This study used "Streptomyces coelicolor" A3(2) as a model organism to study the potential for process intensification in antibiotic production using solid phase growth. This organism produces three chromosomally encoded antibiotics, actinorhodin, prodigiosin, and calcium dependent antibiotic (CDA), and one plasmid encoded antibiotic methylamycin. In this study the production of prodigiosin was the focus for this process intensification. A novel technique was developed in the preparation of the bacterial support (PHP) , which ensures a well-controlled internal architecture. Solid phase growth on this matrix was shown to support the production of prodigiosin and actinorhodin. The growth of "Streptomyces coelicolor" A3(2) on this nano-structured, macro-porous, polymeric support with hierarchic connectivity, generically known as PolyHIPE polymer. Variations of PHP were investigated and compared with conventional flask growth; the production of prodigiosin in PHP was demonstrated to exceed that in flask growth, offering the potential for an order of magnitude improvement in volumetric productivity. A micro-bioreactor was designed to improve spore penetration, the distribution of cells, viability and nutrient supply. Growth was observed by electron microscopy, SEM and TEM. It was operated as a forced inoculation of pre-germinated spore suspension in modified R5 media, this micro-bioreactor enhanced productivity by an order of magnitude. This study shows that bacterial growth rate and secondary metabolism are influenced by the pore size and surface chemistry of the support material. To understand these surfaces and scale dependent phenomena a series of microporous structures with different functionalised surfaces were generated. An initial proteomic study was performed to determine whether solid phase culture had lead to a fundamentally different pattern of growth. One dimensional SDS gel electrophoresis revealed some variations in the pattern of protein bands between samples from growth in flasks and on the different PHP supports. Mass spectrometric analysis by peptide mass fingerprinting, of differentially observed bands, revealed the predominance of elongation factor EF-tu in liquid culture and increases in S-adenosylmethionine synthetase and hypothetical protein SC07276 in solid phase culture.

Bacterial persistence describes the phenotypic variation displayed by clonal bacterial populations which permits a small fraction of cells to survive exposure to antimicrobials. The phenomenon was first described in the 19405 by Joseph Bigger who observed that penicillin could not sterilize a culture of Staphylococcus aureus. The surviving cells were not resistant mutants as they displayed equal susceptibility to penicillin as the parent population upon subculture. Among the various hypotheses which have been proposed to explain this phenomenon, the majority consider persisters to be dormant The prevailing model in Escherichia coli is that this behaviour results from stochastic expression of the toxic portion of chromosomal toxin-antitoxin modules. However, the exact mechanisms underlying this behaviour remain elusive, partly owing to the apparent redundancy of this phenotype. Persistence has been observed in all bacterial species tested to date however, most studies have focused on E. coli. This study aimed to explore the mechanisms of persistence in S. aureus by characterising two transposon insertion mutants identified as being defective in persistence to antimicrobials. The mutants were affected in different ABC transporter-like proteins, AbcA and PapA. The role of ABC transporters in persistence was further evaluated by independently inactivating abcA and papA. The resulting strains exhibited a reduced-persister phenotype to antimicrobials from different classes supporting a role for AbcA and PapA in S. aureus perSistence. The second part of this study sought to investigate the use of mycoplasmae as model systems for investigating persistence. Time-kill studies established that Mycoplasma hominis and Mycoplasma galliseptcium do generate persister cells. As neither organism has toxin-antitoxin modules in their genomes, the results imply that other mechanisms for persistence must exist. These mechanisms were investigated by generating and screening a M, gallisepticum transposon insertion library for genes involved in persistence. Four mutants with reduced persistence, disrupted in spoT, vlhA 1.04, asnA and MGA_0126, were identified.

Isolated mononuclear cells from 20 healthy volunteers were incubated for 4 hours with varying concentrations of the antibiotics either in the absence of presence of PMA, ionomycin and monesin. Both moxifloxacin (0-50μg/ml) and ciprofloxacin (0-100μg/ml) dose-dependently decrease the Th1 signature cytokine IFNγ and IL-4 (a signature cytokine for Th2 cells) (p=0.001) and only IL4 was affected by clarithromycin (p=0.04, 0-125μg/ml). However, the overall ratio of Th1 to Th2 cells was not found to be significantly modulated in cells treated with any of concentrations of the three antibiotics. The findings of the immunomodulatory effects of ciprofloxacin clarithromycin and moxifloxacin strongly implicate transcription factors as major targets of the antibiotics. An investigation of the effect of ciprofloxacin and moxifloxacin on the expression of genes and proteins of leucocytes was undertaken utilising. Affymetrix genechip and protein array analysis. Venous blood was drawn from a healthy volunteer and aliquots were incubated for 4 hours in the presence or absence of 2μ/ml LPS with concentrations of ciprofloxacin (0-50μg/ml) or moxifloxacin (0-20μg/ml). Leuocytes and plasma were isolated from the whole blood and were taken forward to gene and protein array analysis respectively. These revealed that both ciprofloxacin and moxifloxacin are capable of significantly modulating the expressions of a wide range of immune related factors at all the variable concentrations in both endotoxin stimulated and unstimulated whole blood. In particular the array studies revealed that the quinoline drugs were capable of modulating chemoattractant factors which could potentially enhance the inflammatory response and prime cells to migrate to the site of infection, suggesting that early use of the antibiotics may be beneficial. Continued enhanced inflammatory responses could, on the other hand be damaging to patient recovery and survival.

No description available.

The discovery and use of antibiotics in the chemotherapy of bacterial infections has revolutionised medicine as it is today. Unfortunately, the progressive use of antibiotics has promoted the evolution of bacterial defences against these mediators and thus the emergence of antibiotic resistance. Multidrug resistance (MDR) in bacterial pathogens has grown with such rapid progression that it now threatens to compromise the effective chemotherapy of a plethora of diseases. This thesis aspires to elucidate the molecular resistance mechanisms adopted by these bacteria, in order to expand our knowledge and to assist in the development of new therapeutic approaches to circumvent these mechanisms. On this basis, this thesis presents insights into a novel Vibrio cholerae antibiotic resistance, var, operon that encodes a metallo- β -lactamase (Mßl), VarG, and a tripartite ATP-binding cassette-type (ABC-type) transport system, VarACDEF that has substrate specificities for antimicrobial peptides and macrolide antibiotics. Mßls are fast emerging as a primary resistance mechanism, possibly as a consequence of the introduction of newer ß-lactam antibiotics such as the carbapenems in response to increasing Gram-negative bacterial resistance. Fascinatingly, the ABC transporter, through secondary structure predictions, has been envisaged to adopt a tripartite structure similar to the MDR transporter, AcrAB-TolC, from the resistance nodulation and cell division (RND) family. The structural characterisation of this system would be the first such tripartite system to be elucidated and may bring new insights into how Gram-negative bacteria may have evolved to tackle the issue that threatens its existence. The resistance mechanisms in the var Operon are believed to be under the control of a LysR-type transcriptional regulatory protein (LTTR), VarR. LTTR proteins form one of the largest transcriptional regulatory families with extremely diverse functions ranging from amino acid biosynthesis to CO(_2) fixation. VarR binds to three distinct promoter regions, varRG, varGA and varBC located upstream and adjacent to VarG, VarA an AcrA-like membrane fusion protein and VarC a TolC-like outer membrane protein, respectively. VarR has also been shown to act as a repressor at the varRG promoter region in the absence of its substrate. Interestingly, the mechanism of regulation by VarR is strikingly similar to the well documented LTTR, AmpR and serine ß-lactamase AmpC system that are found in many pathogenic bacteria. It could be that V. cholerae has evolved from this regular system and developed a ß-lactamase that would prove more beneficial in light of current selective pressures. Contrary to LTTRs being notoriously recalcitrant to purification due to their low solubility, this thesis reports the successful purification and crystallisation of full-length VarR in the presence and absence of its cognate promoter DNA. Elucidating the structural characteristics of VarR would be the first such regulator associated with MDR in the LTTR family. This would advance the knowledge on the only currently existing full-length crystal structure of a LTTR, CbnR, and will provide further insights into how structural conformations may lead to dissociation from the promoter and induction of gene expression. Understanding the mechanism by which VarR induces expression of these resistance mechanisms is paramount for future strategies to prevent the emergence of MDR microorganisms. Although these mechanisms of MDR maybe elucidated in V. cholerae, the evolutionary relatedness and conservation of structure and function in all families will enable this information to be related to similar systems in alternative bacterial species.

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No description available.

An emerging threat to the efficacy of commonly used β-lactam antibiotics is the bacterial production of metallo-β-lactamases. This family of enzymes, which require Zn²⁺ for activity, is unrelated to the serine β-lactamases, for which inhibitors have been developed. The metallo-β-lactamases are characterized by a broad substrate specificity and are unaffected by the serine β-lactamase inhibitors. Their mechanism is not well understood and there are no clinical inhibitors available. A better understanding of how these enzymes function is crucial if effective inhibitors are to be designed. Whilst all members of the metallo-β-lactamase family require Zn²⁺ ions for activity, they differ in their affinity for Zn²⁺ and the number of Zn²⁺ ions required for catalytic activity. The metallo-β-lactamase from Bacillus cereus is characterized by one higher affinity, and one lower affinity Zn²⁺ binding site (termed Zn1 and Zn2 respectively). Although only one Zn²⁺ ion is necessary for activity, maximum activity is achieved when both are present. Activity is also pH-dependent, decreasing at lower pH values. It has been proposed that a positively charged arginine residue (Arg121) located beneath the Zn2 binding site is responsible for the lower affinity of this enzyme for Zn²⁺ at this site, since other members of the family lacking this arginine residue bind two Zn²⁺ ions with equal, and higher, affinity. This thesis describes crystallographic studies of three B. cereus metallo-β-lactamase mutants, Arg121Cys, Arg121Glu and Arg121His, to investigate the binding of Zn2 in particular, and the mechanism in general. Each mutant has been studied in both mono- and bizinc forms at different pH values. Crystallographic evidence is presented in support of a proposed enzyme mechanism, in which Zn2 binding is flexible. A tri-zinc form of the enzyme is also described. This thesis also presents work relating to the crystallisation. and preliminary X-ray diffraction analysis of a putative phosphinothricin acetyltransferase from Pseudomonas aeruginosa.

No description available.

No description available.