utilizzando oligonucleotidi da 50 e 60-mer, per un totale di 300, appartenenti a 10 diverse classi di antibiotici. La conferma dei risultati è stata ottenuta mediante PCR e sequenziamento. In 9 ceppi di S. thermophilus è stato possibile mettere in evidenza la presenza di almeno uno dei geni tetS ed ermB responsabili della resistenza agli antibiotici Tetraciclina e Eritromicina rispettivamente. / The aim of the present work was to assess the AR diffusion in a total of 70 different strains of Streptococcus thermophilus, collected between 1950 and 2004 and from different environments; in this way we had the possibility to obtain a clear overview of the response of these bacteria to a large variety of antibiotics, having been able to analyze a significant number of different strains, originated from different areas and distributed over a wide time period, since before the use of antibiotics up to the present day. The phenotypic expression has been evaluated by using three different methods: microdilution, E-test and disk diffusion. The genetic analysis was performed using 50 and 60-mer oligonucleotides DNA based micro array for the identification of AR genes; the AR genes represented by the oligonucleotides on the micro array belong to: Aminoglycoside, Extended Spectrum ?-lactamase (ESBL), Chloramphenicol, Macrolide Lincosamides and Streptogramin (MLS) group, Sulfonamide, Tetracycline, Trimethoprim and Vancomycin. tetS and ermB genes were found and sequenced in 4 out of the total of the S. thermophilus investigated. Furthermore we have wanted to establish the genetic location of above-mentioned genes and assess their transfer intra and inter species adopting the conjugation technique in plate.

Humans are generally having a strong, widespread, and negative impact on nature. Given the many ways we are impacting nature and the many ways nature is responding, it is useful to study responses in an integrative context. My thesis is focused largely (two out of the three data chapters) on butterfly species’ range shifts consistent with modern climate change in Canada. I employed a macroecological approach to my research, drawing on methods and findings from evolutionary biology, phylogenetics, conservation biology, and natural history. I answered three main research questions. First, is there a trade-off between population growth rate (rmax) and carrying capacity (K) at the mutation scale (Chapter 2)? I found rmax and K to not trade off, but in fact to positively co-vary at the mutation scale. This suggests trade-offs between these traits only emerge after selection removes mutants with low resource acquisition rates (i.e., unhealthy genotypes), revealing trade-offs between remaining genotypes with varied resource allocation strategies. Second, did butterfly species shift their northern range boundaries northward over the 1900s, consistent with climate warming (Chapter 3)? Leading a team of collaborators, we found that most butterfly species’ northern range boundaries did indeed shift northward over the 1900s. But range shift rates were slower than those documented in the literature for more recent time periods, likely reflecting the weaker warming experienced in the time period of my study. Third, were species’ rates of range shift related to their phylogeny (Chapter 3) or traits (Chapter 4)? I found no compelling relationships between rates of range shift and phylogeny or traits. If certain traits make some species more successful at northern boundary range expansion than others, their effect was not strong enough to emerge from the background noise inherent in the broad scale data set I used.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes. / UOIT

As multidrug resistance in Gram-negative bacilli increases, the old antibiotic colistin has rapidly gained attention as one of few last line treatment options in the form of colistin methanesulfonate (CMS), which is hydrolyzed to colistin both in vitro and in vivo. There is a dearth of knowledge on fundamental aspects of colistin, including pharmacokinetics and optimal dosing regimens. The aim of this thesis was to improve the basis for optimal colistin therapy. To be able to study colistin, an LC-MS/MS assay method was developed which is sensitive, specific and useful in both in vivo and in vitro studies. Using this method we detected a significant loss of colistin during standard laboratory procedures. This loss was characterized and quantified, the hypothesis being that the loss is mainly caused by adsorption to labware. The pharmacokinetics of colistin was studied in two populations of critically ill patients, one with normal renal function and one with renal replacement therapy. Plasma concentrations were assayed with the method above, and population modeling was employed to describe the data. The results include a previously unseen, long elimination half-life of colistin. The data from the population on renal replacement therapy was described without modeling, and showed that both CMS and colistin are cleared by hemodiafiltration. Combination therapy is an approach that is often used when treating patients infected with multidrug-resistant pathogens. The thesis discusses how the joint effect of antibiotics can be measured using colistin and meropenem as a model, and proposes a method for testing antibiotic combinations. Furthermore, a PKPD model was adapted to describe the pharmacodynamics of the combination. In conclusion, a specific and sensitive method for analysis of colistin was developed and the adsorption of colistin to materials was described. The assay method has been well accepted internationally. The pharmacokinetics of colistin and CMS was described in two important patient populations, partly with surprising results that have influenced dosages of colistin worldwide. The pharmacodynamics of combination therapy was investigated and quantified, and the methods applied could be further developed into clinically useful tools for selection of antibiotic combinations.

Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections. In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition.

Clinical S. aureus strains were gathered from four hospitals, two in Turkey (Hacettepe hospital 200 strains and Ankara Hospital 106 strains) and the other two from Libya (Aljalla Hospital 88 strains and Jamahyria Hospital 62 strains). The clinical specimens were collected form different sources including blood, urine, wound, pus, burn, sputum, semen, catheter and aspiration. Patients were aged between 0 to 84 years and from both sexes. Resistance to Methicillin was determined by measuring the Oxacillin MIC / this was done by using the oxacillin E-test, with resistance defined as an MIC of &gt / 2 &micro / g ml. In this study all isolates displayed an Oxacillin MIC of &amp / #8805 / 256&micro / g/ml. The MRSA strains were (56%) in Turkish hospitals, and (59%) in Libyan hospitals. The percentage of the VRSA and VISA in Libyan hospitals was (7%) and (26%) respectively, although the percentage of VRSA in Turkish hospitals was only 2% and there were no intermediately susceptible Staphylococcus aureus (VISA). Besides the MRSA isolates, Coagulase Negative Staphylococcus showing Methicillin resistance was collected from clinical isolates in thirteen patients in Turkish hospitals. In both countries, the majority MRSA isolates were multiresistant to more than five classes of antibiotics including / Ampicillin, Amoxicillin, Tetracycline, Erythromycin and Ciprofloxacin. Most of the MRSA isolates were from blood (68%), wounds (57%) and pus (50%).The results of genetic investigations indicated that the mecA gene was present in the majority of isolates in both countries / the community acquired MRSA type (ccr-BIV) was present in three samples out of thirty in Turkish hospitals and in one case out of twenty in Libyan hospitals / There was no case out of fifty specimens that carry the hospitals acquired MRSA type (ccr-BI, II, III) in both countries. Besides the Methicillin resistance gene, the incidence of Tetracycline resistance gene was quite high (tetM and tetK 50%) in Turkish hospitals isolates, and the prevalence of Panton-Valentine Leukocidin gene was high (PVL 70%) in Libyan hospitals specimens.

Title from PDF of title page. / Document formatted into pages; contains 117 pages. / Thesis (M.S.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: Bacterial source tracking (BST) methods often involve the use of phenotypic or genotypic fingerprinting techniques to compare indicator bacteria such as Escherichia coli isolated from unknown sources against a library of fingerprints from indicator bacteria found in the feces of various known source animals. The predictive capability of a library is based in part on how well the library isolates reflect the true population diversity of indicator bacteria that can potentially impact a water body. The purpose of this study was to compare the behavior of E. coli population structures in the feces of humans, beef cattle and horses across different parameters. Ribotyping and antibiotic resistance analysis were used to "fingerprint", or subtype E. coli isolates. Significantly greater diversity was observed in the E. coli population of horses compared to the human or beef cattle sampled. / ABSTRACT: Subtype sharing between individuals from all host categories was infrequent, therefore the majority of E. coli subtypes were sampled from a single individual. The dominant E. coli populations of nine individuals (three per host source category) were monitored over time, which demonstrated that E. coli subtypes within a host individual vary on a monthly time frame, and an increase in the frequency of subtype sharing was noted between individuals within the same source group over time. The E. coli population of a single human that had just finished antibiotic treatment was studied on a daily basis for one month. The loss of an E. coli subtype with high antibiotic resistance was observed over time, however there was a single dominant E. coli subtype that was present at every sampling event during the entire month. Geographic distinctiveness of E. coli populations was investigated by sampling four herds located in different geographical regions. We observed that E. / ABSTRACT: coli populations are not geographically distinct, but are somewhat individual-specific, as most E. coli isolates had a subtype that was found in a single individual. This study defines factors that should be considered when constructing a successful BST library, and suggests that E. coli may not be the appropriate indicator organism for BST. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

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.