WO2012014244A1

WO2012014244A1 - USE OF TERPINEN-4-ol AS ANTIMICROBIAL AGENT AGAINST BACTERIA OF LEGIONELLA GENUS

Info

Publication number: WO2012014244A1
Application number: PCT/IT2011/000267
Authority: WO
Inventors: Francesca Mondello; Maria Luisa Ricci
Original assignee: Istituto Superiore Di Sanita'
Priority date: 2010-07-26
Filing date: 2011-07-25
Publication date: 2012-02-02
Also published as: IT1401202B1; ITRM20100414A1

Abstract

The present invention refers to the use of terpinen-4-ol, both in aqueous solutions and vapour form, as antimicrobial agent against bacteria of Legionella genus, preferably Legionella pneumophila for disinfecting systems for the distribution of or containing water such as building water distribution systems, cooling towers, spas, small waterlines such as dental unit, medical devices present in hospital and health care facilities or respiratory medical devices from the above bacteria. The invention indicates terpinen-4-ol concentrations and temperature ranges optimal for expression of anti-Legionella activity.

Description

Use of terpinen-4-ol as antimicrobial agent against bacteria of

Legionella genus

The present invention refers to the use of terpinen-4-ol as antimicrobial agent against bacteria of Legionella genus. Particularly, the present invention concerns the use of terpinen-4-ol, both in aqueous solutions and vapour form, as antimicrobial agent against bacteria of Legionella genus, preferably L. pneumophila, for disinfecting systems for the distribution of or containing water such as building water distribution systems, cooling towers, spas, small waterlines such as dental unit, medical devices present in hospital and health care facilities or respiratory medical devices from the above bacteria.

L. pneumophila is the causative agent of two clinical syndromes in humans: a severe pneumonia called Legionnaires' disease (6) and Pontiac fever, a self-limited flu-like illness (7). Legionella is ubiquitous in freshwater environment where it survives as intracellular parasite of free- living protozoa and could be associated with biofilms (8,9). The infection occurs through inhalation or aspiration of Legionella contaminated aerosols, followed by replication in lung alveolar macrophages. The disease arises by the ability of bacteria to replicate inside the phagosome and to escape from lysosomal degradation (10,11). In Europe, Legionella species are the causative agent of about 1.9% of all community-acquired pneumonia cases, 4.9% of those hospitalized, and in 7.9% of those requiring admission to intensive care units (12). Outbreaks of legionellosis have been mostly associated to artificial contaminated environments such as building water distribution systems, cooling towers, spas, and similar. Water distribution systems and aerosol-producing medical devices are main sources of health care-associated legionellosis (13, 14).

Although various physical and chemical disinfection methods have been investigated to reduce Legionella contamination in artificial water sources (15,16,17, 18), environmental control of Legionella is still a great challenge, and the optimal strategy for long-term abatement of bacterial burden remains a problematic issue(19, 20). In light of the above, it is evident the need of new disinfection methods to prevent Legionella colonization in systems for distribution of or containing water which allow to overcome the limits of the so far adopted techniques.

Recently, the essential oils (EOs), their constituents and products of plant secondary metabolism have been evaluated for their antimicrobial activities (1 ,2,3,4,5) in a perspective of their potential therapeutic properties. Among the EOs, Australian tea tree oil (TTO) is one of the most intensely investigated due to the broad spectrum of antibacterial, antifungal, antiviral and other biological activities. In particular, TTO and terpinen-4-ol activities have been assessed against a high number of bacterial species, using standardized or modified methodologies. However, data on in vitro activity of terpinen-4-ol, the main component of TTO, against Legionella pneumophila are lacking, probably also due to the difficulties encountered in standardizing susceptibility methods for this fastidious-to-grow microorganism.

The EOs and components constitute an interesting category of compounds endowed with broad spectrum antimicrobial activity (21 , 22, 23).

In recent years, some studies have highlighted the efficacy of a variety of EOs in vapour state against respiratory tract pathogens (1). One of the first studies on the activity of EOs against L. pneumophila, was performed by Ishimatsu et al. (24). These authors demonstrated by the disk-diffusion method, the arA\-Legionella activity of hinokitiol (beta- thujaplicin), which is a major component of the essential oil of Chamaecypahs obtuse. In a more recent paper, Chang et al. (25) describe the activity of other EOs extracted by Cinnamomum osmophloeum leaves and by different tissues of Cryptomeha japonica against L. pneumophila at 42 °C. In particular, these authors emphasized a potential role for EOs to control Legionella contamination of hot water systems.

Overall, the studies reported so far have been performed with different methods, and different measure outcomes have been employed, with little comparison among them. These differences have strongly influenced the determination of the an\\-Legionella activity of EOs, thus constituting an important limitation in our understanding of their true disinfection potential for Legionella control. Moreover, it appears that in the two previous studies (24,25) on EOs, only one strain of Legionella has been employed.

The Authors of the present invention have now found that terpinen-4- ol shows antibacterial activity against Legionella.

The terpinen-4-ol (trade names/synonyms: (+/-)-4- terpineol; (+/-)-p- menth-1-en-4-ol; (+/-)-para-menth-1-en-4-ol; (+/-)-terpinen-4-ol; 1-terpinen- 4-ol; 1-para-menthen-4-ol; 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-; 4-carvomenthenol; 4-carvomenthenol, natural; 4-methyl-1-(1-methylethyl)- 3-cyclohexen-1-ol; 4-methyl-1-isopropyl-3-cyclohexen-1-ol; 4-terpinenol; 4- terpineol; dl-4-terpineol; menth-1-en-4-ol; para-menth-1-en-4-ol; p-menth- 1-en-4-ol; terpinenol-4; Ci₀Hi₈0; 00203568; RTECS OT0175110 ; CAS NUMBER: 562-74-3; EC NUMBER (EINECS): 209-235-5) used according to the invention is a liquid, colourless to yellow, odor characteristic - mild earthy green with slight wood/pepper aspects, very soluble in alcohol and ether, fairly soluble in water (<0.5%). Terpinen-4-ol is available commercially from the producers Acros Organics BVBA, Geel, Belgium, catalogue number 360020000, 360020250, 36002100097%, from Hangzhou Standard Chemical Co., Ltd. China, from Sanmenxia Xiawei Chemical Co., Ltd. China and from Takasago International Corporation, Japan 2906.14.0000 C020007. Terpinen-4-ol may be produced by synthesis or extracted by fractional distillation of essential oil of Melaleuca alternifolia Cheel (Melaleucol™, from BGR CORPORATION PTY LTD., Level 6, 130 Phillip Street Sydney WALES 2000) or other species of the genus Melaleuca (Myrtaceae), or Leptospermum, Origanum, Cupressus, Chamaecyparis, Juniperus, Elettaria, Myristica, Thymus.

In particular, the present invention concerns the determination of the in vitro activity of terpinen-4-ol against various L. pneumophila serogroups (SG), from different sources (stock cultures, environment, clinical) by using two different methods: the Clinical and Laboratory Standards Institute (CLSI) broth micro-dilution method (26) with slight modifications, and the micro-atmosphere diffusion method (27,28). Furthermore, the conditions of the assay have been improved by careful consideration of oil activity in aqueous phase and preventing evaporation by microplate sealing.

The in vitro activity of terpinen-4-ol, the main component of tea tree oil (TTO), has been tested against 18 strains of L. pneumophila of different serogroup and source of isolation and other Legionella species (data not shown). For this, an established, standard broth micro-dilution method, with slight modifications, and the micro-atmosphere diffusion method were used. Furthermore it has been assessed simple but quite effective sealing procedure in the micro-dilution method to determine the antibacterial activity of terpinen-4-ol against Legionella in aqueous phase. The results showed that L. pneumophila, is sensitive to terpinen-4-ol, with minimal inhibitory concentration (MIC) ranging from 0.06 to 0.125 % v/v, and a bactericidal activity at 0.5% v/v. In addition, terpinen-4-ol vapours exerted critical activity and its action must be controlled for reproducible MIC determination. Finally the authors of this invention have discovered that increasing the temperature of the assay to 40°C and, particularly, 45°C causes a major increase of terpinen-4-ol an\\-Legionella activity compared to the TTO activity suggesting that terpinen-4-ol could be particularly useful in the combination with high temperature in the eradication of Legionella. Therefore, these data suggest that terpinen-4-ol is active as L pneumophila disinfectant for a possible control measure for Legionella water system contamination, especially in spas, in small waterlines or in particular respiratory medical devices. They also indicate a superior advantage of terpinen-4-ol as Legionella disinfectant at temperature in the range of 40-45°C.

It is important to highlight that before the present invention, few strains of L. pneumophila were shown to be susceptible to other kinds of essential oils (24,25) but this is the first report about L. pneumophila susceptibility to terpinen-4-ol, by even accounting for the different methods used to assess antimicrobial properties of EOs in vitro.

In order to test L. pneumophila susceptibility to terpinen-4-ol, the inventors had to made efforts to improving methods to detect and quantify Legionella susceptibility to the above compound, taking into account the difficulties posed by this growth fastidious microorganism for standardized growth inhibition assays and the well-known partition of antimicrobial active terpinen-4-ol mixture between aqueous and vapour phases.

Established standardised procedures and guidelines have been used to assess the antimicrobial activity of different water soluble and nonvolatile antimicrobial agents. Nevertheless, these methods have not been suitable for the evaluation of the antimicrobial activity of poorly soluble and volatile substances of the essential oils, such as terpinen-4-ol, because of their peculiar physical properties (23). Different strategies have been used to overcome this problem, such as, for instance, the addition of solubilising agents to broth and agar test media (34). However, surfactants could decrease the effectiveness of OEs against microbial strains, interfering with the interpretation of results (21 ,35). Thus, various modifications of standardised conventional methods have been proposed, all contributing to show the strong antimicrobial activity of EOs both in liquid and vapour phases (1 ,5,34,35,37,38,39). Nonetheless, appropriate and commonly accepted susceptibility testing methods for the antimicrobial activity of these oils remain to be established, particularly concerning the effect of oil vapours.

According to the present invention, two different assay methods have been compared for the assessment of the antimicrobial activity of terpinen- 4-ol against L. pneumophila, possibly also applying to other EOs, particular to the mother mixture TTO. These conditions particularly considered the potential effects of the diffusion of oil vapours in aqueous phase and preventing evaporation by plate sealing. Previously, vapour effects of EOs were examined in aqueous phase under plate sealing only to determine the antifungal activity and never for antibacterial activity. Thus, a simple but quite effective sealing procedure in the micro-dilution method to determine the antibacterial activity of terpinen-4-ol against L. pneumophila in aqueous phase was evaluated for the first time. Preliminary studies performed with unsealed plates revealed that TTO vapours, which have been shown to be growth-inhibitory against Staphylococcus aureus (40), were lost during incubation. The oil evaporation caused remarkable inconsistencies in antimicrobial effects, with apparently very low MIC values, and rendered somehow ephemeral and irreproducible the exact quantitative determination of the inhibitory effects. The oil vapours were entrapped between the microplate and its lid causing a non-homogeneous distribution of oil concentration in the microplate wells.

On the basis of the above, it is evident the importance of establishing a method to account for vapour diffusion by also studying the direct effect of terpinen-4-ol vapour against L.pneumophila with a micro- atmosphere diffusion method. With this method, the vapour originated by 10 μΙ of terpinen-4-ol caused total growth inhibition, as measured after 2 days of culture, with total killing after 7 days of culture.

In conclusion, the present invention shows that L. pneumophila, quite irrespective of serogroup and source of isolation, is exquisitely sensitive to terpinen-4-ol, a main compound of the TTO mixture, the effect is bactericidal, particularly in the temperature range of 40-45°C. We also showed that the terpinen-4-ol vapour exerts critical activity and its action must be controlled for reproducible MIC determination. As TTO contains multiple components, each with individual properties, it would more practical to make a formulation containing a single active ingredient as potential alternative agent for control of Legionella infections.

Therefore, it is specific object of the present invention the use of terpinen-4-ol as antimicrobial agent against bacteria of Legionella genus, preferably L. pneumophila and other Legionella species such as L. micdadei.L. bozemanii, L. longbeachae, L. gormanii, L. rubrilucens, L. dumoffii. According to the present invention, terpinen-4-ol is intended pure terpinen-4-ol extracted from natural sources (i.e. terpinen-4-ol concentration >97%), as mentioned above, or produced by synthesis. Terpinen-4-ol according to the present invention is not TTO.

According to the present invention, terpinen-4-ol is used particularly for disinfecting systems for distribution of or containing water such as, but not limited to, building water distribution systems, cooling towers, spas, small waterlines, such as dental units, respiratory medical devices present in hospital and health care facilities or other medical devices where contact with L.pneumophila contaminated water can occur. Overall, many other structures could be sanitized by terpinen-4-ol, as listed below:

• air conditioning systems, like humidifiers, nebulizers, spray systems, heat pumps;

• the emergency water systems, such as decontamination showers, eye washing stations and the sprinkler anti-fire systems;

· the intermitting fountains which can be at high risk of contamination;

• the storage cells, normally present in the solar systems for the ACS production (sanitary warm water, whose normal functioning temperature is about 50 °C);swimming pool with massaging water jets;

Especially small waterlines, particular respiratory medical devices or spas represent an important risk to acquire legionellosis due to the often direct or very close aerosol inhalation and the presence in this case of more susceptible people, as documented by the numerous legionellosis outbreaks in these structures (41 ,42,43,44,45).

According to the present invention, terpinen-4-ol can be used in form of aqueous solution or vapour. Terpinen-4-ol in form of aqueous solution or vapour is preferably used at a temperature ranging from 40°C to 60°C, preferably from 40°C to 50°C, more preferably from 40°C to 45°C. The concentration of terpinen-4-ol in aqueous solution can be from 0,001 % v/v, preferably from 0,01% v/v, more preferably from 0,02% v/v to 0,125 % v/v. Particularly, the solution can be used directly on the surfaces of the above systems or terpinen-4-ol can be dissolved in water circulating or contained in the above systems so that to obtain the above suitable concentrations depending on temperature. Higher terpinen-4-ol concentrations can be used, for instance 5% v/v.

The present invention now will be described by way of illustration and not limitation, according to preferred embodiments thereof.

Exemple 1 : Study of the antibacterial activity of terpinen-4-ol Materials and methods

Essential oil-Melaleuca alternifolia Cheel (tea tree)-oil and its components

Although complying with the International Standard ISO 4730 (29) Australian Melaleuca alternifolia (Maiden and Betch) Cheel oil, supplied by Variati (Milan, Italy), was analysed before use for the exact determination of single constituents by gas chromatography (GC-FID) and gas- chromatography-mass spectrometry (GC-MS), as previously reported (30). Terpinen-4-ol and 1,8 cineole, at a concentration of 42.35% and 3.57%, respectively, were the typical constituents which characterize the standard TTO composition, in accord with the prescription of the European Pharmacopeia (31) and International Standard ISO 4730. Terpinen-4-ol, which was purchased from Fluka (Buchs, Switzerland) and 1 ,8-cineole from Sigma-Aldrich (St Louis, MO, USA), were used as positive markers. Both components were >97% pure.

Bacterial strains and culture media

Eighteen L. pneumophila strains from stock collection, clinical and environmental isolate were tested. They included: L pneumophila SG 1 (Lp 1) ATCC 33152, L. pneumophila SG 6 (Lp 6) ATCC 33215, 5 Lp1 clinical isolates, 5 Lp1 environmental isolates, 5 Lp6 clinical isolates and 1 Lp6 environmental isolates. All strains were stored at -70 °C in sterile skimmed milk until use. Subculture of frozen bacteria were grown on buffered charcoal yeast extract agar supplemented with 0.1% alpha- ketoglutarate (BCYE-a, Oxoid, LTD, Basingstoke, England). Plates were incubated at 36±1 °C in humidified air with 2.5% CO₂ for 72 h. E.coli ATCC 25922 was used as quality control.

The micro-dilution test for L. pneumophila and for quality control strain, was performed using buffered yeast extract broth (BYEB) prepared with Bacto yeast extract (Becton Dickinson and Company Sparks, MD, USA) added with Legionella BCYE growth supplement (Oxoid LTD, Basingstoke, England). Tween-80 (Sigma-Aldrich, St Louis, MO, USA) was added to BYEB to facilitate oil solubility (32). After preparing micro- dilution trays with EO solutions the filled trays were sealed in plastic bags and immediately placed in a freezer at≤20 °C until needed.

Micro-dilution method for terpinen-4-ol antibacterial testing against L. pneumophila

L. pneumophila inoculum determination

Preliminary experiments carried out with the standardized inoculum described for antibiotic susceptibility testing according to the CLSI protocol M7-A7 (26) did not allow a realistic, reproducible evaluation of terpinen-4- ol (and as well, the mother mixture TTO) against L. pneumophila. To evaluate the inoculum size, 72 h agar cultures of L. pneumophila were suspended in sterile distilled water to 0.6 optical density measured at 600 nm (≡10⁹ CFU/ml), vortex-mixed and diluted to give the following concentrations: ≤10⁵, 10⁶, 10⁷, 10⁸ CFU/ml. 50 μΙ of each diluted suspension were added in duplicate in two-fold dilutions of TTO or terpinen-4-ol-solutions to give approximate concentrations of 10⁴, 10⁵, 10⁶, 10⁷ CFU/well (final volume 100 μΙ/well) in 96-well microtitre plates. The final TTO and terpinen-4-ol concentrations tested ranged from 0.0078% to 4% v/v. The quality control strain used in this study was as described in CLSI standard protocol. The viable inoculum was determined by CFU enumeration (in BCYE-a agar medium). Experiments were performed with the final inoculum size approximating 5x10⁴ CFU/ml for E.coli ATCC 25922.

TTO and terpinen-4-ol solubilisation

Preliminary experiments were performed to evaluate the minimal Tween 80 concentration necessary to solubilise TTO and able to maintain L. pneumophila viability. Serial dilution of Tween 80 in BYE-oc broth from 1% to 0.0015% v/v concentration were performed in duplicate in 96-well microtitre plates. 10⁸ bacteria per ml were inoculated at a final concentration of -2.5x10⁶ cells/well. After culture of 72 h at 36±1 °C in 2.5% CO₂ atmosphere, optical density was measured spectrophotometrically at 600 nm, and the number of viable Legionella cells in each well was determined by CFU counting in BCYE-a agar medium. Determination of minimum inhibitory and bactericidal concentration Susceptibility testing to TTO and terpinen-4-ol were performed according to the CLSI broth micro-dilution method, with the following modifications: 1 ) BYEB instead of Muller-Hinton broth; 2) emulsifier Tween 80; 3) inoculum size 10⁸ instead of 10⁵ CFU/ml; 4) use of a sterilised Transparent Microplate Sealer (TMS) during incubation for 72 h. TTO and terpinen-4-ol were diluted using BYEB in the presence of Tween 80 at 0.001 % v/v. Aliquots of 50 μΙ of two-fold dilutions of TTO or terpinen-4-ol solutions were dispensed in 96-well microtitre plates. The final concentration of the essential oil and component ranged from 0.0078% to 4% v/v for TTO and terpinen-4-ol. For inoculum 72 h agar cultures of L. pneumophila were suspended in sterile distilled water to 0.6 optical density measured at 600 nm (≡_10⁹ CFU/ml), vortex-mixed and diluted to give 10⁸ CFU/ml. 50 μΙ of each diluted suspension (10⁸ CFU/ml) was added in duplicate in two-fold dilutions of TTO or terpinen-4-ol -solutions to give final concentration of ~2.5 x10⁶ cells/well. Microtiter plates were then incubated at 36±1 °C with 2.5% CO₂ for 72 h covered with a sterilised Transparent Microplate Sealer (TMS) (AMP Llseal, Greiner Bio-one, Germany). The control of bacterial growth was performed in TTO and terpinen-4-ol -free medium added with 0.001 % v/v Tween 80. Each tray included a sterility column (eight wells) non inoculated.

The minimum inhibitory concentration (MIC) values were defined as the lowest TTO or terpinen-4-ol concentration showing a growth inhibition of 100% compared with the oil-free L. pneumophila control growth as resulted spectrophotometrically or visually. The minimum bactericidal concentration (MBC) values were determined by plating 10 μΙ from each wells with no apparent growth onto BCYE-cc agar medium and incubated at temperature ranging from 36° to 45 °C with 2.5% CO₂, After 72 h the viability was assessed. Each experiment was carried out in duplicate. MBC were defined as the lowest oil concentration resulting in the death of

99.9% of the initial inoculum.

Agar diffusion method BCYE-α agar medium added with sterile Tween 80 (0.01 % v/v) was used to assay the susceptibility of L. pneumophila reference strains, clinical and environmental Lp1 and Lp6, to terpinen-4-ol in this assay. Approximately 1x10⁸ CFU/ml were used to lawn inoculate solid media. A 6 mm in diameter paper disc (Oxoid, LTD, Basingstoke, England) was placed on the inoculated agar surfaces and impregnated with of 10 μΙ of pure terpinen-4-ol. All plates were sealed with two sheets of parafilm (Pechiney Plastic Packaging, USA), incubated at 36±1 °C with 2.5% C0₂ and observed after two and seven days to check whether the effects were static or bactericidal. BCYE-a agar with Tween 80 (0.01 %), but no oil, was used as positive growth control. Each test was performed in duplicate and the mean values of the growth inhibition zone were determined. The inhibitory diameter was measured by means a ruler and when no growth occurred on the entire area of the plate the inhibitory zone was recorded as >90 mm, which was the inside diameter of the dish.

Micro-atmosphere diffusion method

A slightly modified agar diffusion method was used to estimate the terpinen-4-ol activity on L. pneumophila reference, clinical and environmental strains Lp1 and Lp6, in vapour phase, (27,28). A micro- coverglass (Prestige, Italy, 22 mm x 22 mm) was attached with a drop of commercial enamel on the upper lid of a Petri dish. Subsequently a 6 mm in diameter paper disc (Oxoid, Unipath LTD, Basingstoke, England) moistened with 10 μΙ of pure terpinen-4-ol was put at the centre of this slide. The surface of the disc was at a distance of about 4 mm from the growth surface of the test organism. The upper lid was then inverted, sealed with parafilm and incubated at 36±1 °C with 2.5% CO₂ and observed after two and seven days. Some strains were incubated with this method at 40°C and 45°C with 2.5% CO₂ and observed after seven days. BCYE-a agar with Tween 80 (0.01 %) in absence of oil, was used as positive growth control. The space inside the sealed Petri dish was calculated to be ~60 cm³ of air. Each test was performed in duplicate and the mean values of the growth inhibition zone were determined after two and six days. The results were given as described above in agar diffusion

Results

Effect of Tween 80 on L. pneumophila growth

In order to exclude the interference of the detergent in our assay we carried out preliminary experiments in our working condition to establish the correct solubilising but non-inhibitory concentration of Tween 80. In fact, and as already described (33), various detergents could have an inhibitory effect against some Legionella species depending on the concentration. The inhibitory activity of Tween 80 against the growth of L. pneumophila SG1 and SG6 was tested with Tween 80 concentrations ranging from 1% to 0.0015% v/v. It was observed that concentrations higher than 0.06% inhibited the growth of all strains tested; at lower concentrations (between 0.06% and 0.015% v/v) of Tween 80 viable bacteria were still present as determined by CFU counting. Since no toxic effect was shown by lower Tween 80 concentrations, thus all further experiments were carried out with a Tween 80 concentration of 0.001 % v/v.

Anti-Legionella activity of terpinen-4-ol

Before examining the activity of terpinen-4-ol against all the different strains of Legionella, experiments were performed with micro- dilution method using repeated tests with L. pneumophila SG 1 or 6 reference strains In these experiments, initially performed without TMS, we noticed a very large inter-experimental variation, coupled with very low MIC values. Consequently, we resorted to the use of a TMS, which was placed on each individual well, thus excluding vapour diffusion spreading over the wells with different terpinen-4-ol besides TTO concentrations. By this modification, the repeated MIC of L. pneumophila serotype 1 and 6 constantly measured to 0.25% v/v. Similarly, the highly variable MBC in repeated experiments in unsealed wells, conformed to 0.5% v/v under sealing conditions. Thus, all further determinations of MIC and MBC were performed with all strains under microplate sealing. Table 1 shows the activity of terpinen-4-ol, in comparison with TTO, both as MIC and MBC, against each single strain of Lp.

Table 1

Activity of tea tree oil and terpinen-4-ol against individual Legionella pneumophila (Lp) strains with micro-dilution method and sealing of microtiter well

Test organisms Tea tree oil Terpinen-4-ol

MIC % (v/v) MBC % MIC % (v/v) MBC %

1. Lp1 ATCC 33152 0.25 0.5 0.125 0.5

2. Lp1 1/3224* 0.25 1.0 0.125 0.5

3. Lp1 2510* 0.25 0.5 0.06 0.5

4. Lp1 41/2883* 0.25 0.5 0.125 0.5

5. Lp1 3260* 0.125 0.5 0.125 0.5

6. Lp1 4357* 0.25 0.5 0.125 0.5

7. Lp1 73** 0.25 0.5 0.125 0.5

8. Lp1 66** 0.25 0.5 0.125 0.5

9. Lp1 55/3646** 0.25 1.0 0.125 0.5

10. Lp1 2258** 0.25 0.5 0.125 0.5

11. Lp1 3261** 0.25 0.5 0.125 0.5

12. Lp6 ATCC 33215 0.25 0.5 0.125 0.5

13. Lp6 11/2378* 0.5 0.5 0.125 0.5

14. Lp6 51/3380* 0.25 0.5 0.125 0.5

15. Lp6 3265* 0.25 0.5 0.125 0.5

16. Lp6 3374* 0.25 0.5 0.125 0.5

17. Lp6 3303* 0.25 0.5 0.25 0.5

18. Lp6 4405** 0.25 0.5 0.125 0.25 "clinical isolate; ** environmental isola

Table 2 compares the MIC₅₀ and MIC₉₀ of the two serogroups isolates. In both tables, the uniformity of MIC values at 0.25% v/v and the MBC values at 0.5% v/v is noticed, with rare exceptions, and no differences either between the two serogroups or between the environmental and the clinical isolates.

Table 2

Cumulative activity of terpinen-4-ol against Legionella pneumophila (Lp) isolates with micro-dilution method and sealing of microtiter well

Organisms n MICso MICgo MIC range MBCso MBCgo MBC range

% v/v % v/v % v/v % v/v %v/v % v/v

Lp1^§ 11 0.125 0.125 0.06-0.125 0.5 0.5 0.5

Lp6^5§ 7 0.125 0.125 0.125-0.25 0.5 0.5 0.25-0.5 (1 ATCC 33152 reference strain + 5 clinical and 5 environmental strains) ^§§(1 ATCC 33215 reference strain +5 clinical and environmental strains) n, num

Table 3 shows in independent experiments the comparison of TTO and terpinen-4-ol an\\-Legionella activity at temperatures of 40°C and 45 °C, showing the markedly superior terpinen-4-ol activity compared to TTO at these temperatures with respect to 36°C.

Table 3. Activity of tea tree oil and terpinen-4-ol against individual Legionella pneumophila (Lp) strains with microdilution method and sealing of microtiter wells at different temperatures.

40°C 45°C

Test organisms TTO TERP TTO TERP

% v/v % v/v % v/v

MIC MBC MIC MBC MIC MBC MIC MBC

1. Lp1 66** 0.50 0.37 0.0078 0.125 0.17 0.37 <0.0078 O.0078

2. Lp6 11/2378* 0.37 0.08 0.08 0.08 0.26 0.06 <0.0078 <0.0078

3. Lp1 ATCC33152 0.42 0.50 0.26 0.06 0.04 0.34 NG NG

4. Lp6 ATCC33215 0.50 0.83 0.03 0.31 0.19 0.20 <0.0078 <0.0078

5. Lp1 3224* 0.50 0.50 0.0078 0.23 0.03 0.33 <0.0078 O.0078

6. Lp6 4405** 0.43 0.50 0.01 0.06 0.16 0.17 <0.0078 <0.0078

*clinical isolate; ** environmental isolate

Data are the average of three experiments

NG: no growth of this Legionella strain at 45°C

Micro-atmosphere diffusion method

The experiments above suggested the importance of terpinen-4-ol vapours in the anW-Legionella activity. To further confirm this issue, we tested terpinen-4-ol and TTO activity in a micro-atmosphere diffusion method, which allowed the contact of only the TTO or terpinen-4-ol vapours with the bacterial cells (see Materials and Methods). As shown in Table 4, after two days total inhibition of growth was observed (the diameter of the inhibition area equals the Petri dish diameter). After 7 days, the inhibition was still total for all strains, except for Lp6 ATCC, suggesting that the vapour released by a pure terpinen-4-ol solution is sufficient alone to kill the bacteria under the conditions of the micro- atmosphere tests. Data obtained by a standard disk diffusion method, showed total inhibition of L. pneumophila growth at 2 days, but some growth at 7 days (data not shown).

Table 4

Micro-atmosphere diffusion method: terpinen-4-ol vapours effect on Legionella pneumophila (Lp) serogroups growth

TERPINEN-4-OL

Microrganisms

Control Two days contact⁵ Seven days contact⁵

Lp1 ATCC 33152 90 90 Lp1 2510* 0 90 90

Lp1 3260* 0 90 90

Lp6 ATCC 33215 0 90 18

Lp6 11/2378*

0 90 90

Lp6 3374*

0 90 90

Lp6 54/3645**

0 90 90

⁵ Inhibition zone in mm

* clinical isolate; ** environmental isolate In addition, Table 5 shows the comparative effects of TTO and terpinen-4-ol in the temperature range of 36 to 45°C for three selected strains of Legionella pneumophila. It is quite evident that temperature increase causes a dissociation between TTO and terpinen-4-ol anti- Legionella activity markedly in favour of the latter, at least in some representative strains, as assessed by the diameter of growth inhibition. Table 5.

Micro-atmosphere diffusion method: tea tree oil and terpinen 4-ol vapours effects on Legionella pneumophila (Lp) after seven days contact² at different temperatures.

45°CD 40°C 36°CD

Test organisms TERP TTO TERP TTO TERP TT

Lp1 ATCC 33152 NG NG 90 30 90 20

Lp6 ATCC 33215 90 90 20 20 20 15

Lp6 4405** 90 38 ND 20 48 20

Lp1 66** 77 40 40 20 48 17

Lp6 11/2378* 90 90 90 30 90 25

Lp1 3224* 90 90 30 30 30 15

⁸ inhibition zone in mm; data are the average of three experiments

^*clinical isolate; ^** environmental isolate

NG: no growth of this Legionella strain at 45°C

ND: not determined References

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Claims

1) Use of terpinen-4-ol as antimicrobial agent against bacteria of Legionella genus.

2) Use according to claim 1 , wherein said bacteria are chosen from the group consisting of Legionella pneumophila, L. micdadei, L. bozemanii, L. longbeachae, L. gormanii, L. rubrilucens, L. dumoffii.

3 ) Use according to anyone of the claims 1-2, for disinfecting systems for distribution of or containing water.

4 ) Use according to claim 3, wherein the systems for distribution of or containing water are chosen from the group consisting of building water distribution systems, cooling towers, air conditioning systems, emergency water systems, intermitting fountains, storage cells, swimming pool with massaging water jets, spas, small waterlines, medical devices present in hospital and health care facilities or respiratory medical devices.

5) Use according to claim 4 wherein the air conditioning systems are chosen from the group consisting of humidifiers, nebulizers, spray systems, heat pumps.

6) Use according to claim 4, wherein the emergency water systems are chosen from the group consisting of decontamination showers, eye washing stations, sprinkler anti-fire systems.

7 ) Use according to anyone of the previous claims, wherein isolated terpinen-4-ol is used in form of aqueous solution or vapour.

8 ) Use according to claim 7, wherein isolated terpinen-4-ol in form of aqueous solution or vapour is used at a temperature ranging from 40°C to 60°C, preferably from 40°C to 50°C, more preferably from 40°C to 45°C.

9 ) Use according to claim 7, wherein the concentration of isolated terpinen-4-ol in aqueous solution is from 0,001% v/v, preferably from 0,01% v/v, more preferably, from 0,02% v/v to 0,125 % v/v