1 Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF; 2 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3TL, UK; 3 Ciba Specialty Chemicals Inc., PO Box 1266, D-79630 Grenzach-Wyhlen, Germany
Received 26 November 2004; returned 12 January 2005; revised 7 February 2005; accepted 15 March 2005
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Abstract |
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Methods: The effect of triclosan on fatty acid synthesis in a triclosan-resistant Escherichia coli and its sensitive counterpart and in Pseudomonas aeruginosa was investigated by measuring acetate incorporation into total lipid followed by analysis of fatty acid methyl esters by gas chromatography. Concurrently, the bactericidal effect of triclosan against these bacterial strains was assessed.
Results: Triclosan inhibited fatty acid biosynthesis in all the strains tested. However, for triclosan-resistant E. coli (MIC > 1000 mg/L) the concentration required to achieve inhibition was higher than that required for the susceptible counterpart. These concentrations did not significantly affect cell survival in any of the strains tested.
Conclusions: This study shows that the inhibition of fatty acid biosynthesis by the bisphenol might be involved in its growth-inhibitory action and that other mechanisms are involved in its lethal effect. In addition, although microorganisms with a high triclosan MIC were still susceptible to the inhibitory effect of the bisphenol on fatty acid biosynthesis, a higher concentration of the compound was required. This suggested that triclosan bioavailability was different in these strains.
Keywords: enoyl reductase , mechanism of action , bacteriostatic , bactericidal
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Introduction |
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Following the identification of triclosan's inhibition of enoyl reductase, other genes coding for enoyl reductases have been identified, namely fabK in Streptococcus pneumoniae and P. aeruginosa,7 fabL in Bacillus subtilis,5 and InhA in Mycobacterium tuberculosis and Mycobacterium smegmatis.8 While InhA is susceptible to triclosan,8 both FabK and FabL are resistant.5,7
Such inhibition of fatty acid synthesis by triclosan was also identified in higher organisms such as Plasmodium falciparum,9,10 the protozoan that causes malaria, and Toxoplasma gondii.11 Both these organisms, as members of the apicomplexan, contain a type II fatty acid synthase.12 More recently, triclosan was reported to inhibit a type I fatty acid synthase (a multifunctional form characteristic of mammals) in breast cancer cells,13 although generally such enzymes are thought to be insensitive.
Biocides have usually been described to possess multiple target sites in microbial cells, the inhibition or destruction of which results in bacteriostatic and/or bactericidal effects. Triclosan is particularly unique, as at low concentrations the bisphenol has been considered to have a single target site in several microorganisms.14 This has led to controversies as to whether such a selective mode of action of triclosan could lead to the emergence of bacteria resistant to the bisphenol and other antimicrobials by modification of the enoyl reductase enzyme.
The aim of this investigation was to assess the effect of triclosan on fatty acid synthesis in triclosan-susceptible, -resistant and -non-susceptible bacterial strains, and to attempt to relate the inhibition of enoyl reductase to bacterial viability.
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Materials and methods |
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The triclosan-susceptible E. coli ATCC 8739 (MIC0.1 mg/L; subsequently referred to as E. coli TM0) and its tolerant counterpart (MIC > 1000 mg/L; subsequently referred to as E. coli TM3, courtesy of Dr G. McDonnell, Steris Corporation, Cleveland, OH, USA), and the triclosan-non-susceptible P. aeruginosa ATCC 1544215
were used in this study. E. coli TM3 was obtained by serially subculturing E. coli TM0 in increasing concentrations of triclosan (G. McDonnell, K. Haines, D. Klein & D. Pretzer, personal communication).
All strains were grown at 37°C in M9 minimal media (Sigma, Poole, UK) prepared with added MgSO4 (Fisher Chemicals, Loughborough, UK) and D-glucose (BDH Chemicals, Poole, UK). Stock cultures were kept in tryptone soya agar (TSA; Oxoid, Basingstoke, UK) at 4°C and were renewed every fortnight. Two sequential subcultures in M9 were carried out prior to any experiment.
Inhibition of fatty acid synthesis
Five millilitres of an overnight culture (containing 1 x 108 to 5 x 108 cfu/mL) in M9 were added to 5 mL of fresh pre-warmed media at 37°C containing 20 µL of [1-14C]acetate (specific activity of 2.15 GBq/mmol; Amersham International, Amersham, UK) and different concentrations of triclosan (Ciba Specialty Chemicals, Grenzach-Wyhlen, Germany). The bisphenol was prepared using dimethyl sulphoxide (DMSO; Sigma) as a co-solvent to a maximum of 1% (v/v) and equivalent amounts of DMSO were added to all cultures to guard against non-specific effects. After 15 s, and every 25 min thereafter, a 1 mL sample was taken and centrifuged at 6000 rpm (3000 g) for 3 min (MSE Microcentaur, Sanyo, Japan). The supernatant was discarded and the pellet resuspended in 1 mL of distilled water. Ten microlitres of the suspension was used for scintillation counting measured in a KLB Wallac, 1209 Rackbeta liquid scintillation counter (Wallac Oy, Finland) using Optifluor (Perkin Elmer, Boston, MA, USA) as scintillation cocktail. Quench correction was made by the external standard channels ratio method as indicated in the manufacturer's manual.
Prior to the investigation of the effect of triclosan on fatty acid synthesis, the uptake of acetate into these bacteria was investigated. Samples were prepared as described above, in the absence of triclosan. After 15 s and every 25 min thereafter, samples were taken and spun down as described above. An aliquot (0.5 mL) of the supernatant was retained for scintillation counting and the cell pellet was resuspended in 1 mL of distilled water.
Lipid extraction
Lipid extraction was carried out using a method first described by Garbus and colleagues.16,17 Cell pellets were suspended in 1 mL water and 3.75 mL chloroform-methanol (1:2 v/v; both obtained from Fisher Chemicals) was added. After thorough mixing, the solution was left for 15 min at room temperature (25°C), after which 1.25 mL chloroform and 1.25 mL Garbus buffer (2 M potassium chloride in 0.5 M phosphate buffer at pH 7.4; Fisher Chemicals) were added. After thorough mixing, the suspensions were left to stand at room temperature for 15 min and then centrifuged at 1500 rpm (1000 g) for 10 min (Baird & Tatlock Auto Bench centrifuge with fixed angle rotor). The procedure provided a very efficient extraction of membrane lipids including very polar materials.16 The lower phase (containing lipids) was removed and dried under a stream of oxygen-free nitrogen gas (BOC Gases, Guildford, UK). This portion, which comprised the total lipid extract, was finally resuspended in 1 mL of chloroform. Aliquots were then taken, the chloroform evaporated and radioactivity determined by scintillation counting in Optifluor.
Bactericidal effect of triclosan
In order to assess the relationship between fatty acid synthesis and bacterial growth, cultures were prepared as described previously without the radiolabelled acetate but with the same triclosan concentrations. After 15 s and every 30 min thereafter, samples were taken and neutralized using 9 mL of the neutralizer (lecithin 3 g/L; polysorbate 80 30 g/L, L-histidine 1 g/L, sodium thiosulphate 5 g/L in tryptone soya broth; CEN 1499; 1997).18 The potential bacterial toxicity and the ability of the neutralizer to quench the activity of triclosan was tested prior to this investigation and found to be satisfactory (data not shown).19 After a minimum of 5 min contact with the neutralizer, samples were serially diluted in phosphate buffer solution (Sigma) and spotted in triplicate onto TSA as described elsewhere.18 After an overnight incubation at 37°C, spots that contained between three and 30 colonies were counted and the bacterial concentration was expressed as cfu/mL.
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Results |
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Total uptake of radioactive acetate into the three bacterial strains is shown in Figure 1. In each case uptake was initially rapid and then plateaued. The final values of incorporated acetate against the initial concentration of acetate were about 32.76%, 32.65% and 23.74% for E. coli TM0, TM3 and P. aeruginosa, respectively. All bacteria had a high value at the initial time point. Nominally this was 15 s but, because cells had to be centrifuged down from the medium the actual time was nearer 3.5 min. Such initial values (at time 15 s) indicated an early rapid flux of [1-14C]acetate, particularly in the triclosan-susceptible E. coli TM0 (Figure 1). Radiolabelled acetate was incorporated at a slower rate in the triclosan-resistant mutant TM3 over the first 60 min (Figure 1).
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The range of triclosan concentrations investigated here depended upon the bacterial cells. For the triclosan-susceptible E. coli TM0, concentrations of 0, 0.1 and 1 mg/L were investigated, whereas concentrations of 0, 20 and 40 mg/L were used for the other two strains.
Figures 24 present viable counts and [1-14C]acetate incorporation into lipid depending upon triclosan concentration for the three bacterial strains studied. It was clear that triclosan inhibited the incorporation of radioactive acetate into lipid in a concentration-dependent manner in all the microorganisms tested.
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The triclosan-resistant E. coli TM3 showed a similar population growth pattern (Figure 3) to the parent strain. Incorporation of radioactivity into lipids was also broadly comparable. Much higher concentrations of triclosan were used and growth was prevented at both 20 and 40 mg/L. At 20 mg/L triclosan, lipid labelling was reduced by 55% and, in a similar fashion to the intermediate concentration used from E. coli TM0 (Figure 2) after 100 min labelling appeared to plateau. The highest concentration of triclosan (40 mg/L) effectively prevented lipid labelling and bacterial growth.
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Discussion |
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In all of the bacterial strains tested it was possible to inhibit lipid labelling completely. However, much higher concentrations of triclosan were needed to produce such inhibition in the resistant E. coli strain and in the inherently non-susceptible P. aeruginosa.
At intermediate concentrations of triclosan it was possible to achieve some lipid labelling, but in these cases there was no increase in bacterial population as measured by viable cell numbers. This suggests that, if inhibition of fatty acid synthesis is the primary site of action for triclosan, its complete inhibition does not necessarily correlate with a bactericidal effect, but rather with a bacteriostatic one. Triclosan is likely to have another important site(s) of action that causes lethality independent of the effect on fatty acid biosynthesis. Likewise, it was notable that when complete inhibition of lipid labelling was achieved following challenges with higher concentrations of the bisphenol, viable counts were not significantly reduced over the experimental period. Thus, complete inhibition of fatty acid biosynthesis, via enoyl reductase, does not immediately produce lethality. It is clear that at these concentrations, triclosan acts as a bacteriostatic agent. These experiments were conducted over a 150 min period only, but one may speculate that if lipid synthesis was prevented over a long period of time then membranes could be neither made nor renewed, and the microorganisms would eventually die.
Our data show that, provided the concentrations of triclosan are appropriate, it is possible to produce similar effects on viable counts and fatty acid synthesis in all bacteria whether they are susceptible, have acquired resistance or are inherently non-susceptible. As others have noted,7,20 resistance to high concentrations of triclosan could be associated with the properties of enoyl reductase, but also, and more probably, by a reduction in the effective intracellular concentration of triclosan. Our results clearly showed that the bacteriostatic activity of triclosan is associated with an inhibition of membrane biogenesis. However, the inhibition of fatty acid synthesis does not account for any short-term bactericidal activity. These results might question the use of enoyl acyl reductase as a target for non-biocidal antimicrobial design.
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Acknowledgements |
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