Chemical fractionation of phosphorus-32 labelled cells of Micrococcus lysodeikticus treated with chlorhexidine

Abstract

A method for the chemical fractionation of Micrococcus lysodeikticus into "cold trichloroacetic acid soluble", alcohol soluble, ribonucleic acid and residual fractions is described. The results of applying this method to untreated and chlorhexidinetreated 32P labelled cells are discussed. In untreated cells the 3zP content of the "cold trichloroacetic acid soluble" fraction, which constitutes the metabolic pool, increases at a rate dependent on temperature increase and there is a corresponding decrease in the a2P content of the ribonucleic acid fraction. At the highest temperature, 40", the increase of 32P in the pool is followed by a decrease due to leakage from the pool. f i e release of 32P from chlorhexidine-treated cells maintained at l o , 20", 30" and 40" has been measured after 0.5, 2 and 13 hr. There is an initial rapid release which is entirely from the "cold trichloroacetic acid soluble" fraction and a slower secondary release from the ribonucleic acid fraction. This secondary release is almost completely inhibited at 1" and by high (64 and 128 pglml) chlorhexidine concentrations.HE release of radioactive compounds from phosphorus-32 labelled T cells of Micrococcus lysodeikticus treated with chlorhexidine occurs in two stages. The initial rapid release (1-2 hr) has been attributed to cell membrane damage allowing the leakage of small molecules from the cells, and the slower secondary release to autolytic breakdown in the damaged cells (Rye & Wiseman, 1964). The antibacterial action of chlorhexidine has now been further investigated by studying the effect of temperature on the patterns of release and by a chemical fractionation of untreated and chlorhexidine-treated cells. Experiment a1The materials, conditions of culture and harvesting were as previously described (Rye & Wiseman, 1964) except that the culture medium used was 1% tryptone agar containing 0-.5pc/ml of sodium p h~s p h a t e -~~P .Suspending medium. ~/ 1 5 phosphate buffer pH 7.2 or MI40 trishydroxymethylaminomethane (Tris)-HC1 buffer pH 7.2.Reaction mixtures. Bacterial suspensions and equal volumes of buffer or chlorhexidine solutions were maintained at the required temperature for 1 hr before mixing. The final cell concentrations were 1.5 x 10lO/ml. Materials. CHEMICAL FRACTIONATION OF CELLS AND MEASUREMENT OF RADIOACTIVITY 1.Release of 32P into the suspending medium. 4 ml samples from each reaction mixture were taken at various time intervals and centrifuged at 5000 rpm for 5 min. The supernatant fluids were removed, recentrifuged and retained.

The Comparative Effect of Chlorhexidine and Cetrimonium Bromide on Erythrocyte Membranes

“…In isotonic media the addition of chlorhexidine in amounts less than 30 nmol per 107 cells gives a haemolysis, which follows a distinct biphasic pattern. Initially a rapid release of haemoglobin is observed followed by a secondary phase of very slow release, a phenomenon similar to that described for other types of cells (HUGO & LONGWORTH 1964& 1966RYE & WISEMAN 1964ELFERINK & BOOIJ 1974). The presence of higher concentrations of chlorhexidine i. e. more than 30 nmol per 107 cells provokes an increase of the secondary release leading to a confluence of the two phases.…”

“…Chlorhexidine (chlorhexidinum (NFN): 1,l'-hexamethylene bis (5-(4-chloro-pheny1)biguanide)) has been shown to exert concentration dependent effects on the plasma membrane of some bacteria (HUGO & LONGWORTH 1964& 1966RYE & WISEMAN 1964 and yeast cells (ELFERINK & B~I J 1974) causing leakage of cytoplasmic constituents. It has also been shown that chlorhexidine affects the membranes and membrane-bound enzyme activities of microsomes, mitochondria, lysosomes and peroxisomes (CHRIS- TENSEN & JENSEN 1974;CHRISTENSEN ef a/.…”

The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic constituents, dehydrogenase activity and cell walls of Escherichia coli and Staphylococcus aureus

“…This initial leakage is followed, in the presence of low concentrations of chlorhexidine, by a secondary release. In the presence of high concentrations of the drug this secondary release is inhibited (Hugo & Longworth, 1964a;Rye & Wiseman, 1965). Mean single survivor time data indicate that chlorhexidine is more effective as a bactericide at concentrations which inhibit the secondary release (Hugo & Longworth, 1964a).…”

The Effect of Chlorhexidine on some Enzyme Activities of Rat Liver Lysosomes and Peroxisomes

“…It has also been shown that chlorhexidine affects the plasma mem-brane of some bacteria making them permeable to ions etc. (HUGO & LONG-WORTH 1964RYE & WISEMAN 1964& 1965. We therefore found it of interest to examine whether chlorhexidine also influences other lipid membranes and the present paper reports on findings concerning the effect of chlorhexidine on rat liver lysosomes and peroxisomes and their associated activities.…”