Genetic changes that correlate with the pine-oil disinfectant-reduced susceptibility mechanism of Staphylococcus aureus.

AIMS: To identify factors associated with the Staphylococcus aureus pine-oil disinfectant-reduced-susceptibility (PD(RS)) mechanism and to describe one possible PD(RS) model. METHODS AND RESULTS: Comparative genomic sequencing (CGS) and microarray analysis were utilized to detect mutations and transcriptome alterations that occur in a S. aureus PD(RS) mutant. Mutant analysis, antimicrobial gradient plates, growth studies and 3-hydroxy-3-methylglutaryl coenzyme A synthase assays were then performed to confirm the biological consequences of the 'omics' alterations detected in a PD(RS) mutant. CGS uncovered three mutations in a PD(RS) mutant in a(n): alcohol dehydrogenase (adh), catabolite control protein A (ccpA) and an NADPH-flavin oxidoreductase (frp). These mutations lead to increased growth rates; increased transcription of an NAD-dependent D-lactate dehydrogenase gene (ddh); and increased flux through the mevalonate pathway. PD(RS) mutants demonstrated reduced susceptibility to bacitracin and farnesol, and one PD(RS) mutant displayed upregulation of bacA, a bacitracin-resistance gene. Collectively, this evidence demonstrates altered undecaprenol metabolism in PD(RS) mutants. CONCLUSIONS: The PD(RS) mechanism proposed results from increased catabolic capabilities and increased flux through the mevalonate pathway as well as altered bactoprenol physiology. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel mechanism that bacteria utilize to overcome the killing effects of PD formulations is proposed that is unique from the PD(RS) mechanism of the enterobacteraciae.

The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil.

Mutants of Escherichia coil strain AG100 exhibiting the multiple antibiotic resistance (Mar) phenotype demonstrated a greater level of tolerance to tea tree oil (TTO) compared with the parent strain. The ability of TTO to kill all E. coil strains studied was greater at 37 than at 30 degrees C. Growth of parent strain AG100 in the presence of salicylate, which induces the mar operon leading to the Mar phenotype, also increased tolerance to TTO. Escherichia coli Mar mutant YL1 demonstrated greater tolerance to antimicrobial terpenes found in TTO and did not leak K+ as rapidly in the presence of TTO when compared with its parent strain AG100. Attempts to isolate Mar mutants of Staphylococcus aureus using tetracycline gradients proved unsuccessful. However, when grown in the presence of salicylate, S. aureus strain BB255 demonstrated greater tolerance to TTO and did not leak K+ as rapidly in the presence of TTO compared with this strain grown without additions. This evidence demonstrates that bacterial Mar phenotypes increase tolerance to the killing action of TTO. This work also adds indirect evidence that the target of TTO is the cell membrane.

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil).

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli.

Concentrations of tea tree oil (TTO) which inhibit or decrease growth of Escherichia coli also inhibit glucose-dependent respiration and stimulate the leakage of intracellular K+. Stationary phase cells are more tolerant to these TTO effects than exponential phase cells.

Effects of tea tree oil on Escherichia coli.

Tea tree oil (TTO) stimulates autolysis in exponential and stationary phase cells of Escherichia coli. Electron micrographs of cells grown in the presence of TTO showed the loss of electron dense material, coagulation of cell cytoplasm and formation of extracellular blebs. Stationary phase cells demonstrated less TTO-stimulated autolysis and also had greater tolerance to TTO-induced cell death, compared to exponentially grown cells. It was also revealed that subpopulation of stationary phase cells demonstrated increased tolerance to TTO-bactericidal effects.