The antibacterial activity of triclosan-impregnated storage boxes against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Shewanella putrefaciens in conditions simulating domestic use

Josephine J. Braid and Martin C. J. Wale,*

Department of Microbiology & PHLS Antimicrobial Susceptibility Surveillance Unit, Queens Medical Centre, Nottingham NG7 2UH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antimicrobial resistance has increased over the past decade causing concern for public health. Domestic antimicrobial products containing triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether), a broad-spectrum antibacterial agent, were introduced in 1997 and have become popular among consumers. Cross-resistance to other antibacterial agents has been suggested as a possible consequence of their widespread use. Triclosan-impregnated plastic storage boxes were tested for activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Shewanella putrefaciens in various conditions, including some designed to simulate usual storage conditions. Results showed inhibition up to a factor of 106 of bacteria grown in direct contact with triclosan-impregnated plastic at 30 and 22°C, but not at 4°C. Triclosan resistance was not found to increase after repeated exposure in triclosan-impregnated boxes. Further investigation into the effect of triclosan-impregnated products on bacteria will increase understanding of domestic antimicrobial products and implications of their overuse.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The past decade has seen an increase in bacteria resistant to multiple antimicrobial agents, giving rise to growing concern for public health. Organisms insusceptible to over 10 different antibiotics or chemotherapeutic agents are not unusual.1 Causes may include the widespread, often inappropriate, use of antibacterial agents, especially broad-spectrum drugs, and incomplete compliance with basic infection control practices such as handwashing.2 In hospitals, antimicrobial resistance leads to longer stays and hence greater morbidity and mortality, and requires the use of more expensive drugs. In the community, resistant organisms cause both sporadic infections and outbreaks, and resistance is now common in organisms such as Salmonella spp.3 Almost 50% of antimicrobial usage in the developed world, possibly more in the developing world, is thought to be unnecessary, with agricultural use being a notable contributor.4 In the light of growing public awareness and fear of communicable infections, commercial interests have identified an opportunity for sales of household goods with antibacterial properties. There are suggestions that overuse of these products may lead to more widespread resistance to therapeutic antibacterial agents.5

Since the launch in 1997 of the triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether)-impregnated Microban range in Sainsbury's supermarkets, the availability in the UK of products claiming antibacterial protection has increased rapidly. In the USA, a similar trend has been driven by increased public awareness and fear of microbial infections.5 A wide range of domestic products incorporating these agents is now available, including dishcloths, food boxes, toothbrushes, washing-up liquid and hand-washing gels. Manufacturers claim these products give ‘permanent protection against bacteria’.6 However, there is little independent scientific evidence of either efficacy or possible adverse effects.

Triclosan is a bisphenol widely used for antisepsis and in anti-plaque agents.7 Triclosan is particularly active against Gram-positive bacteria. Its efficacy against Gram-negative bacteria and yeasts can be enhanced by formulation.7 The patent for triclosan-impregnated plastic reveals that the concentration of triclosan ranges from c. 0.1% to 5% by weight of the plastic composite.8 Triclosan inhibits bacterial fatty acid synthesis at the enoyl–acyl carrier protein reductase (EACPR) (FabI) step.9 The EACPR catalyses the final regulatory step in the fatty acid synthase cycle: the reduction of a carbon–carbon double bond in an enoyl moiety that is covalently linked to an acyl carrier protein.10 The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to the EACPR.11 The EACPR is also the target for the antituberculosis drug isoniazid,12 and there is evidence of crossresistance between triclosan and isoniazid.13

The fatty acid biosynthetic (Fab) pathway is an excellent target for antibacterial agents. It plays a pivotal role in providing metabolic precursors for several important cellular functions, including cell wall biogenesis (phospholipids, lipopolysaccharides and lipoproteins) and the synthesis of acylated homoserine lactones required for virulence factor gene expression.14

Identification of a ternary FabI–NAD+–triclosan complex reinforces the conclusion that FabI is a specific intracellular target for triclosan.9 The finding that triclosan permeabilizes the bacterial envelope may have encouraged the widespread and increasing use of triclosan in consumer personal care products, based on the reasoning that bacteria would not acquire resistance to a non-specific membrane disrupter. But agents that block fatty acid biosynthesis disturb membrane assembly by stopping phospholipid production, hence evidence that triclosan has non-specific membrane effects is insufficient. McMurry et al.15 found evidence that triclosan has a specific cellular target: triclosan-resistant strains of Escherichia coli have missense mutations in the FabI gene, and the reported effects of triclosan on membrane structure and function result from its specific inhibition of fatty acid biosynthesis at the FabI step.

The ability of E. coli to acquire genetic resistance to triclosan and related compounds through mutations in the FabI gene indicates that the widespread use of this drug could lead to the appearance of resistant organisms.16


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Experiments were carried out in liquid phase, involving direct contact between broth and the triclosanimpregnated plastic, and in solid phase, with organisms growing on blood and nutrient agar plates in triclosan-impregnated boxes. Antimicrobial susceptibility changes were determined with Etests (AB Biodisk, Solna, Sweden) and a disc diffusion method17 (a definitive version of reference 17 has now been published, see reference 20). Five bacterial species were tested, including three clinical isolates (Staphylococcus aureus, E. coli and Pseudomonas aeruginosa) and two NCTC strains [Bacillus cereus (NCTC 7464) and Shewanella putrefaciens (NCTC 10736)]. Strains were exposed to triclosan at 30, 22 and 4°C as appropriate. Solid phase experiments were carried out on blood agar and nutrient agar, to simulate growth on meat and non-meat products, respectively.

Liquid phase experiment

The two triclosan-impregnated storage boxes each had a volume of 0.2 L. The ‘washed box’ was washed in a dishwasher and the ‘unwashed box’ was rinsed with water. A plastic box (Addis, Swansea, UK) of equal volume was used as a control for plastic in the boxes, and growth controls were grown in sterile universal bottles.

A 103 cfu/mL suspension of bacteria in 10 mL of nutrient broth was placed in the four different conditions (washed and unwashed triclosan-impregnated boxes, unimpregnated box and glass universal). An initial sample was taken to confirm the inoculum density. Samples of broth incubated at 30°C were taken after 24 h, and those incubated at 22 and 4°C at 72 h. These samples were mechanically plated out using a spiral plater, incubated overnight at 37°C, then colonies were counted using the spiral plate counter (Don Whitley Scientific Ltd, Shipley, UK). The series was repeated twice for experiments carried out at 30 and 22°C, and three times at 4°C.

Solid phase experiment

The triclosan-impregnated storage boxes had a volume of 9 L, with one washed and the other unwashed, and the plastic control box (Addis) had a 10 L volume. For control conditions the agar plate was placed in the incubator.

A 103 cfu/mL suspension of bacteria in Ringer's solution was spiral plated out on blood and nutrient agar plates. Petri dish lids were removed, and plates were left in the boxes with the box lids sealed in the four different conditions: unwashed and washed triclosan-impregnated boxes, normal box and control (not in a box). Samples grown at 30°C were left for 24 h, at 22°C for 72 h and at 4°C (S. putrefaciens only) for 10 days. Colonies were counted using the spiral plate counting machine. All experiments were repeated at least twice.

Re-exposure protocol

Re-exposure was carried out by using samples grown in unwashed triclosan-impregnated boxes as the source of the inoculum for subsequent exposures. E. coli was tested at 30, 22 and 4°C, S. aureus and B. cereus at 30°C only, and S. putrefaciens at 22°C only, with samples taken for colony counting as described above.

In the liquid phase experiment, nutrient broth from the unwashed box was used as the source of the inoculum; this was carried out two or three times (i.e. three or four exposures in total; see Table 1Go). In the solid phase experiment, the blood agar sample from the unwashed box was re-plated on to blood agar and nutrient agar, and re-exposed as described above. This was carried out 10 times at 30°C for all organisms except S. putrefaciens (i.e. 11 exposures), six times at 22°C for all organisms (seven exposures) and twice at 4°C for S. putrefaciens only (three exposures).


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Table 1. Effect of exposure to triclosan on bacteria growing in nutrient broth (liquid phase)a
 
Antimicrobial susceptibility testing

Susceptibility testing of S. aureus, E. coli and P. aeruginosa was undertaken after repeated exposure in the solid phase. Etests for methicillin, amoxicillin, cefotaxime, gentamicin, ciprofloxacin and co-trimoxazole were carried out. The susceptibility of P. aeruginosa to ceftazidime was tested using the BSAC standardized disc sensitivity testing method.17


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A difference in viable count of <10-fold was regarded as within experimental limits and thus not significant.

Liquid phase

Growth in liquid phase experiments, where the nutrient broth came into direct contact with the triclosanimpregnated plastic in a single exposure, was inhibited 105- to 107-fold at 30°C in the triclosan box for E. coli, S. aureus and B. cereus. At 22°C, 105-fold growth inhibition was found for E. coli, but not S. putrefaciens. No inhibition was seen for E. coli at 4°C. These results are summarized in Table 1Go, and the counts for E. coli are presented in Figure 1Go.



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Figure 1. E. coli counts after first exposure to triclosan in liquid phase. Temperature and incubation times as indicated. BA, blood agar; NA, nutrient agar. Symbols: {blacksquare}, unwashed box BA; , unwashed box NA; {square}, washed box BA; , washed box NA; ,normal box BA; , normal box NA; , control BA; , control NA.

 
Following repeated exposure in liquid phase, growth was inhibited by >10-fold in the triclosan boxes at 30°C for E. coli, S. aureus and B. cereus. At 22°C, growth of E. coli was inhibited 104-fold, and that of S. putrefaciens 10-fold. Repeated exposure of E. coli at 4°C produced no observed effect, and P. aeruginosa was unaffected at any temperature. The counts for E. coli are presented in Figure 2Go.



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Figure 2. E. coli counts after repeated exposure to triclosan in liquid phase. At 30 and 22°C organisms were subcultured twice in the presence of triclosan after first exposure. At 4°C subculture was carried out three times after first exposure. Temperature and incubation times as indicated. BA, blood agar; NA, nutrient agar. Symbols: {blacksquare}, unwashed box BA; , unwashed box NA; {square}, washed box BA; , washed box NA; , control BA; , control NA.

 
No significant difference was observed between growth in unwashed and washed triclosan-impregnated boxes.

Solid phase

In the solid phase experiment bacteria were grown on plates placed in a triclosan-impregnated box, thus being exposed to the atmosphere in the box rather than coming into direct contact with the triclosan-impregnated plastic. Both washed and unwashed triclosan boxes were used and compared with non-impregnated boxes and no box (growth on a plate placed directly in the incubator), and growth was compared on both nutrient agar and blood agar (to simulate non-meat and meat products, respectively).

At 22°C, 10-fold inhibition of growth was observed for S. aureus and S. putrefaciens following single exposure in triclosan boxes on nutrient agar. This difference no longer occurred following repeated exposure.

No inhibition was observed when organisms were grown on blood agar at any temperature. No inhibition was observed for any organism tested at 30°C. Growth of S. putrefaciens at 4°C was unaffected by single or repeated exposure to triclosan on either nutrient or blood agar. No difference was observed between growth in unwashed triclosan-impregnated boxes and growth after repeated dishwasher washing. These results are summarized in Table 2.Go


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Table 2. Effect of exposure to triclosan on bacteria growing in solid phasea
 
In addition, suppression of growth in the area of the plate adjacent to the triclosan-impregnated box wall was observed for S. aureus grown on blood agar plates at 30°C for 24 h (Figure 3Go). This was a consistent finding; the experiment was repeated four times.



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Figure 3. Photograph of S. aureus grown on blood agar at 30°C for 24 h, illustrating suppression of growth on the area of the plate close to the triclosan-impregnated box wall. (a) S. aureus grown in unwashed triclosan-impregnated box. (b) S. aureus grown in washed triclosan-impregnated box. (c) Control, S. aureus grown in unimpregnated box. (d) Control, S. aureus not grown in plastic box. The triclosan-impregnated box wall would have been along the left-hand side; in the 9 L boxes only one wall is impregnated.

 
Antimicrobial susceptibility testing

In all cases, repeated exposure to triclosan made no difference to susceptibility to clinically used antibacterial agents for any of the organisms tested.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The inhibitory effect of triclosan in the triclosanimpregnated plastic was temperature dependent. This was shown in both the liquid and solid phase experiments, but particularly in the liquid phase, where growth inhibition was greatest at 30 and 22°C, and not evident at 4°C. At lower temperatures, movement of triclosan molecules through the plastic, and release from it, would be expected to be slower, indicating that a lower vapour pressure would be generated, and thus less inhibition of bacterial growth would be expected. The growth rate of bacteria is also lower at lower temperatures, thus rendering them less susceptible to agents acting on cell membrane synthesis. Repeated exposure to triclosan made no significant difference to these effects.

Washing the triclosan-impregnated boxes did not alter the effects observed on microbial growth. This supports the manufacturer's claims that the effects do not diminish as the product is used. Although the boxes were not tested to the end of their ‘useful lives’, any reduction in effect would be expected to be most marked early in the life of the product.

Growth was inhibited in the triclosan-impregnated plastic in the liquid phase experiment for all bacteria except P. aeruginosa, although no inhibition was seen for E. coli at 4°C. In the solid phase experiment at 22°C, growth inhibition was observed on nutrient agar of S. aureus and S. putrefaciens following first exposure in triclosan boxes. No difference in colony counts was observed on blood agar, but the colonies tended to be smaller. Presumably, this is owing to the extra nutritive factors in blood agar. P. aeruginosa is usually resistant to triclosan, because although it expresses the target, EACPR, wild-type P. aeruginosa expresses an efflux system (the MexAB-OprM system).18

We are unable to account for the consistent finding (illustrated in Figure 3Go) of suppressed growth of S. aureus incubated at 30°C on the side of a blood agar plate directly adjacent to the triclosan-impregnated wall in the large boxes (in these boxes only one wall is impregnated). If triclosan achieves sufficient vapour pressure in a 9 L box to inhibit growth—which seems implausible—why does this not affect all the plates in the box equally and evenly?

Susceptibility testing after repeated exposure on solid media shows no evidence of increased resistance to the panel of therapeutic antibacterial agents tested. This reassuring finding indicates that the use of triclosanimpregnated products will not contribute to the burden of increasing antimicrobial resistance by affecting exposed organisms directly. However, widespread use in the domestic situation would be expected to select for different, more resistant strains, rather than more resistant variants of the same strain, in a manner similar to selection of Pseudomonas and Serratia spp. in triclosan handwashes.19 This would have a potentially serious impact, but would not be detected using the methodology described here.

In conclusion, triclosan-impregnated antibacterial storage boxes do inhibit growth of the bacteria tested at 30 and 22°C, but not at 4°C, and this effect is most marked for growth in liquids in direct contact with the plastic. The effect is similar in magnitude to that of refrigeration. Resistance to common clinically used antibacterial agents was not demonstrated after the strains tested here had been exposed to triclosan; this does not rule out selection of other resistant strains in day-to-day usage. Triclosan-impregnated storage boxes are thus potentially useful for storage of food for short periods (e.g. lunch boxes or overnight) where refrigeration is not possible. Continued research into this new area of domestic antibacterial agents and the possible implications of its widespread use in other domestic products is important.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Dagmar Biegon, Dr Suzanne Surman and Dr John Lee for their advice on technical aspects of this work. This work was funded entirely by the Department of Microbiology, Nottingham University Medical School.


    Notes
 
* Corresponding author. Tel: +44-115-970-9048; Fax: +44-115-970-9019; E-mail: mwale{at}cdsctrent.phls.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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10 . Levy, C. W., Roujeinikova, A., Sedelnikova, S., Baker, P. J., Stuitje, A. R., Slabas, A. R. et al. (1999). Molecular basis of triclosan activity. Nature 398, 383–4. [ISI][Medline]

11 . Ward, W. H. J., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E. et al. (1999). Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan. Biochemistry 38, 12514–25. [ISI][Medline]

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Received 8 March 2001; returned 15 May 2001; revised 20 August 2001; accepted 5 September 2001