Fatty Acid Supplementation Reverses the Small Colony Variant Phenotype in Triclosan-Adapted Staphylococcus aureus: Genetic, Proteomic and Phenotypic Analyses

Abstract

Staphylococcus aureus can develop a small colony variant (SCV) phenotype in response to sub-lethal exposure to the biocide triclosan. In the current study, whole genome sequencing was performed and changes in virulence were investigated in five Staphylococcus aureus strains following repeated exposure to triclosan. Following exposure, 4/5 formed SCV and exhibited point mutations in the triclosan target gene fabI with 2/4 SCVs showing mutations in both fabI and fabD. The SCV phenotype was in all cases immediately reversed by nutritional supplementation with fatty acids or by repeated growth in the absence of triclosan, although fabI mutations persisted in 3/4 reverted SCVs. Virulence, determined using keratinocyte invasion and Galleria mellonella pathogenicity assays was significantly (p < 0.05) attenuated in 3/4 SCVs and in the non-SCV triclosan-adapted bacterium. Proteomic analysis revealed elevated FabI in 2/3 SCV and down-regulation in a protein associated with virulence in 1/3 SCV. In summary, attenuated keratinocyte invasion and larval virulence in triclosan-induced SCVs was associated with decreases in growth rate and virulence factor expression. Mutation occurred in fabI, which encodes the main triclosan target in all SCVs and the phenotype was reversed by fatty acid supplementation, demonstrating an association between fatty acid metabolism and triclosan-induced SCV.

Figure 1

Colony size of P0 and SCVs (P10) with and without the addition of fatty acids. All P10 SCVs showed reduced colony size compared to P0. When the media supplemented with fatty acids, all SCV colony size reverted to pre-exposure size (P0). *Significant change (p < 0.05) compared to P0 and FA stands for media supplemented with fatty acids (Tween 80). Results are means and standard error from representative colonies (n = 3).

Figure 2

Survival curve of four SCVs (NCTC 13277, ATCC 43300, SAR17 and Newman) and SA2831 (non-SCV). All SCVs P10 strains exhibited reduced larval lethality compared to P0 with exception of NCTC 13277. ATCC 43300, Newman and SAR17 P10 exhibited a significant reduction in relative pathogenicity, and PX10 pathogenicity reverted to P0 level with exception of SAR17, which remain attenuated even when triclosan was removed (PX10). *Significant change (p < 0.05) compared to P0. Results are means from three separate experiments.

Figure 3

Proteins altered in 3 SCVs (Newman, ATCC 43300 and SAR17) and non-SCV (SAR2831) compared to P0 categorised based on the function or pathway. Newman exhibited the highest number in protein expression alteration while ATCC 43300 and SAR 17 showed changes in 26 and 48 proteins, respectively.

Figure 4

Keratinocytes invasion assay of P0, P10 and PX10 in four SCVs (NCTC 13277, ATCC 43300, SAR17 and Newman) and SA2831 (Non-SCV). ATCC 43300, SAR17, Newman and SAR2831 P10 exhibited a significant reduction in recovered bacteria and number of intracellular reverted to unexposed level when triclosan was removed. *Significant change (p < 0.05) compared to P0. Results are means and standard error from three separate experiments with two technical replicates.