Elucidation of the molecular basis of pseudoresistance to antibiotics in Staphylococcus aureus

The discovery of antibiotics in the early part of the 20th century made possible for the first time the treatment and cure of bacterial infections, and heralded a new era for humanity. However, the utility of these compounds has been progressively eroded as bacteria have evolved to resist their effects. Increases in antibiotic resistance prevent effective treatment of bacterial infections, and are associated with significantly greater illness and death of patients. Consequently, major strain is placed on the healthcare system and the adverse effects are felt throughout society as a whole. This is especially true for what is arguably the most important ?superbug?, Staphylococcus aureus. This organism causes a wider array of infections than any other bacterium, and the number of effective drugs available to treat the infections it causes is rapidly dwindling. Ordinarily, S. aureus becomes resistant as a result of permanent changes to its genetic make-up that allow it to grow in the presence of antibiotics. However, all S. aureus strains frequently display ?pseudoresistance? (PR) to antibiotics, a state in which the bacteria are unable to grow but are not killed, allowing them to resume growth when antibiotic treatment stops. The most important of these PR mechanisms are biofilms and antibiotic tolerance. The former describes films of bacteria growing on biological or inert surfaces (e.g. human tissue, indwelling medical devices), while the latter describes a type of PR which can be achieved by more than one route. These PR mechanisms are extremely important, since they occur frequently (e.g. biofilms occur in two-thirds of human infections, and tolerance is seen in two-thirds of strains isolated from patients with a S. aureus heart-valve infection), and both result in treatment failure with antibiotics. This project aims to understand exactly how these PR mechanisms prevent antibiotics from effectively killing bacteria. This information is essential for guiding the development of strategies and/or drugs that could allow PR to be circumvented. Dissection of the molecular basis of PR will shed light on bacterial processes that can be blocked or perhaps themselves be targeted by new antibacterial drugs. The potential for developing strategies aimed at preventing the formation of intractable biofilms on medical devices such as intravenous catheters, an extremely common cause of hospital infection, will be particularly valuable.

The ?superbug? Staphylococcus aureus causes serious hospital infections which are becoming increasingly difficult to treat as a consequence of mounting resistance to currently available antibiotics. ?Classical? antibiotic resistance, which is conferred by heritable genetic determinants and enables bacteria to grow in the presence of antibiotics, has been extensively studied and is relatively well understood. However, S. aureus frequently displays ?pseudoresistance? (PR) to antibiotics, a state in which staphylococci are growth-inhibited but become refractory to the killing effects of antibiotics. Consequently, PR prolongs the length of antibiotic treatment regimens and accounts for a significant proportion of therapeutic failures. Several PR mechanisms are evident in S. aureus, and include those that are intrinsic to the organism in particular physiological states (e.g. stationary-phase persistence, PR during biofilm growth) and those that are acquired by some strains (genotypic tolerance). This project aims to understand, at a molecular level, the mechanisms underlying these staphylococcal PR strategies, a prerequisite for developing approaches aimed at overcoming or circumventing them. This will be achieved by the identification and characterization of S. aureus mutants displaying altered PR properties. A near-saturation transposon insertion (TI) library will be generated in S. aureus SH1000, in part using a novel, custom transposon carrying a strong staphylococcal promoter, which, in addition to null-mutants, will enable the recovery of hypermorphs and hypomorphs (over- and underexpressor phenotypes). Approximately 20,000 individual TI mutants will be screened for loss of intrinsic PR in the biofilm state and during stationary-phase persistence, and for the development of acquired (genotypic) tolerance. Loci impacting PR will be identified by rescue cloning of TI sites, and the molecular mechanisms underlying PR in these strains examined by complementary expression profiling techniques (DNA microarray and proteomic analysis) alongside the wild-type strain. Clinical isolates and chemically-mutagenised S. aureus strains displaying genotypic tolerance (GT) will also be analysed by expression profiling, and the roles of individual candidate GT genes subsequently examined by artificially-regulated gene expression. Elucidation of the molecular basis of PR not only constitutes crucial, fundamental, biological research that will add significantly to our current understanding of bacterial growth and physiology, but is essential for guiding the development of medical strategies and novel drugs that could allow PR to be circumvented.