No description available.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the majothu~ampathogens and is a common cause of hospital-associated infection in immunocompromised patients and individuals with open wounds. However, infections affecting the healthy and young have increased in the past few decades and are a rising clinical concern. Understanding the genetic diversity of S. aureus can therefore result in a better understanding of the pathogenesis, spread and evolution of S. aareus, which in turn can lead to the development of effective infection control measures. In the present study, molecular methods were utilised to investigate differences in the distribution and variability of bacterial genes present between successful and unsuccessful lineages of MRSA. Lineages were assigned based on multilocus sequence typing (MLST) clonal complexes and successful lineages were defined as those lineages that have given rise to multiple epidemic MRSA done. In addition to well-established molecular methods such as MLST and staphylococcal cassette chromosome mec typing, a novel fluorescent amplified fragment length polymorphism assay was developed to identify regions of genetic heterogeneity between and within lineages. These differences were attributed to lineage-specific sequence variation and differences in the distribution of mobile genetic elements (MOEs) between lineages. These genetic differences were investigated in relation to functional differences within the bacterium, to explore the,role of cell functions encoded by these regions in the emergence and prevalence (or spread) of dominant genetic lineages. This study indicates a combination of factors play a role in the success of major MRSA lineages. The genetic diversity amongst core regions and MOEs appear to alter antimicrobial resistance and virulence factors that are vital to their success, as they enable the rapid adaption of lineages to their environment.

No description available.

No description available.

No description available.

No description available.

Staphylococcus aureus is the most frequently isolated pathogen in hospitals and community and methicillin-resistant S. aureus (MRSA) prevalence has increased in Saudi Arabian hospitals from 2% in 1989 to 33% in 1998. In this study, the prevalence of healthcare associated infections and the predominant organisms was investigated through survey of the infection control departments in three hospitals in Jeddah, namely King Abdul Aziz University Hospital (KAUH), King Fahad Hospital (KFH), and the Maternity and Children Hospital (MCH). Two hundred and seven MRSA isolates were collected from the three respective laboratories and their multiple antibiotic resistance (MAR) profiles identified. Twenty three strains were selected for further investigation using Pulse Field Gel Electrophoresis (PFGE) and Spa typing based on MAR profile and demographic data. In addition, the antimicrobial activity of some Saudi herbs and spices were assessed in vitro and in vivo as possible alternative treatments for MRSA. Data showed there was a similarity in infection control policies across the three hospitals. Collective data from all hospitals showed that MRSA was frequently isolated from wounds (36.7%) and respiratory tract infections (30.4%). They were resistant to penicillin (100%), oxacillin (100%), erythromycin (62.9%), gentamicin (51.2%), trimethoprim-sulfamethoxazole (54.6%) and ciprofloxacin (50.2%). All strains were sensitive to vancomycin, teicoplanin and rifampicin. Ten MAR profiles were identified. Two MAR profiles were endemic in KAUH and KFH and resistant to six out of nine antibiotics. One MAR profile was endemic in MCH and showed resistance to five out of nine antibiotics. The majority of strains were PFGE type 1 predicted as EMRSA 1 and spa type t-363, t-037 and these were seen in the eight of the ten MAR profiles. The minimal inhibitory concentration, minimal bacteriocidal concentration and time kill assays of aqueous and alcoholic extracts of Pimenta dioica and Punica garantum singly or combination were active at 512mg/ml for 90% of MRSA strains. Gas chromatography-mass spectrometry analysis confirmed the major components in both extracts were in the phenolic group. An in vivo study using both extracts and combinations as an aqueous wash and ointment showed some potential but would not be a suitable replacement for chlorhexidine or fucidin as decontamination treatment. Future work on determining the correct concentration for in vivo use needs to be undertaken.

Since the initial association of the Panton Valentine leucocidin (PVL) toxin with highly virulent strains of Staphylococcus aureus found in the community, a firm epidemiological link has been established between the PVL encoding genes and community-acquired strains of both meticillin resistant (CA-MRSA) and susceptible (CA-MSSA) Staphylococcus aureus. While most research has predominantly concentrated on the genotyping of CA-MRSA strains, PVL-MSSA appear to pose an increasing public health risk. Currently though, there exists a dearth of epidemiological data on PVL-MSSA strains, particularly with regard to the lukS and lukF genes which encode the PVL toxin. This first aim of the present study therefore was to explore the genetic diversity of a group of PVL-MSSA clinical local isolates in order to contribute to the limited current data and provide insight into the evolution and emergence of PVL-MRSA isolates. In addition, as current typing systems are cumbersome, time consuming and expensive, this present study was also aimed at the development of a rapid high resolution melt (HRM) typing system for the characterisation of PVL-positive isolates. The PVL toxin is encoded for by two highly conserved adjacent genes (lukF and lukS) which are co-transcribed. Variations in these genes correlate with a strain’s genotype. Therefore, the present study set out to genotype isolates based on the four major SNPs at positions 527 and 663 of the lukS gene and 1396 and 1729 of the lukF gene. The final aim of the present study was the development of an enzyme linked immunosorbent assay (ELISA) system (for the detection and quantification of both PVL and alpha haemolysin) that has potential application in clinical diagnosis and as a research tool. Characterisation of a collection of UK PVL-MSSA isolates by MLST and spa typing which is presented in the present study, showed a genetic similarity to circulating PVL-MRSA strains, with 94.7% of the isolates belonging to CA-MRSA related genetic backgrounds (ST1, ST22, ST30, ST772 and ST88). Three novel spa types (t6642, t6643 and t6769) and a novel ST (ST1518) were however detected in this population. Furthermore, the presence of identical PVL phages and haplotypes in the PVL-MSSA isolates to those previously described in PVL-MRSA isolates point strongly at the role these strains may play as precursors of emerging lineages of clinical significance. The HRM assay developed in this study was able to accurately genotype PVL-positive isolates based on the double allelic variations in both the lukS and lukF genes. The high degree of sensitivity of this technique was clearly demonstrated by its ability to differentiate between the lukS A527/G663 and G527/T663 genotypes which theoretically should have the same melt temperature. Despite the issues in data interpretation, which arose following attempts to improve the discrimination of this technique by the addition of a third locus (spa), the technique still showed potential as a useful tool for the rapid genotyping of PVL-positive isolates. While HRM was useful in rapidly detecting and genotyping PVL-positive isolates, the actual level of protein production of both PVL and HLA toxins could only be determined following the development and validation in the present study of a simple competitive ELISA platform which exploited the high affinity biotin/streptavidin interaction to improve sensitivity. This technique would be especially useful in settings which lack the specialised equipment required for genetic studies like HRM. In summary, in addition to contributing to the limited epidemiological information about PVL-MSSA strains and demonstrating a clear role for these strains in the evolution of PVL-MRSA strains, the present study has developed two distinct methods to aid the study of S. aureus PVL producing strains which are becoming a significant healthcare problem worldwide. The present study will contribute to our understanding of these strains and to the development of intervention strategies to curb their spread and threat.

S. aureus is a natural commensal of the human host and an opportunistic pathogen that causes a wide range of diseases. Biofilm formation is an important S. aureus virulence determinant. S. aureus colonisation of medical devices and host tissues as a biofilm can impede treatment as genetically and metabolically diverse cells in the different layers of the film can prevent the penetration or activity of the therapeutic agent used. Biofilm formation is a multi-factorial process which can be influenced by many environmental factors. A major environmental stress encountered by bacteria in vivo is severe iron-restriction. However, pathogenic bacteria can use low iron concentrations as a signal to up regulate factors responsible for virulence. This work demonstrates for the first time that S. aureus biofilm formation is iron regulated. It demonstrates that biofilms formed in low iron are dependant on the proteins Eap and Emp which are also iron regulated and are positively regulated by the ferric uptake regulator, Fur. This work has also identified that PNAG, which currently has a controversial role in biofilm formation, was capable of compensating for the loss of Eap and Emp in low iron when it was over expressed, but that wild type levels of PNAG appeared to have a limited role. Nevertheless, the ica operon responsible for the production of PNAG was essential for the expression of Eap and Emp. In addition, Sae, Fur, Agr, SarA and Hfq were shown to all have interlinking roles in the expression of these and other proteins associated with virulence and low iron biofilm formation. The identification of the importance of Eap and Emp in low iron biofilm formation and the regulatory network controlling their expression may have implications on the development of new therapeutic agents essential for the prevention and treatment of S. aureus infection.

Staphylococcus aureus is a pathogenic bacterium that causes a wide spectrum of human diseases and is a leading cause of nosocomial infection in the UK. Metal homeostasis is an important aspect of bacterial biology as transition metals such as copper and iron are required as enzyme cofactors but can also be toxic to cells at high concentrations. These metal homeostasis systems can be important for virulence. However, several important aspects of S. aureus metal homeostasis remain to be defined. This project focuses on novel S. aureus iron/Fur gene regulation and copper homeostasis. Fur is a well-described DNA binding repressor protein, found in many pathogenic bacteria. In S. aureus, Fur has been seen to both activate and repress genes in iron replete and iron restrictive conditions, and there is also Fur independent iron regulation. However, the regulatory mechanisms involved remain undefined. This investigation into novel iron regulation identified a new S. aureus iron regulator, LysR. lysR expression was found to be auto-regulated and activated by Fur in low iron. Phenotypic analysis suggested a possible role for LysR in the control of genes of the histidine utilisation pathway, as well as oxidative stress resistance. Two copper responsive operons have been found in S. aureus; copAZ and copB/mco. However, many important aspects of the S. aureus response to copper remain undefined. In this study, copper tolerance was shown to vary between strains and ATCC 12600 was identified as the first hyper copper-tolerant S. aureus, due to a transferable copper-resistance plasmid. A new S. aureus regulator, CsoR, was found to control the copper response of copAZ and both chromosomal and plasmid encoded copB/mco. Finally, this data shows that H2O2 scavenging is an essential S. aureus copper resistance mechanism and that extracellular surface copper toxicity is important in S. aureus.