The effects of chlorpromazine on the outer cell wall of Salmonella typhimurium in ensuring resistance to the drug

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Abstract

Chlorpromazine (CPZ), a compound employed for the management of psychosis, has a wide ranging antibacterial activity. The growth of Salmonella typhimurium, while highly resistant to CPZ (MIC >100 mg/l), was initially inhibited during the first 8–16 h of exposure to concentrations of CPZ below the MIC. During this period of transient susceptibility, the distribution of ribosomes was markedly altered in a concentration dependent manner; the rough cell wall was transformed into a smooth form. The protein composition of the outer cell wall of 55 kDa was markedly decreased, whilst there was an increased number of high molecular weight proteins. After 16 h of exposure to sub-MIC levels of CPZ, the inhibitory effect of the drug was no longer apparent whereas the effects noted on the cell wall were retained. These Salmonella were, as the control, agglutinated by O antigen specific antibody. Whereas agglutination of the control Salmonella was blocked by the presence of CPZ at concentrations that induced the cell-wall effects, agglutination of CPZ exposed-Salmonella for periods in excess of 16 h was not blocked by any concentration of CPZ. These results suggested that eventual resistance to CPZ was dependent upon changes induced by CPZ at the cell wall level. The results also suggested that the CPZ binds to the 55 kDa protein and that such binding interfered with the recognition of the O antigen by antibody.

Introduction

Chlorpromazine has antimicrobial activity against a wide range of microorganisms [1]. Determination of the susceptibility of Salmonella spp. by classical methods (18–24 h cultures) to CPZ indicates that the organism is very resistant; concentrations of >100 mg/l are required to inhibit complete growth. Continuous optical monitoring of Salmonella cultures containing sub-inhibitory levels of CPZ ranging up to 100 mg/l indicate that the growth of the organism is markedly affected during the first 8–16 h of exposure (unpublished observations). As CPZ affects the ultrastructure of the cell wall of susceptible bacteria [2], the effect on the cell wall of Salmonella typhimurium exposed to different sub-MIC levels of CPZ was studied using electron microscopy, electrophoretic analyses of outer cell wall proteins, and agglutination that involves the outer cell wall constituents. Changes during the period of transient susceptibility were noted.

Section snippets

Materials and methods

The organism studied was Salmonella typhimurium (ATTC # 14028 obtained from Difco, USA). The cultures were prepared in Trypticase Soy Broth (TSB) to an optical density of 0.5 at 578 nm.

Electron microscopy

The organism was grown overnight in TSB and when the cultures reached an OD of 0.5 (at 578 nm), aliquots of 100 μl were transferred to 10 ml tubes containing 0.85% saline. From the saline-containing tubes, 100 μl were transferred to separate quadruplicate sets of 50 ml TSB tubes containing varying concentrations of chlorpromazine. The cultures were incubated at 37°C and the OD determined at 4, 8, 12, 16, 20 and 24 h.

The minimum inhibitory concentration (MIC) of CPZ was taken as the

Results

The effects of various concentrations of CPZ during a 24 h culture is presented in Fig. 1. Concentrations >75 mg/l significantly inhibit the growth of S. typhimurium during the first 8–16 h of exposure; after which time, the inhibitory effects were gradually lost. Thin layer chromatography of the supernatant of all CPZ containing cultures indicated no degradation of CPZ during the culture periods up to 48 h (data not shown).

The effects of CPZ concentrations on the ultrastructure of Salmonella

Discussion

Chlorpromazine was found to have an inhibitory effect on the growth of S. typhimurium during the first 8–16 h of culture, after which time the organism became resistant to the presence of very high concentrations of drug. The transient effect was preceeded by changes in the distribution of ribosomes, the cell wall and outer cell wall composition. The distribution of ribosomes became highly heterogenous, the cell wall lost its rough texture appearance and the major 55 kDa protein of the outer

Acknowledgements

We thank Tom Bergan (Oslo, Norway) for his valuable comments and advice.

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