Staphylococcus aureus mutants and clinical isolates with reduced susceptibility to the biocide triclosan differ in phenotype and genotype

Objectives: In view of the requirements posed by licensing process of disinfectants according to the EC biocide directive, we evaluated the possibility to devise an in vitro assay for testing the bacterial resistance to the biocide Triclosan. Triclosan is a synthetic bisphenol compound and active ingredient in many human hygiene biocidal products, targeting specifically the bacterial fabI gene encoding a enoyl-[acyl-carrier-protein] reductase. Methods: Starting from five S. aureus reference strains 32 mutant were selected in vitro by culturing strains in medium containing triclosan. Mutants were both selected by single exposure on selective media or by cicling on increasing conditions of the biocide. The phenotypes and fabI sequences were determined in all mutants and compared to data from of 76 clinical S. aureus strains showing decreased susceptibility to Triclosan. Results: The frequency of selection of S. aureus mutants in vitro for triclosan was between 2x10^9 and 3x10^10 depending on the strain. The single passage mutants showed an MIC of 4-8 ug/ml and MBC of 8-16 ug/ml, while the multi-passage mutants all showed a MIC of 16-32. In contrast the clinical isolates had a variable range of MIC values (0.125 to 4 ug/ml). All except three in vitro selected mutants showed mutations in the fabI coding region. In contrast only half of the clinical isolates showed mutations in fabI. Except for two mutations, none of the mutations selected in vitro matched to those found in clinical strains. Conclusion: Both the MIC and MBC profiles and the mutations of in vitro selected triclosan resistant mutants differed from those detected in clinical isolates with reduced susceptibility to triclosan. In addition our data clearly indicate that there are additional and yet undescribed mechanisms of resistance to triclosan in clinical isolates of S. aureus. Summarised these data indicate that for S. aureus an in vitro test for prediction of resistance development to triclosan is not feasible and, in any case, would yield results of no clinical relevance.