Inhibitory effect of polymyxin B on prodigiosin biosynthesis in Serratia marcescens.

Sir: Prodigiosin or prodigiosin-like antibiotic pigments are tripyrrole compounds synthesized by Serratia marcescens, Vibrio psychroerythrus, Pseudomonas magnesorubra and Alteromonas rubra as well as several genera of actinomycetes.1.2) Although the structures of several of the pigments are known, the site of localization and biosynthesis is less than clear.3,4) Studies of inhibition of prodigiosin formation in nonproliferating cells of S. marcescens by certain antibiotics and antimetabolites indicated that macromolecular synthesis might be involved in the biosynthesis.5,6) These macromolecules might serve as binding sites for the prodigiosin molecules or their intermediates.6,7) The fact that pigment formation was more sensitive to inhibition than growth of cells led to the suggestion that prodigiosin biosynthesis might involve pathways other than those primarily required for cellular growth.8,9) In the course of our studies on the effect of polymyxin B on outer membranes of S. marcescens,10.11,12) We observed that polymyxin B exerted an inhibitory effect on prodigiosin biosynthesis without disturbing cellular growth. S. marcescens 08, a chromogenic polymyxin B resistant strain (MIC> 1,000 pg/ml) was used in our studies. Inhibition of pigment formation was monitored by both the agar-diffusion technique and spectrophotometric analysis. S. marcescens 08 was cultured in 10 ml of Nutrient Broth (Difco) at pH 7 and incubated at room temperature for 8 hours. An aliquot of 0.1 ml of the bacterial suspension was spread on each of the Trypticase Soy Agar (TSA) plate at pH 7. To the sterilized disc blands (6.5 mm in diameter), concentrations of 5,000, 500, 400, 300, 250, 150, 100, 50, 25, 12.5 and 0 pg/ml of polymyxin B sulfate (Aerosporin, Burrough Wellcome) were added. The antibiotic discs were placed on the surface of the seeded agar plates. Each concentration of polymyxin B was carried out in quadruplicates. The plates were then incubated at room temperature for 48 hours. Diameters of pigment inhibition zones were measured and expressed to the nearest mm. In order to quantify the actual reduction of prodigiosin biosynthesis S. marcescens 08 was grown in the presence of 12.5 pg/ml of polymyxin B (the MIC at which pigment was inhibited). Four hundred milliliters of a 48-hour culture (in light) were harvested and extracted with acetone, followed by partition with petroleum ether according to the method of WILLIAMS, et a1.13) The water-free petroleum ether extract was evaporated to dryness and reconstituted in 5 ml of dichloroethane for spectrophotometric analysis in a Beckman ACTA VI Spectrophotometer at 537 nm.