K-075. Azoreductase Activity of Skin Bacteria

R. L. Stingley, H. Chen, C. Cerniglia;
Natl. Ctr. for Toxicological Res., Jefferson, AR.

Azo dyes, characterized by one or more azo bonds, represent a major class of synthetic colorants used in tattooing, cosmetics and other consumer products. These dyes are metabolized by bacteria to colorless aromatic amines, some of which are carcinogenic. An azoreductase gene (azo1) was previously identified in the skin bacterium Staphylococcus aureus. Purified Azo1 metabolizes a number of azo dyes, including Methyl Red (MR), Orange II (Or II), Ponceau S and Ponceau BS. A rapid assay method to monitor dye reduction spectrophotometrically was developed using a SpectraMax Plus 384 plate reader. Reduction of MR and Or II by 26 bacterial species associated with skin was monitored at 430 nm and 482 nm, respectively, and growth was measured simultaneously at 550 nm. Reduction assays were performed at least in triplicate with no-dye controls for bacterial growth. All 26 skin bacteria were able to reduce MR to some degree by 24 h. Patterns of fast (0.5 to 1.5 h), intermediate (3.5 to 9 h) and slow (11 to 14.5 h) reduction were observed among those that completely reduced MR. Only 1 species was unable to reduce Or II to any degree by 24 h, and only 3 species were able to completely reduce Or II within 24 h. MR reduction in S. aureus started with early exponential growth and in S. epidermidis and Pseudomonas aeruginosa with mid-exponential growth. Reduction of Or II, Ponceau S (520 nm) and Ponceau BS (502 nm) started during late exponential-early stationary growth for each of these species. In Southern hybridizations a S. aureus azo1 probe bound to the genomic DNA of 11 out of 12 other Staphylococcus species. A blastn search revealed that the genomes of 2 of the 11 staphylococci that hybridized with azo1, S. epidermidis and S. haemolyticus, contain hypothetical genes with 80% and 83% sequence identity to azo1, respectively. Among the staphylococci that contained a gene similar to azo1 based on Southern hybridization, the rate of MR reduction varied from 1 h to 9 h, suggesting that the regulation of this gene varies among the species, that the dye uptake varies among the species, or that azo1 is not the only gene involved in azo dye reduction.