Triclosan inhibition of fatty acid synthesis and its effect on growth of Escherichia coli and Pseudomonas aeruginosa

Margarita Gomez Escalada1, J. L. Harwood2, J.-Y. Maillard1,* and D. Ochs3

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


* Corresponding author. Tel: +44-29-2087-9088; Fax: +44-29-2087-4149; Email: maillardj{at}cardiff.ac.uk

Received 26 November 2004; returned 12 January 2005; revised 7 February 2005; accepted 15 March 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To assess the effect of triclosan on fatty acid synthesis and to relate the inhibition of enoyl reductase to bacterial viability.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the discovery of the inhibition of enoyl reductase (FabI) by triclosan in Escherichia coli,1 this bisphenol has been found to inhibit the enzyme in other microorganisms such as Pseudomonas aeruginosa2 and Staphylococcus aureus.3,4 The FabI family of enoyl-ACP reductases forms non-covalent, high-affinity ternary complexes with triclosan and NAD(P)+, thus preventing the enzyme from participating in the biosynthetic pathway.5 This inhibition is achieved by triclosan binding to the enoyl reductase active site at a site adjacent to the nicotinamide ring of the nucleoside co-factor. Triclosan's phenol ring forms a face-to-face interaction with the nicotinamide ring, allowing extensive interaction.6

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test organisms

The triclosan-susceptible E. coli ATCC 8739 (MIC{approx}0.1 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Uptake of acetate onto the cell

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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Figure 1. Uptake of radioactive acetate into the bacterial strains studied. Circles, E. coli TM0; squares, E. coli TM3; triangles, P. aeruginosa. Each plot indicates the mean ± SD of three repeats.

 
Triclosan inhibition of fatty acid biosynthesis and its effect on cell viability

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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Figure 2. Uptake of radioactivity into total lipid in E. coli TM0. Average of three repeats in viable counts (solid lines) and average of two repeats in [1-14C]acetate incorporation into lipid (broken lines) (error bars indicating SD have been removed for clarity). Triclosan concentrations: controls (triclosan absent), squares; 0.1 mg/L, triangles; 1.0 mg/L, circles.

 


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Figure 4. Uptake of radioactivity into total lipid in P. aeruginosa. Average of three repeats in viable counts (solid lines) and average of two repeats in [1-14C]acetate incorporation into lipid (broken lines) (error bars indicating standard deviation have been removed for clarity). Triclosan concentrations: controls (triclosan absent), squares; 20 mg/L, triangles; 40 mg/L, circles.

 
In the parent strain E. coli TM0 (Figure 2), the incorporation of radioactivity into total lipids in the absence of triclosan rose steadily throughout the period of the experiment. At the same time, the number of bacteria increased from 4.91 x 104 to 1.14 x 105 cfu/mL. Triclosan at 0.1 mg/L prevented growth and reduced radiolabel incorporation into lipids by ~70% within a 75 min period. After 75 min, unlike cultures in the absence of triclosan, there was no further labelling of lipids. Triclosan 1 mg/L both prevented lipid labelling and inhibited bacterial growth (Figure 2).

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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Figure 3. Uptake of radioactivity into total lipid in E. coli TM3. Average of three repeats in viable counts (solid lines) and average of two repeats in [1-14C]acetate incorporation into lipid (broken lines) (error bars indicating standard deviation have been removed for clarity). Triclosan concentrations: controls (triclosan absent), squares; 20 mg/L, triangles; 40 mg/L, circles.

 
For P. aeruginosa, high concentrations of triclosan were also used. Although the cultures without triclosan showed significant growth and continuous incorporation of radioactivity into lipid (Figure 4), both 20 and 40 mg/L prevented bacterial growth. At 20 mg/L triclosan, lipid labelling was poor but then increased between 50 and 75 min, after which it plateaued. This period was just before cell division appeared to take place in control cultures. Even so, at 75 min lipid labelling of 20 mg/L triclosan-treated cultures was half of controls. Triclosan at 40 mg/L was a strong inhibitor of both lipid labelling in, and growth of, P. aeruginosa.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Triclosan has been shown previously to inhibit fatty acid synthesis.1,3,9 This was confirmed in the present study by the inhibition of incorporation of radioactivity from [1-14C]acetate into lipid.

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.


    Acknowledgements
 
It is the policy of this journal not to add a deceased contributor in the author list. We want to acknowledge the contribution of Professor A. D. Russell to this manuscript. Sadly, Professor Russell passed away in September 2004. We would like to thank all the members of the lipid biochemistry group at Cardiff University, particularly Darren Baker, Janis Weeks and Irina Guschina, for their help during the course of this work. We would also like to thank Ciba Specialty Chemicals for their financial support, in the form of a research studentship to M.G.E.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . McMurry LM, Oethinger M, Levy SB. Triclosan targets lipid synthesis. Nature 1998; 394: 531–2.[CrossRef][ISI][Medline]

2 . Hoang T, Schweizer HP. Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (Fab I): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. J Bacteriol 1999; 181: 5489–97.[Abstract/Free Full Text]

3 . Heath RJ, Li J, Roland GE et al. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by Triclosan and hexachlorophene. J Biol Chem 2000; 275: 4654–9.[Abstract/Free Full Text]

4 . Slater-Radosti C, Van Aller G, Greenwood R et al. Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus. J Antimicrob Chemother 2001; 48: 1–6.[Abstract/Free Full Text]

5 . Heath RJ, Su N, Murphy CK et al. The enoyl-[acyl carrier protein] reductases, FabI and FabL, from Bacillus subtilis. J Biol Chem 2000; 275: 40128–33.[Abstract/Free Full Text]

6 . Levy CW, Roujeinikova A, Sedelnikova S et al. Molecular basis of triclosan activity. Nature 1999; 398: 384–5.

7 . Heath RJ, Rock CO. A triclosan-resistant bacterial enzyme. Nature 2000; 406: 145–6.[CrossRef][ISI][Medline]

8 . McMurry LM, McDermott P, Levy SB. Genetic evidence that InhA of Mycobacterium smegmatis is a target for Triclosan. Antimicrob Agents Chemother 1999; 43: 711–3.[Abstract/Free Full Text]

9 . Bhat GP, Surolia N. Triclosan and fatty acid biosynthesis in Plasmodium falciparum: New weapon for an old enemy. J Biosci 2001; 26: 1–3.[ISI][Medline]

10 . Surolia N, Surolia A. Triclosan offers protection against blood stages of malaria by inhibiting enoyl ACP reductase of Plasmodium falciparum. Nat Med 2001; 7: 167–73.[CrossRef][ISI][Medline]

11 . McLeod R, Muench SP, Rafferty JB et al. Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of Apicomplexan Fab I. Int J Parasitol 2001; 31: 109–13.[CrossRef][ISI][Medline]

12 . Heath RJ, White SW, Rock CO. Inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics. Appl Microbiol Biotechnol 2002; 50: 695–703.

13 . Liu B, Wang Y, Fillgrove KL et al. Triclosan inhibits enoyl-reductase of type I fatty acid synthase in vitro and is cytotoxic to MCF-7 and SKBr-3 breast cancer cells. Cancer Chemother Pharmacol 2002; 49: 187–93.[CrossRef][ISI][Medline]

14 . Maillard J.-Y. Bacterial target sites for biocide action. J Appl Microbiol 2002; 92: 16S–27S.[CrossRef][ISI][Medline]

15 . Russell AD. Similarities and differences in the responses of microorganisms to biocides. J Antimicrob Chemother 2003; 52: 750–63.[Abstract/Free Full Text]

16 . Garbus J, DeLuca HF, Loomans ME et al. The rapid incorporation of phosphate into mitochondrial lipids. J Biol Chem 1963; 238: 59–63.[Free Full Text]

17 . Smith KL, Douce R, Harwood JL. Phospholipid metabolism in the brown alga, Fucus serratus. Phytochem 1982; 21: 569–73.[CrossRef]

18 . CEN (Comité Européen de Normalisation, European Committee for Standardization). British Standard EN 1499: Chemical Disinfectants and Antiseptics—Hygienic handwash—Test Method and Requirements (phase 2, step 2). CEN, Brussels, 1977.

19 . Maillard J.-Y, Messager S, Veillon R. Antimicrobial efficacy of biocides tested on skin using an ex-vivo test. J Hosp Infect 1998; 40: 313–23.[CrossRef][ISI][Medline]

20 . Chuanchuen R, Karkhoff-Schweizer RR, Schweizer HP. High level triclosan resistance in Pseudomonas aeruginosa is solely due to efflux. Am J Infect Control 2003; 31: 124.[CrossRef][ISI][Medline]





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