Hexachlorophene (HCP) inhibits both endogenous and exogenous respiration (oxygen uptake) and o are present, but not a, the inhibition occurs on the oxygen side of cytochrome b . Exogenous menadione, an analogue of menaquinone, reverses the inhibition in both circumstances. The primary lethal action of HCP thus appears to be respiratory i,nhibition at a site within the membrane-bound part of the electron transport chain.Hexachlorophene [2,2'-methylenebis(3,4,6-trichlorophenol); HCP] has been in use as an antimicrobial agent for about 25 years, yet the primary basis of its lethal action remains uncertain. Four general mechanisms have been suggested: (i) generalized protein denaturation; (ii) membrane disruption; (iii) membrane damage leading to leakage of small cytoplasmic solutes, without membrane disruption; and (iv) respiratory inhibition. The first three of these mechanisms are known consequences of HCP action, but only at doses greater than the minimal amount required to kill cells of a susceptible test bacterium, Bacillus megaterium (8,16,21). Consequently, these mechanisms appear secondary.The inhibition of respiration as a possible primary action was indicated because much of the HCP taken up by intact cells of B.megaterium is localized in the protoplast membrane (21) where many respiratory enzymes are located (20). Furthermore, certain dehydrogenases and cytochromes of B. subtilis, Escherichia coli, and mammalian systems are inhibited by HCP (13,14). At high concentrations, HCP is I This paper was assigned journal article no. 6075 from the Michigan Agricultural Experiment Station.an effective inhibitor of respiration in mitochondria isolated from brain or liver and, at low concentrations, apparently uncouples oxidative phosphorylation (7).Consequently, we undertook study of the effects of HCP on oxygen uptake or electron transport in intact cells, isolated membranes, and solubilized enzymes in the same bacterial system as used previously (8,16,21). The results indicated that inhibition in the membrane-bound portion of the electron transport chain accounted for the primary lethal action of the drug for the bacterium.
MATERIALS AND METHODSOrganisms and growth conditions. Cells of the obligately aerobic, asporogenous KM strain of B. megaterium were grown to the late exponential phase in 2% Oxoid peptone broth (Flow Laboratories, Rockville, Md.) at 30 C, as previously described (8). For membrane isolations, 10-liter lots of the broth containing 0.3 g of Dow antifoam per liter were used in a fermentor (model MF-14; New Brunswick Scientific Co.). The fermentor was operated at 30 C with an aeration rate of 10 liter/min and a stirring rate of 250 rpm. Where indicated, the medium was buffered at pH 7.0 with 0.01 M sodium potassium phosphate buffer; otherwise, the pH was periodically adjusted to 7.0 with concentrated HCl during growth.
