The Gram positive bacterium Staphylococcus aureus is a major cause of human disease in industrialized countries. This multifaceted pathogen is adapted to thrive in a variety of host niches, including the intracellular compartment. S. aureus rapidly develops antibiotic resistance, and infections due to resistant clones pose a global threat, calling for novel therapeutic approaches. The ability to exploit host nutrients and efficiently metabolize these resources for growth is paramount for bacterial pathogenesis. Understanding the nutritional and metabolic determinants that underpin bacterial virulence may lead to the identification of novel antimicrobial targets. This thesis investigates carbon nutrition and metabolism of community-acquired methicillin resistant S. aureus (CA-MRSA) USA300, a widely spread, hyper virulent multi-resistant strain. The dependence of S. aureus on carbohydrates for growth was considered first. In vitro studies in supplemented chemically defined media showed that sugar phosphates, such as hexose phosphates and glycerol phosphates, promote staphylococcal growth more efficiently than glucose. Deletion mutations were introduced to the two putative sugar phosphate transporter genes present in the S. aureus genome, uhpT (hexose phosphate permease) and glpT (glycerol phosphate permease). Phenotypic analysis of USA300 mutants and heterologous expression of the transporters in a previously described Listeria monocytogenes Δhpt mutant, totally unable to use sugar phosphates, confirmed that S. aureus UhpT and GlpT have different substrate specificities. Whilst both can transport glycerol monophosphate (excluding glycerol-2-phosphate) and the organophosphate antibiotic fosfomycin, hexose monophosphates are only imported via UhpT. Since sugar phosphates are only present in significant amounts inside living tissues, particularly the intracellular compartment, the role of S. aureus UhpT and GlpT in pathogenesis was investigated by constructing a double deletion mutant. The ΔuhpTΔglpT USA300 mutant was used to infect several relevant mammalian cell lines. In the conditions tested, it was found that UhpT and GlpT played no role in the intracellular replication of S. aureus. By contrast, Listeria exploits sugar phosphates from the host cell cytosol via the homologous hexose phosphate transporter, Hpt, to maximise replication and enhance virulence. The distinct requirement of sugar phosphates for intracellular proliferation may reflect intrinsic differences in carbon nutrient dependence between the two organisms. It was confirmed that S. aureus can efficiently use other readily available carbon sources for growth, such as amino acids. In contrast, Listeria is strictly dependent upon sugar-derived carbon for growth, due to an incomplete tricarboxylic acid cycle. Whilst the double ∆uhpT∆glpT mutation had no effect in S. aureus, expression of staphylococcal uhpT or glpT restored wild-type intracellular growth in the L. monocytogenes ∆hpt mutant. Taken together, the results illustrate that sugar phosphate permeases have a contextual role in bacterial virulence, where the background in which the genes are expressed determine their contribution as a virulence factor. The intracellular dynamics of S. aureus was also explored using immunofluorescence microscopy. It was observed that, during epithelial cell infection, USA300 remains enclosed in a membrane-bound vacuole. This localisation may form a barrier to cytosolic sugar phosphates and potentially explain the absence of effect of the sugar phosphate permease deletions in intracellular proliferation. Preliminary characterisation of the S. aureus containing vacuole (SACV) was performed and it was found to be positive for the Rab7 late-endosomal GTPase and for trans-Golgi markers. This suggests that SACVs converge at the Golgi apparatus. Interestingly, a USA300 mutant lacking the global regulatory system agr was unable to proliferate intracellularly and did not acquire Rab7 or Golgi markers. Since the Δagr mutation did not cause any impairment in carbon source dependent growth, these preliminary data suggest that modification of the SACV by Agr-regulated effectors may play a key role in modulating cellular processes that control staphylococcal intracellular survival and/or replication. Evidence presented in this thesis provides a platform for further exploration of S. aureus host cell nutrient dependence and the mechanisms that drive replication.

Infection is one of the factors that may contribute to non-healing of chronic wounds; the presence of antibiotic resistant bacteria serves to exacerbate the problem due to limited treatment options. Bacteria utilise several mechanisms to survive exposure to antibiotics, including synthesis of deactivating enzymes, target modification or substitution, changes to membrane permeability, upregulation of efflux pumps and the formation of a biofilm. Quorum sensing is a density-dependent mechanism of bacterial cell to cell communication that can be instrumental in co-ordinating biofilm initiation. Hyperbaric oxygen therapy (HBOT) is an option offered to some patients with chronic wounds, including diabetic foot ulcers. Evidence suggests that HBOT can reduce the incidence of major amputation in these patients. As well as the direct toxicity of increased tissue oxygenation on anaerobic bacteria HBOT may also increase levels of reactive oxygen and nitrogen species in the wound environment. This study aimed to investigate the effects of hyperoxia and oxidative damage on three specific mechanisms of antibiotic resistance: the activity of penicillinase, an antibiotic deactivating enzyme synthesised by bacteria; the activity of quorum sensing signalling molecules (AHLs); and biofilms and their associated bacteria. It also analysed the population dynamics of, primarily, bacteria in diabetic foot ulcers during HBOT, by the use of molecular analysis tools such as PCRDGGE. The presence of fungal species was investigated in wounds prior to HBOT and in two wounds at two points during HBOT. This study found that hydrogen peroxide, hypochlorous acid and peroxynitrite reduced the activity of penicillinase in vitro. Hypochlorous acid reduced the activity of a range of AHLs in vitro but not in vivo. Oxygen concentration did not have any impact on biofilm mass, nor did it significantly affect the ability of an oxidant-generating enzyme to reduce live bacterial cells within a biofilm. The population dynamics of bacterial species identified in all the wounds were complex and did not undergo identifiable changes during HBOT. Fungal species were identified in all wounds prior to HBOT, though different profiles were observed in the two wounds investigated during HBOT. These results suggest that oxidants could play a role in the attenuation of antibiotic resistance in chronic wound bacteria. It is unclear whether HBOT alters the population dynamics of non-healing wound microflora

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Bacterial resistance against antibiotic treatments is a global concern and resistance to almost every known antibiotic has already been reported. There is thus a significant need for the development of novel antimicrobial drugs. In addition to probiotic traits, certain bacteria have the ability to produce antimicrobial peptides, referred to as bacteriocins. Lantibiotics, a group of small ribosomally synthesized bacteriocins, recently gained interest for their application in the medical field. Lantibiotics have a very specific structure, including lanthionine rings, that stabilise the peptides. Due to their small size and specific action, these peptides reach specific sites of infection without affecting the composition of the host’s natural microbiota. As with any therapeutic agent, antimicrobial peptides are also prone to in vivo degradation, binding, clearance via immune action and development of bacterial resistance. Nisin F, a class Ia lantibiotic produced by Lactococcus lactis subsp. lactis F10, has already shown activity against the well-known pathogens Stapylococcus aureus, Listeria monocytogenes and various antibiotic resistant strains. The aim of this study was to assess the antimicrobial activity of nisin F against systemic S. aureus infections in mice and possible immune responses elicited by the peptide. A single administration of nisin F to the peritoneal cavity protected mice from S. aureus infection for at least 15 min. After continuous administration, the peptide showed no significant antimicrobial activity against S. aureus. The peptide did, however, convey some degree of protection to infected mice by stimulating a pro-inflammatory action through lymphocyte protection. When administered to uninfected mice, nisin F had an immune boosting effect via interleukin (IL)-6 and IL-10 without being detrimental to the host. The ex vivo effects of nisin F was compared to nisin A, a natural nisin variant, and Nisaplin®, a commercially purified form of nisin A. None of the three peptides inhibited the functional capacity of leukocytes in terms of 1L-1β en IL-6 production, not even in the presence of an external stimulus (lipopolysaccharides from Escherichia coli). Cytotoxicity was detected in response to high dosages of nisin F. Serum inhibited the antimicrobial effect of nisin F and nisin A, but Nisaplin® remained unaffected. Nisin F was applied against systemic infection for the first time and the immunological effect of the peptide was investigated. Nisin F partially protected mice against S. aureus infections through immunomodulatory effects. This study provided valuable knowledge on the in vivo application of nisin F. With further optimization of nisin F preparation and application systems, the peptide might be more effective against in vivo infections. / AFRIKAANSE OPSOMMING: Bakteriële weerstand teen antibiotika wek wêreldwyd kommer en weerstand teen amper elke bekende antibiotikum is reeds aangemeld. Daar is dus 'n groot behoefte vir die ontwikkeling van nuwe antimikrobiese middels. Bykomend tot probiotiese eienskappe, het sekere bakterieë die vermoë om antimikrobiese peptiede, bekend as bakteriosiene, te produseer. ‘n Groep klein ribosomaal-gesintetiseerde bakteriosiene, lantibitiotika, is onlangs vir mediese toepassing oorweeg. Lantibiotika beskik oor 'n baie spesifieke struktuur, insluitend lantionien ringstrukture, wat die peptied stabiliseer. Weens hul klein grootte en spesifieke aksie is hierdie peptiede daartoe in staat om spesifieke areas van infeksie te bereik sonder om die gasheer se natuurlike mikrobepopulasie te beïnvloed. Soos met enige terapeutiese middel, is bakteriosiene ook geneig tot in vivo afbreking, binding, klaring via die immuunsisteem en ontwikkeling van bakteriële weerstand. Nisien F, 'n klas Ia lantibiotikum, deur Lactococcus lactis subsp. lactis F10 geproduseer, het reeds aktiwiteit teen die bekende patogene Stapylococcus aureus, Listeria monocytogenes en verskeie antibiotika-weerstandige stamme getoon. Die doel van hierdie studie was om die antimikrobiese aktiwiteit van nisien F teen sistemiese S. aureus infeksies in muise te bepaal, asook die moontlike immuunreaksies wat die peptied mag veroorsaak. 'n Enkele toediening van nisien F het muise vir ten minste 15 min teen S. aureus beskerm. Na deurlopende administrasie het die peptied geen beduidende antimikrobiese aktiwiteit teen S. aureus getoon nie. Die peptied het egter 'n mate van beskerming aan geinfekteerde muise verleen deur ‘n pro-inflammatoriese aksie te inisieer deur limfosiet beskerming. Met toediening aan gesonde diere, het nisien F 'n immuunversterkende effek teweeg gebring via interleukin (IL)-6 en IL-10 vlakke, sonder nadelige uitwerking op die gasheer. Die ex vivo effek van nisien F is ook vergelyk met nisien A, 'n natuurlike variant van nisien, asook Nisaplin®, 'n kommersieël-gesuiwerde vorm van nisien A. Nie een van die drie peptide het leukosiete se funksionele kapasiteit in terme van 1L-1β en IL-6 produksie inhibeer nie, selfs nie in die teenwoordigheid van ‘n eksterne stimulus (lipopolisakkariede van Escherichia coli) nie. Seltoksisiteit is na blootstelling aan hoë dosisse van nisien F waargeneem. Serum het die antimikrobiese effek van beide nisien F en nisien A geïnhibeer, terwyl die werking van Nisaplin® nie beïnvloed is nie. Nisien F is vir die eerste keer teen sistemiese infeksies ingespan en die immunologiese impak van die peptied is ondersoek. Nisien F het gedeeltelike beskerming aan muise met S. aureus infeksies verleen deur die immuunsisteem te versterk. Die resultate het ‘n waardevolle bydrae gelewer tot die in vivo toediening van nisien F. Met verdere optimisering van nisien F voorbereiding en toedieningsisteme, mag die peptied moontlik meer effektief teen in vivo infeksies aangewend word. / The National Research Foundation (NRF) of South Africa for financial support and funding of the research

<p>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.</p><p>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.</p><p>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.</p>

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.

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Device associated infections are a growing problem in the field of orthopaedics and dentistry. Bacteria adhering to implant surfaces and subsequent biofilm formation are challenging to treat with systemic administered antibiotics. Functionalization of implant surfaces with therapeutic coatings that are capable of inhibiting bacterial adhesion are therefore considered as a straight forward strategy to treat and prevent implant related infections. In this thesis, the use of crystalline, arc deposited TiO2 and biomimetic hydroxyapatite (HA) coatings were evaluated with respect to their potential as antibacterial surface modifications for bone-anchored implants. UV light induced photocatalysis of anatase dominated TiO2 coated surfaces was shown to provide a bactericidal effect against S. epidermidis under clinically relevant illumination times and doses. Major parts of the drug release work carried out was based on biomimetic HA (HA-B) coated fixation pins. The analysis of the coating characteristics revealed that the nanoporous structure of HA-B coatings in addition to the chemical composition and surface charge are essential parameters that influence the drug carrier performance. Loading by adsorption was demonstrated to be a feasible approach to quickly incorporate antibiotics. The controlled release of antibiotics was shown to facilitate bactericidal effects against S. aureus over application-relevant time periods, even when exposed to biomechanical forces during insertion into bone model materials. Antibiotic incorporation during coating growth was shown to promote somewhat longer drug release time periods than those obtained using adsorption loading. In summary, functionalization of implant surfaces with bioactive and biocompatible coatings is a promising concept to impact the clinical success for bone-anchored applications. The additional feature of optional, on-demand antibacterial properties of these coatings through either on-site drug release or photocatalytic antibacterial treatment is advantageous for the prevention and effective treatment of devices-associated infections. Both strategies provide an immediate response to the implant contamination by bacteria and are believed to contribute towards minimizing the origin of post-surgical infections, while at the same time improving the interfacial stability between implant and bone.

Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-29 21:18:18.966

Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.