Evaluation of metabolism of azo dyes and their effects on Staphylococcus aureus metabolome

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (similar to 90x) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.