The Isolation ofStaphylococcus aureusTea Tree Oil-Reduced Susceptibility Mutants

Abstract

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.

Proteomic and Metabolomic Analyses of a Tea-Tree Oil-Selected Staphylococcus aureus Small Colony Variant

“…Two different subunits of DNA polymerase, DnaN (DNA polymerase III β-subunit) and DnaX (DNA polymerase III γ and τ subunits), and nucleotide metabolism proteins PurA (adenylosuccinate synthetase) and Tdk (thymidine kinase) were all decreased in SH1000-TTORS-1 ( Table 1). The production of AtpG (F0F1 ATP synthase γ-subunit) was also decreased in SH1000-TTORS-1 and an intergenic mutation was identified between the last gene of the ATP synthase operon (atpC) and the hypothetical gene SAOUHSC_02339 in SH1000-TTORS-1 [30] (Table 1).…”

“…TTO can also select for S. aureus SCVs that display reduced susceptibility to TTO in addition to ethanol, isopropanol, and individual TTO antimicrobial terpenes. Compared to the parent strain, a TTO-selected SCV was also characterized by a small cell size and a thinned cell wall [30]. Genomic analysis of the TTO-selected SCV revealed the presence of two intragenic and 19 intergenic mutations [30].…”

“…Compared to the parent strain, a TTO-selected SCV was also characterized by a small cell size and a thinned cell wall [30]. Genomic analysis of the TTO-selected SCV revealed the presence of two intragenic and 19 intergenic mutations [30]. One of the intragenic mutations was located in acpP [30], which encodes the essential acyl carrier protein (ACP) required for fatty acid biosynthesis [31].…”

“…Genomic analysis of the TTO-selected SCV revealed the presence of two intragenic and 19 intergenic mutations [30]. One of the intragenic mutations was located in acpP [30], which encodes the essential acyl carrier protein (ACP) required for fatty acid biosynthesis [31]. To our knowledge, this is the first report of an antimicrobial-selected acpP mutation, and possibly the only reported example of an acpP mutation in S. aureus.…”

Increased Susceptibility of Staphylococcus aureus Small Colony Variant to Tea Tree Oil: Comment on Cuaron et al. 2014

“…In their recent article, Cuaron et al describe isolation of Staphylococcus aureus with a reduced susceptibility to tea tree oil (TTO) and with certain features of a small colony variant (SCV; Cuaron et al, 2014).…”