Enhancement of plasmid curing by 9-aminoacridine and two phenothiazines in the presence of proton pump inhibitor 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone

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Abstract

Plasmid-containing bacteria often cause serious therapeutic failure during the treatment of infectious diseases. The selection of resistant-mutant strains and the transfer of mobile genetic determinants (such as plasmids and transposons) of resistance promote increased antibiotic resistance. In the last 30 years the antiplasmid effect of acridine dyes, ethidium bromide, sodium dodecyl sulphate and phenothiazines was described. The main aim of this study was to test the mechanism of the antiplasmid effect of promethazine and 9-aminoacridine on doxycycline-resistant enteric bacteria. The antiplasmid effects of promethazine and 9-aminoacridine were studied on plasmid elimination of native plasmid DNA and plasmid DNA isolated from drug-treated cells of plasmid-containing Escherichia coli, Citrobacter freundii and Enterobacter cloacae. The effects of some phenothiazines on plasmid profiles of bacterial strains isolated from urinary tract infections were analysed by agarose gel electrophoresis. Various complex of plasmid DNA were identified in the presence of promethazine, trifluoperazine and 9-aminoacridine in the agarose gel electrophoresis. Doxycycline resistance of tested enteric bacteria was the target of ‘curing’ in the presence of promethazine and trifluoperazine. The frequency of elimination of tetracycline resistance was low despite the formation of antiplasmid compounds complex with isolated plasmid DNA. Tetracycline resistance plasmid was isolated and re-transformed. The plasmid curing effects of promethazine, trifluoperazine and 9-aminoacridine were increased in the presence of a trifluoroketone proton pump inhibitor on E. coli K12 LE140 strain in a model experiment. We propose that the inefficient penetration of antiplasmid compounds could be responsible for the weak plasmid-curing effect in some clinical isolates and that membrane active, calmodulin- and proton pump inhibitors may be combined for plasmid curing in antibiotic-resistant bacteria.

Introduction

Multiple antibiotic resistance to useful classes of antibiotics often causes serious therapeutic failure in the treatment of infectious diseases. The selection of resistant mutant strains from the patient's own flora during antibiotic treatment or the transfer between bacteria of mobile genetic determinants of resistance (plasmids and transposons) promote antibiotic resistance [1], [2].

Bacterial plasmids can be eliminated from a portion of the population but could not be totally eradicated. In the last 30 years the antiplasmid effect of acridine orange, ethidium bromide, sodium dodecyl sulphate and phenothiazines has been described [3]. The F'lac plasmid of Escherichia coli K12 LE140 has served as a convenient model for studying the plasmid curing effect. It has been shown that promethazine is effective in the elimination of different plasmids [4], [5], [6], whereas other phenothiazine derivatives and benz(c)acridines showed antibacterial activity only. Whereas some amino-acridines have more active antibacterial effect than imino-acridines, imino-acridines have a more powerful antiplasmid effect than the amino-derivatives [7], [8], [9], [10], [11].

The tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor site. Tetracyclines are broad-spectrum agents, exhibiting activity against a wide range of gram-positive, gram-negative bacteria, atypical organisms such as chlamydia, mycoplasmas, rickettsiae, and protozoan parasites. The increasing incidence of bacterial resistance to tetracyclines has resulted in efforts to establish the mechanisms by which genetic determinants of resistance are transferred between bacteria [12].

The main aim of our experiments is to test the mechanism of the antiplasmid effects of promethazine and trifluoperazine on doxycycline-resistant enteric bacteria such as E. coli, Citrobacter freundii and Enterobacter cloacae strains isolated from urinary tract infections. A correlation between the chemical structure and the antiplasmid effect of some tricyclic compounds and the co-operative effect of proton pump inhibitor on the plasmid curing effect of promethazine, 9-aminoacridine and trifluoperazine were also studied. The plasmid curing effect of promethazine, trifluoperazine and 9-aminoacridine in the presence of a trifluoroketone, an inhibitor of the E. coli K12 LE140 proton pump [13], [14], [15] received special consideration.

Section snippets

Materials

Pipolphen®(promethazine) [PMZ] (EGIS Pharmaceutical Company, Budapest, Hungary), 9-Aminoacridine hydrochloride monohydrate, 98% (9AA) (Aldrich-Chemie, D-7924 Steinheim), trifluoperazine dihydrochloride (TFP) (SIGMA Chemical Co., St.Louis, USA), doxycycline (Chinoin Pharmaceutical Company, Budapest, Hungary), 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18) were kindly provided by Professor Masami Kawase (Faculty of Pharmaceutical Sciences, Josai University, Japan)

Bacterial strains

The following bacterial

Results

Ten clinical isolates were investigated for elimination of tetracycline resistance. These bacterial strains were grown in the presence of various concentrations of promethazine and trifluoperazine. After a 24 h incubation the MIC values were obtained (Table 1). The samples showing growth at concentrations below the MIC (sub-MIC) were tested for Tcr plasmid containing and Tcr plasmidless bacteria by plating them onto MTY nutrient agar. Following 24 h of incubation the colonies from master plates

Discussion

The widespread use of tetracyclines has led to increased frequency of resistance of pathogenic bacteria by genetic acquisition of tet genes [20]. All tet efflux genes code for membrane-associated proteins that export tetracycline from the cell by a proton pump that extrudes the drug against a concentration gradient. The efflux genes of gram-negative bacteria are normally associated with large plasmids most of which are conjugative [12], [21].

The plasmid curing activity of promethazine and

Acknowledgements

This study was supported by COST Action B16 and the Ph.D. programme of SZTE Medical Microbiology.

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