Tea tree oil-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. TTORS mutant revertants (RV) were also isolated from the TTORS mutants following growth in drug free media and all RV strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that a SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared to parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in a SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.
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