Many home cleaners (HC) are biocide/disinfectant mixtures contain plant essential oil extracts, detergents, alcohols and other constituents. Our laboratory has demonstrated that HCs can select for Staphylococcus aureus strains with increased HC tolerance (HC t) and reduced susceptibility to the important anti-staphylococcal drug vancomycin. Experiments have also determined that in-vitro selected vancomycin-intermediate S. aureus (VISA) demonstrate increased tolerance to a HC. Collectively this evidence indicates that the mechanisms and genetics of vancomycin resistance and HC t are linked. Presently, the identity of the HCt and all the VISA determinants are unknown. We have also determined that two interacting global regulators are involved with the HC t mechanism in S. aureus. Our major goal now is to identify the HC t determinants of S. aureus and chromosomal loci associated with the HC-stress response and HC t "regulon''. We propose to 1. Characterize the HC t-phenotype and investigate the action of HCs on cell wall/membrane ultrastructure. 2. Identify HC t-determinants and the Hc t regulon via: i. analyzing sequences and regulation of two chromosomal global regulators; ii. direct cloning and selection of HC t-determinants; iii. focusing on chromosomal loci controlled by global regulators and involved with reduced vancomycin susceptibility using a DNA subarray; and iv. investigating alterations in the S. aureus transcriptome following HC t mutation utilizing S. aureus genomic DNA arrays. 3. Determine if HC t mutants are present among clinical S. aureus isolates and determine if HC t phenotype can be used to detect S. aureus with reduced susceptibility to vancomycin. We hypothesize that HC tolerance in S. aureus is mediated by mutations in a global regulator operon/loci. Since our evidence indicates that the mechanisms and genetics of vancomycin resistance and HC t are linked, we expect to find HC t loci that are also involved with the VISA mechanism of S. aureus. Whats more, we intend to develop a novel assay for the detection of low-level vancomycin resistance among clinical isolates. This project will also clarify the role of global regulation in the bacterial response to biocides that target multiple bacterial gene products and processes.