LI E R. 1998. Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and general antimicrobial agents. They have been shown to be active against fungal cell walls, associated membranes and bacterial transport processes. Investigations were undertaken into the effect of these antimicrobial agents on substrate catabolism and intracellular ATP levels using an oxygen electrode and luciferin-luciferase technology, respectively. Results indicate that, while both compounds are poor inhibitors of substrate catabolism, sub-inhibitory concentrations of biocide greatly reduces intracellular ATP levels in both Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. This is thought to be due to the action of NaPT and ZnPT on the Gram-negative bacterial membrane. INTRODUCTIONSodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively, Fig. 1) are the sodium salt and zinc chelate of 1-hydroxy-2-pyridinethiol (Shaw et al. 1950 ;Albert et al. 1956 ;Hyde and Nelson 1984). Both NaPT and ZnPT have been shown to possess a wide spectrum of antimicrobial activity against fungal and bacterial species (Pansy et al. 1953 ;Albert et al. 1956 ;Hyde and Nelson 1984 ;Khattar et al. 1988). They are widely used as cosmetic preservatives (Nelson and Hyde 1981 ;Hyde and Nelson 1984) and are incorporated as anti-dandruff agents in shampoos (Fredriksin and Feargeman 1983 ;Hyde and Nelson 1984). They also possess excellent metal chelating properties and ZnPT is active as a metal complex (Davies 1985 ;Fenn and Alexander 1988).Previous work has suggested that this group of compounds is membrane active. This is indicated by the inhibition of uptake of several unrelated substrates in both bacteria and fungi (Chandler and Segel 1978;Friedman 1981;Khattar et al. 1988 ;Khattar and Salt 1993) and the observed depolarization of the transmembrane electrical potential in Neurospora crassa (Ermolayeva et al. 1995). The effects of an antimicrobial agent on substrate transport and related metabolism may be used as indicators of the membrane activity of the test agent (Gilbert et al. 1991). In turn, these effects may be reflected as a reduction in intracellular ATP levels (Harold Dundee, Bell Street, Dundee, DD1 1HG,.© 1998 The Society for Applied Microbiology 1972). Regardless of substrate transport, a direct membrane effect by a biocide may decrease intracellular ATP levels via disruption of the transmembrane proton motive force (PMF) and related processes. This has been exhibited with several membrane active agents including fentichlor, PHMB, alexidine and chlorhexidine (Bloomfield 1974 ;Chopra et al. 1987 ;Chawner and Gilbert 1989).This study describes the activity of NaPT and ZnPT on substrate transport and catabolism in Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548 and the effects of these agents on bacterial intracellular ATP levels at sub-inhibitory and minimal inhibitory concentrations (MIC). MATERIALS AND METHODS Organisms and chemicalsStock cultures of Es...

A .J . D I NN IN G , I .S . I. AL -AD HA M , P . A U ST IN A ND P. J . C OL L IE R. 1998. Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and metal chelating agents. They are commonly assayed using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). However, a simple quantitative colorimetric assay has not been previously reported for these compounds. This paper describes the development of a spectrophotometric assay for the quantification of the pyrithiones which is based on the chelation of copper (II) ions by the biocides. This assay was developed in order to facilitate the determination of the distribution of these biocides in the Gram-negative bacteria Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. Sodium pyrithione was exhibited only in the cytosol of E. coli and Ps. aeruginosa. Zinc pyrithione, however, was assayed in the cytosol of both bacteria and was found in the cell envelope of Ps. aeruginosa. These findings suggest that the pyrithione biocides are active within bacterial cells as well as at the cell membrane.