1. Identification of bacterial genes involved in antibiotic resistance using whole genome screens

Antibiotic resistance in disease-causing bacteria has spread, resulting in the inability to treat some bacterial infections, which can lead to the deaths of patients. Bacteria can become resistant to antibiotics using different mechanisms, only some of which have been characterised. A better understanding of how bacteria resist antibiotic action will assist in the making of new antibiotics, may provide new ways of controlling resistance, and help us to predict when resistance to a particular antibiotic is likely to arise, allowing a pre-emptive switch to a different antibiotic for disease treatment. At the Sanger Institute, by using the latest DNA sequencing technology to analyse very large collections of bacterial mutants, we can firstly identify those components of the bacterial cell that are essential for life. These provide candidate targets for the development of new antibiotics. Secondly, we can measure every non-essential component of the bacterial cell for its contribution to antibiotic resistance. We have performed a pilot study which has shown that the method also tells us which cellular components contribute to the bacterial cells? sensitivity to antibiotics. This information tells us how it may, in the future, be possible to make resistant bacteria susceptible once again to antibiotics. It also gives us a more complete understanding of how bacteria resist antibiotics, and in general provides clues as to the role of the bacterial cell components, as the role of most components is not fully understood and the role of many is unknown. It is proposed to perform these experiments on a number of disease causing bacteria: the large mutant collections needed are already available for Salmonella Typhi which causes typhoid fever, and Salmonella Typhimurium which causes food poisoning. In addition, mutant collections will be made in MRSA (a strain of Staphylococcus aureus), Enterobacter cloacae, and E. coli, all of which cause infections acquired by patients whilst in hospital, and are particularly dangerous for individuals who are already ill with other medical conditions. Many antibiotics exist, but there are relatively few different types. Using one of each type will maximise our understanding of antibiotic resistance mechanisms for significantly less effort and expense. Once the data have been generated, they will be made available, in an easy to understand form, on the Sanger Institute web site where they will provide a freely and widely available resource for researchers and any other interested parties.

Antibiotic resistance in bacterial pathogens has spread, resulting in therapeutic failures in the treatment of some bacterial infections. A number of antibiotic resistance mechanisms are known, however, resistance is dependent on other, complementing mechanisms that have been termed ?innate resistance,? such as efflux systems which eject antibiotics from the bacterial cell. Recent work, and our proof of principle study, has identified genes not previously known to contribute to antibiotic resistance, and has indicated that other unknown resistance mechanisms exist. These numerous resistance mechanisms have been termed the bacterial ?resistome.? Transposon Directed Insertion-site Sequencing (TraDIS) combines high through-put Illumina sequencing and very large pools of transposon mutant libraries to simultaneously identify the site of every transposon insertion. This firstly identifies essential gene candidates, representing approximately 10% of the genome, in which transposon mutations are absent. These essential genes indicate candidate targets for the development of new antibacterial agents. Secondly, TraDIS allows every non-essential gene, approximately 90% of the genome, to be assayed simultaneously, which has not been achievable previously. Thus, by growing the mutant pool in sub-lethal concentrations of an antibiotic, the relative contribution of each non-essential gene to tolerance or sensitivity to the antibiotic may be measured. The entire non-essential gene set may then be listed in order of genes most important for resistance, through genes of no importance to those genes whose functions contribute to sensitivity. It is proposed to perform this type of experiment using two serotypes of Salmonella for which large transposon mutant libraries are already available; Typhi, which causes typhoid fever, and Typhimurium which causes food poisoning. In addition, transposon mutant collections will be generated for a methicillin resistant Staphylococcus aureus, an E. coli strain from a case of septicaemia, and Enterobacter cloacae, all of which cause nosocomial infections, and are particularly dangerous for immunocompromised and intensive care patients. A number of different antibiotics will be tested. This potentially provides new ways of combating resistance: resistant strains may be rendered sensitive using compounds that inhibit targets involved in resistance, and by compounds that induce expression of those genes products that contribute to sensitivity. In addition, the data will provide clues as to which resistance mechanisms may be operating in strains where the mechanism has not been identified. All the data will be made available on the Sanger Institute web site and will provide a useful resource for researchers engaged in antibiotic research and development.