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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism’s susceptibility to tea tree oil and to its normally inert hydrocarbons, p-cymene and γ-terpinene.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES

Tea tree oil, the essential oil of Melaleuca alternifolia, has long been used as a topical antiseptic and a range of medically significant bacteria and fungi are susceptible to its effects ( Markham 1999). Although the oil comprises a variable and complex mixture of over 100 components, its composition has been described as being approximately a 50/50 blend of oxygenated and nonoxygenated monoterpenes ( Brophy et al. 1989 ).

Tea tree oil and other antimicrobially active essential oils act as membrane damaging agents ( Cox et al. 1998 ; Knobloch et al. 1988 ). However, in spite of their broad-spectrum antimicrobial action these essential oils are often ineffective against some strains of Pseudomonas aeruginosa ( Carson and Riley 1994; Chand et al. 1994 ; Nguyen et al. 1994 ; Pattnaik et al. 1995 ).

This tolerance is not surprising, since Ps. aeruginosa possesses an intrinsic resistance, which is associated with the nature of its outer membrane, to a wide range of biocides ( McDonnell and Russell 1999). The outer layer of the Gram-negative outer membrane is composed primarily of lipopolysaccharide. This forms a hydrophilic permeability barrier which provides protection against the effects of toxic agents ( Nikaido and Vaara 1985). However, a wide range of polycationic substances can increase the susceptibility of different Gram-negative bacteria, including Ps. aeruginosa, to the effects of biocides by acting as outer membrane permeabilizers ( Hancock and Wong 1984; Vaara 1992).

In this study we have used the permeabilizing action of polymyxin B nonapeptide (PMBN) to examine the contribution of the outer membrane of a tea tree oil-tolerant strain of Ps. aeruginosa. PMBN interacts with the surface lipopolysaccharide layer of the Gram-negative outer membranes without releasing lipopolysaccharide or outer membrane protein. Evidence for its permeabilizing effect on the Ps. aeruginosa outer membrane has been provided by sensitization to hydrophobic antibiotics and increased uptake of hydrophobic probe molecules such as nitrocefin and N-phenylnaphthylamine ( Vaara 1992).

The antimicrobial activity of tea tree oil can be principally attributed to terpinen-4-ol, which makes up about 40% v/v of the oil ( Southwell et al. 1993 ). However, approximately 50% of the content of tea tree oil consists of monoterpenes that display low antimicrobial activities. We have also used PMBN to investigate the extent to which the inactivity of these compounds is related to an inability to penetrate the outer layer of the Gram-negative outer membrane.

MATERIALS and METHODS

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES

Organism and medium composition

Pseudomonas aeruginosa NCTC 6749 was passaged twice in iso-sensitest broth (ISB) and aliquots from overnight cultures were used as the inocula for all assays, except minimum inhibitory and minimum bactericidal concentrations (MIC/MBC), which used 5-h cultures.

Tea tree oil and monoterpenes

Tea tree oil (Main Camp batch 6801) was a gift from Main Camp Tea Tree Oil (Ballina, Australia). The oil complied with ISO 4730 (1996). The terpinen-4-ol and 1,8-cineole contents were 39·8% and 4·5%, respectively. Monoterpenes were purchased from Aldrich Chemical Company (Castle Hill, Australia).

Minimum inhibitory and minimum bactericidal concentrations

Six sets of serial doubling dilutions of tea tree oil or components were prepared in sterile tubes containing ISB (final volume of 1 ml). Inocula were prepared by diluting exponential phase cultures of Ps. aeruginosa (to give approximately 106 cfu ml−1) in either ISB only or ISB containing 10 µg ml−1 PMBN and pre-incubating for 10 min to allow absorption of PMBN. Assay tubes were inoculated with 1 ml of cell suspension to give triplicate sets of dilutions with and without PMBN. Control tubes contained no tea tree oil or monoterpene. All tubes were then incubated at 37 °C for 18 h on an orbital shaker at 200 r.p.m. and assessed visually for turbidity. The MIC was determined as the lowest concentration of the test substance in which at least two of the three tubes remained free of bacterial growth. Following this, 20 µl from all non-turbid tubes was subcultured into 2 ml ISB and incubated for 24 h at 37 °C. MBC was then determined as the minimum concentration of the test agent in which two out of three tubes demonstrated no survivor regrowth.

Viability assays

Assay flasks containing 20 ml cell suspension in ISB (approximately 5·0 × 106 cfu ml−1) and, where required, 10 µg ml−1 PMBN, were set up on magnetic stirrers at room temperature (22 °C). Flask contents were stirred slowly for 10 min to allow absorption of PMBN, after which time tea tree oil was added at the required concentration to all flasks except controls. One control contained PMBN and cells, and the other cells only. The stirring rate was then adjusted to ensure adequate mixing of the tea tree oil. The ‘cells only’ control was sampled immediately, serially diluted and plated onto tryptone soy agar (TSA; Oxoid, Basingstoke, UK) to determine cell density. After 60 min at room temperature, aliquots (1 ml) were removed and added to neutralizing broth (tryptone soy broth, 30 g l−1, Oxoid; neutralized liver digest, 30 g l−1, Oxoid; lecithin, 10 g l−1, Defiance, Sydney, Australia) which had been prewarmed to 32 °C. These were left for 10 min at room temperature, cooled in a water bath (13 ± 2 °C) and then serially diluted. Viable counts were determined after preparing TSA pour plates and incubating at 37 °C for 72 h.

The effects of p-cymene and γ-terpinene on viability were determined as described above, except that aliquots were removed from flasks for plating after 0, 30, 60 and 120 min of exposure.

RESULTS and DISCUSSION

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES

In this study the presence of the outer membrane permeabilizer, PMBN increased the antimicrobial activity of tea tree oil, γ-terpinene, p-cymene and 1,8-cineole against the normally tolerant Ps. aeruginosa strain NCTC 6749. Table 1 shows that including PMBN reduced the MIC/MBC values of tea tree oil and these monoterpenes against this strain to levels displayed by more tea tree oil-sensitive species ( Gustafson et al. 1998 ; Mann and Markham 1998). PMBN on its own had no effect on growth in control tubes (data not shown). Similarly, viability assays reveal that rate of tea tree oil-induced inactivation was more extensive in the presence of PMBN ( Fig. 1). Again, PMBN had no effect on viability in the 60-min assay period.

Table 1.  Effect of 10 µg ml−1 PMBN on the MIC/MBC of tea tree oil and some of its monoterpene components against Ps. aeruginosa NCTC 6749
MIC/MBC (v/v percentage)
Test componentNo PMBNWith PMBN (10 µg ml−1)
Tea tree oil4·0/8·00·25/0·5
γ-terpinene> 8·0/ > 8·01·0/2·0
p-cymene > 8·0/ > 8·00·5/1·0
1,8-cineole> 8·0/ > 8·00·5/0·5
image

Figure 1. Effect of 10 µg ml−1 PMBN on the viability of Ps. aeruginosa NCTC 6749 after exposure to tea tree oil for 60 min. Inoculum density was 3·7 × 106 cfu ml−1 for flasks containing PMBN and 6·5 × 106 cfu ml−1 for flasks without PMBN. Results are the mean of duplicate samplings from single flasks. (●) – tea tree oil only; (○) – tea tree oil + PMBN. There was no appreciable decline in cell numbers, in controls with and without PMBN, over the test period. Limit of detection is 10 cfu ml−1. Error bars represent standard deviations

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In the absence of PMBN, the increase in the extent of Ps. aeruginosa inactivation ( Fig. 1) was progressively reduced as tea tree oil concentration exceeded 2% v/v. For example, the extent of inactivation over a 60-min period in 10% (v/v) tea tree oil was only slightly more than 1 log unit greater than that in 2% (v/v). This is consistent with the tea tree oil-induced decline in Ps. aeruginosa viability being a diffusion-limited process. Increased monoterpene diffusion through a permeabilized outer membrane provides a simple explanation for the effect of PMBN on the extent of cell death ( Fig. 1).

In a series of disc diffusion experiments, Carson and Riley (1995) found that, of eight major components of tea tree oil tested, only terpinen-4-ol was active against Ps. aeruginosa NCTC 10622. Non-oxygenated monoterpenes had virtually no activity against any of the test organisms. Accordingly, our MIC data indicate that p-cymene, γ-terpinene and 1,8-cineole do not inhibit Ps. aeruginosa even at 8% (v/v), the maximum concentration tested. However, in the presence of PMBN, the MIC values of these compounds fell to levels that are similar to that of terpinen-4-ol (0·5% v/v, unpublished observation). The potentiating effect of PMBN was also evident in viability assays which show that adding either γ-terpinene or p-cymene at 0·1% (v/v) to flasks containing permeabilizer produced a decline in viability ( Fig. 2).

image

Figure 2. Effect of 10 µg ml−1 PMBN on the antimicrobial activity of 0·1% (v/v) γ-terpinene and 0·1% (v/v) p-cymene against Ps. aeruginosa NCTC 6749. Inoculum density was 1·0 × 105 cfu ml−1. Data shown is the mean of duplicate samplings from a single assay flask, with standard deviations shown as error bars. (●) –γ-terpinene; (○) –γ-terpinene + PMBN; (▪) –p-cymene, (□) –p-cymene + PMBN

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The increased activity of these ‘inactive’ components against permeabilized cells infers that increased diffusion through the outer membrane permits their accumulation in cytoplasmic membranes to toxic levels. That is, these compounds are normally inactive because they are unable to effectively penetrate the outer membrane. Log Pow is considered an important determinant of toxicity. Log Pow, the logarithm of the partition coefficient of a particular solvent between n-octanol and water phases is considered an important determinant of toxicity ( Aono and Inoue 1998). Generally, the more lipophilic a compound, the greater its affinity for cell membranes and the greater its toxicity. However, solvents with log Pow values > 5 are not generally toxic to micro-organisms. The low aqueous solubility of these solvents, in spite of their high lipophilicity, is thought to prevent them from reaching toxic levels in cell membranes. The relationship between Gram-negative outer membrane permeability and toxicity reported here, highlights the fact that monoterpene uptake will be determined by both its aqueous solubility and the permeability of the outer envelope of the target micro-organism.

In conclusion, the findings presented here demonstrate that the outer membrane of Ps. aeruginosa NCTC 6749 provides an essential contribution to its tolerance to tea tree oil. Other mechanisms such as active efflux of tea tree oil components, inhibition of porin production and modifications to membrane structure ( Isken and de Bont 1998) might also be involved. However, it is clear that such mechanisms are insufficient to ensure survival in the absence of an intact lipopolysaccharide diffusion barrier, even in relatively low concentrations of tea tree oil.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES

This work was wholly funded by the Australian Tea Tree Oil Research Institute (ATTORI), Lismore, Australia.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS and METHODS
  5. RESULTS and DISCUSSION
  6. Acknowledgement
  7. REFERENCES
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  • Brophy, J.J., Davies, N.W., Southwell, I.A., Stiff, I.A., Williams, L.R. (1989) Gas chromatographic quality control for oil of Melaleuca Terpinen-4-ol Type (Australian Tea Tree). Journal of Agricultural and Food Chemistry 37, 1330 1335.
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