Staphylococcus aureus is a worldwide leading cause of skin and soft tissue, bone and joint, and bloodstream infection. Despite this, S. aureus is also a harmless commensal in about one third of the population, although carriage is a risk factor for subsequent disease. S. aureus has evolved resistance to several antibiotics, including meticillin, resulting in meticillin-resistant S. aureus (MRSA), which in the UK largely consists of two epidemic lineages. In spite of much research, substantial aspects of the epidemiology and biology of S. aureus are still poorly understood. In investigating the S. aureus host-organism relationship, this thesis has three aims. To explore the interface between community and hospital-acquired S. aureus; to investigate the carriage dynamics of S. aureus in the community; and to use population genetic methods to study epidemic hospital associated S. aureus lineages. Case-control studies comparing hospital and community-acquired MRSA revealed that the majority of UK MRSA remains healthcare associated, with community-acquired MRSA reliably identified in only 0.2% of individuals. However, an additional 0.2% of individuals also carried "feral" MRSA with molecular characteristics identical to hospital-associated strains, but in hosts with no healthcare risk factors. To further investigate S. aureus carriage dynamics in the community, a carriage study was designed to collect detailed host factor information and correlate this with S. aureus carriage over time. In the study 32% of participants carried S. aureus of which the majority carried for over one year. Younger age was associated with transient carriage, including S. aureus acquisition in individuals who were initially negative. Finally, whole-genome sequencing of two epidemic S. aureus lineages indicated rapid clonal expansion of MRSA and clear geographic and temporal genetic structure. One particularly closely related cluster of strains may provide a genetic explanation for an MRSA outbreak in Brighton.

Staphylococci form micro-communities, known as biofilms, on the surface of inserted medical devices leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. Bacteria in the biofilm are enclosed within an endogenously-produced exopolymeric matrix, which protects them from antimicrobial agents and host immune responses. Prolonged antibiotic therapy and device removal are often required to resolve such infections. Thus, there is a need for new therapeutics but the structural mechanisms of biofilm formation that could assist their development remain unknown. SasG and Aap are highly homologous proteins from Staphylococcus aureus and Staphylococcus epidermidis, respectively, that promote cell-to-cell accumulation during biofilm formation. They are cova:lently attached to the cell-wall and form extended fibrils on the bacterial surface. Both proteins consist of an N-terminal A domain followed by aB region, composed of tandemly arrayed 128 residue repeats. The repeats, which show very high levels of sequence identity, have been proposed to mediate intercellular accumulation through Zn2+ -dependent homo-dimerisation. The present work is a biochemical, biophysical and structural dissection of the biofilm-forming protein SasG from S. aureus that addresses these issues. Zn2+ was found not to cause SasG to dimerise suggesting other components of the biofilm matrix, such as teichoic acids, might eo-mediate the intercellular aggregation. NMR spectroscopy in combination with in vitro folding studies demonstrated that each 128-residue sequence repeat is in fact comprised of a G5 domain of 78 amino acids and a smaller sub-domain of 50 amino acids (herein called E), which is unstable in isolation but folds cooperatively in the context of a C-terminal G5. X-ray crystallography revealed that G5 and E domains are arranged head-to-tail and share the same fold (two single-layer ~- sheets connected via a collagen-like triple-helical motif), which lacks a compact hydrophobic core. This domain arrangement, coupled with interlocking G5-E and E-G5 inter-domain interfaces, results in a contiguous, elongated, apparently rigid, monomeric structure, which explains the fibrillar nature of SasG at the bacterial cell surface. Despite all these unusual structural features, multi-domain SasG constructs exhibit thermodynamic stabilities comparable with other globular proteins of similar size. This work provides the first structural insight into staphylococcal biofilm-associated proteins and a paradigm for the formation of fibrils on the 100 nrn scale from a single polypeptide chain. It presents a novel protein fold, which results in elongated yet thermodynamically stable domains via a 'distributive hydrophobicity' mechanism, distinct to the familiar compact hydrophobic core of globular proteins. Finally, formation of two domains by conserved sequence repeats might provide a simple solution to avoid protein misfolding when a tandem arrangement of highly similar sequences is biologically advantageous.

Staphylococcus aureus is an important human pathogen and is a common cause of hospital -associated infections. It has the ability to cause a wide range of diseases, facilitated by a plethora of virulence factors. Resistance to antibiotics has played an important role in the recent evolution of this bacterium, allowing it to persist in the face of antibiotic treatment. The ever- increasing levels of resistance to multiple classes of antibiotics, has earned MRSA the name "superbug". However this antibiotic resistance comes at a biological cost. The aim of this thesis were to examine the negative effect of the methicillin resistance encoding type II SCCmec element on cytotoxicity and investigate the lack of an effect caused by the type IV SCCmec element We found that; The type II SCCmec element is implicated in the reduced cytotoxicity of type Il HA-MRSA. Expression of the mecA gene from the SCCmec element is directly responsible for this effect. High levels of mecA expression are required to produce the loss of cytotoxicity . Loss of cytotoxicity is caused by a lack of Agr quorum system activity. The cell wall of highly resistant type Il HA-MRSAs prevent quorum system signalling. Reduced cytotoxin expression in type IJ HA-MRSAs leads to reduced virulence in a murine model of sepsis. The relatively low level of mecA expression from the type IV element explains the maintenance of cytotoxicity in type IV CA-MRSAs. By increasing the level of mecA expression in type IV CA-MRSA we can reduce cytotoxin production. By addition of sub-inhibitory concentrations of oxacillin to the growth environment of type IV CA-MRSAs we can reduce cytotoxicity and reduce infection related morbidity in an invertebrate model of infection.

Gram-positive bacteria of the genus Staphylococcus are known commensals of the human skin and mucosa, but also opportunistic pathogens especially for immunocompromised patients. The rapid development of antibiotic multiresistance poses a great problem for the treatment of staphylococcal infections and results in an ongoing need for novel antibiotic targets. The first part of this work addressed the phenotypic and genetic characteristics of a set of S. epidermidis samples which had been isolated from a leukaemia patient over the course of an eventually fatal infection. Molecular typing methods (PFGE and MLST) were used to identify a single S. epidermidis strain of the widespread sequence type ST2 as the cause of infection. The strain displayed a high phenotypic variability during the infection with regard to oxacillin resistance and biofilm formation. The underlying mechanism is most likely the high genetic variability of this particular S. epidermidis clonal lineage. The same genomic deletion observed in vivo could be triggered in an in vitro experiment and occurred with a very high frequency (10-²) The second part of this thesis concerned the characterisation of a recently identified putative T-box transcriptional control system for methionine biosynthesis genes. The nucleotide sequence alignment revealed high homologies between staphylococcal species and predicted structural elements associated with Bacillus T-box systems. In vitro binding assays with this met leader RNA and tRNA Met demonstrated the binding interaction between the two RNA species and mutational studies of the T-box motif revealed the importance of single nucleotides for tRNA binding. Transcript levels of the met leader RNA and the downstream genes were shown to vary between different staphylococcal strains, but to be generally induced under methionine starvation. From various S. aureus RNase mutants investigated mainly RNase Y, RNase J2 and RNase III seem to be involved in met leader RNN mRNA processing and degradation.

Staphylococcus aureus is an opportunistic pathogen in UK hospitals and MRSA is a major concern due to burgeoning multi-drug resistance. However these resistance genotypes have not accumulated into one fully-resistant MRSA clone. MRSA from St. George's Healthcare NHS trust (SGHT) over a period of ten years were investigated. Antibiotic resistance phenotypes, genotypes and lineage types of infective MRSA isolates, incidence of MRSA, records of antibiotic prescribing both before and after a change in antibiotic stewardship and implementation dates of infection control interventions were combined. These data established that a decline in MRSA incidence correlated with a decrease in prescription of fluoroquinolones and cephalosporins at SGHT and no other recorded infection control intervention. CC22 MRSA became the dominant lineage of MRSA at SGHT from 2006 onwards, this coincided with the sudden acquisition of new antibiotic resistances by this lineage and the ability to gain and lose these resistances in subsequent years. These changes did not correlate with a change in antibiotic stewardship and could not be associated with any other infection control intervention. No MRSA were fully drug resistant, the majority were multi-drug resistant. However resistance did not accumulate within any lineage over time. Antibiotic resistance genes in MRSA are often carried on mobile genetic elements (MGEs). Resistance genotypes of infective MRSA suggested that carriage of resistance genes differs between lineages. Barriers to horizontal gene transfer (HGT) between lineages such as restriction-modification systems have been previously characterised using laboratory isolates of S. aureus and restriction-deficient mutants. The effect of these barriers on the spread of naturally occurring MGEs between clinical MRSA has 3 • not been measured. The efficiency of barriers to HGT between clinical MRSA lineages was assessed by developing an in vitro model conjugation. A conjugative gentamicin resistance plasmid was transferred at high frequency within a lineage, but at low frequency between lineages. Barriers to HGT were also assessed by attempting to develop an in vitro model of transduction. Phage from isolated from local water sources were used to transfer antibiotic resistance genes within and between lineages of clinical MRSA by generalised transduction. Transduction was detected between clinical MRSA of different lineages at low frequency, however transductions were broadly unsuccessful. Further investigation is warranted as generalised transduction appears to be an important mechanism by which antibiotic resistance genes are exchanged among MRSA. The presence of efficient barriers to HGT in clinical MRSA contributes to our understanding of why antibiotic resistance has not been able to accumulate in MRSA and why we have not seen a fully drug resistant S. aureus. ", •

Invasion of host cells by S. aureus is likely to play an important role during colonisation and infection. Studies have shown S. aureus invasion to occur via an interaction between its fibronectin binding proteins (FnBPs) and the host integrin a5~J utilising fibronectin (Fn) as a bridging molecule. The reports of S. aureus invasion vary in a number of ways and a number of factors are likely to influence invasion. The aim of this study was to investigate factors involved in determining S. aureus invasion of epithelial cells. The presence of a stable sub-population with enhanced invasive capability has previously been identified for the oral bacterium Porphyromonas gingivalis. In this study consecutive antibiotic protection assays were used in an attempt to determine whether such a sub-population structure exists in S. aureus cultures. Initial findings indicated that S. aureus cultures may contain an invasive sub-population but that this was transient and not stable. This was later attributed to technical aspects of the assay, in particular variation in multiplicity of infection (MOl) between the first and second rounds of invasion. When a MOl of 100 was maintained throughout, S. aureus cultures did not appear to contain invasive sub-populations. This work also highlighted a number of other methodological factors in the antibiotic protection assay, which is most commonly used, including intracellular growth, growth phase of bacteria, MOl and host cell confluence. S. aureus invasion is highly variable between strains and host cell lines. Expression and availability of FnBPs and a5~J are likely to be factors in determining the level of invasion. However, invasion did not correlate completely with levels of Fn-binding and expression of FnBPs, indicating additional factors are involved. The multi functional adhesin Iron-regulated surface determinant protein A (lsdA), was identified as a potential invasin due to its ability to bind Fn and its expression under conditions expected to pertain in vivo. Despite binding Fn, IsdA was not found to facilitate invasion. However, growth of S. aureus in minimal media produced slightly and reproducibly enhanced invasion and may therefore influence expression of other factors involved in facilitating invasion. The level of expression of a5~1 was found to be comparable in an oral, nasal and skin cell line and is therefore unlikely to be responsible for variation in S. aureus invasion. Invasion was enhanced in all cell lines when carried out in suspension; conditions that would allow access to a5~1 molecules even if they had been distributed unevenly on the cell surface when growing on polystyrene. Such polarity, however, did not account for the variation seen in invasion between cell types though further work showed that the nasal epithelial cells exibited significantly reduced binding to Fn. Since Fn acts as a bridging molecule between a5~1 and S. aureus, presence of non-active integrins would explain the difference in invasion seen. In summary, this work confirms Fn-binding capability by host cells is important in bacterial adhesion and invasion of cells but shows that the traditional proteins involved in Fn binding (e.g. FnBP) do not explain fully the different invasive behaviours of S. aureus strains. There is, therefore, redundancy in this functionality. II

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Central to the regulation of the diverse array of virulence factors encoded by the opportunistic human pathogen Staphylococcus aureus is the accessory gene regulator (agr) operon. The agr operon contains two transcription units; RNAII, encoding a quorum-sensing system including the master transcriptional activator AgrA, and RNAIII, the effector RNA molecule that regulates virulence gene expression. RNAII and RNAIII are transcribed from the divergent promoters P2 and P3, respectively. Due to the sub-optimal spacer length of the P3 promoter, it is widely believed that transcription from P3 is dependent on AgrA. A fully native S. aureus in vitro transcription system was setup to provide the first insight into the molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, results revealed that, in the absence of AgrA, RNA polymerase (RNAp) can interact with P2 and P3 equally well, however the transcription-competent open promoter complex (RPo) forms more readily at P2 than P3. AgrA was demonstrated to increase the occupancy of P2 and P3 by RNAp, to increase transcription initiation at both promoters, with a more pronounced effect at P3. The P3 promoter is unusual as it has a genuine extended -10 motif and a near to consensus -35 promoter element. "Scrambling" of the -35 element or shortening of the sub-optimal spacer to the optimal 17 nucleotides both significantly increased transcription initiation at P3, by facilitating the rate of isomerisation of the initial RNAp-P3 complexes to the RPo, in a strictly extended -10 promoter element dependent manner. Therefore, two possible mutually inclusive mechanisms by which AgrA activates transcription at the agr operon P3 promoter are proposed. In addition, the involvement of the transcription factor SarA on transcription from p2 and P3 was investigated. Overall, this study provides the first molecular insights into how transcription is regulated at the agr operon in S. aureus.

Τα ανθεκτικά στη μεθικιλλίνη στελέχη σταφυλοκόκκου παραμένουν ένα σοβαρό πρόβλημα στις ενδονοσοκομειακές λοιμώξεις παγκοσμίως, εξαιτίας της πολλαπλής αντοχής και της ικανότητάς τους να διασπείρονται πολύ γρήγορα μέσα σε νοσηλευτικά ιδρύματα προκαλώντας υψηλή νοσηρότητα και θνητότητα. Ιδιότητα των στελεχών αυτών είναι η τάση που έχουν να συγκεντρώνουν επιπλέον στοιχεία γενετικού υλικού που φέρουν γονίδια αντοχής σε αντιβιοτικά και τα οποία δεν προέρχονται μόνον από στελέχη του ιδίου γένους. Επιδημίες παρατηρούνται ιδιαίτερα σε Μονάδες Εντατικής Θεραπείας ενηλίκων και νεογνών, σε Μονάδες Μεταμόσχευσης και Χειρουργικές Μονάδες. Σκεφθήκαμε και προσπαθήσαμε να συλλάβουμε το θέμα αυτό σε μια δυναμική εξελικτική διαδικασία, μελετώντας την επιδημιολογία πολυανθεκτικών στελεχών σταφυλοκόκκου που απομονώθηκαν από κλινικά δείγματα ασθενών του Περιφερειακού Πανεπιστημιακού Νοσοκομείου Πατρών, με μοριακές τεχνικές. Η μελέτη οργανώθηκε και πραγματοποιήθηκε στο Εργαστήριο Μικροβιολογίας του Ιατρικού Τμήματος Πανεπιστημίου Πατρών και καλύφθηκε οικονομικά από το πρόγραμμα «Καραθεωδορή υπ’αριθμόν 1953». / -