ITRM20100414A1 - USE OF TERPINEN-4-OL AS AN ANTIMICROBIAL AGENT AGAINST LEGIONELLA PNEUMOPHILA. - Google Patents
USE OF TERPINEN-4-OL AS AN ANTIMICROBIAL AGENT AGAINST LEGIONELLA PNEUMOPHILA. Download PDFInfo
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- ITRM20100414A1 ITRM20100414A1 IT000414A ITRM20100414A ITRM20100414A1 IT RM20100414 A1 ITRM20100414 A1 IT RM20100414A1 IT 000414 A IT000414 A IT 000414A IT RM20100414 A ITRM20100414 A IT RM20100414A IT RM20100414 A1 ITRM20100414 A1 IT RM20100414A1
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- WRYLYDPHFGVWKC-SNVBAGLBSA-N 4-Terpineol Natural products CC(C)[C@]1(O)CCC(C)=CC1 WRYLYDPHFGVWKC-SNVBAGLBSA-N 0.000 title claims description 48
- 241000589242 Legionella pneumophila Species 0.000 title claims description 47
- 229940115932 legionella pneumophila Drugs 0.000 title claims description 12
- 239000004599 antimicrobial Substances 0.000 title claims description 9
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- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/20—Gaseous substances, e.g. vapours
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2307/00—Location of water treatment or water treatment device
- C02F2307/14—Treatment of water in water supply networks, e.g. to prevent bacterial growth
Description
Uso di terpinene-4-olo come agente antimicrobico contro Legionella pneumophila Use of terpinen-4-ol as an antimicrobial agent against Legionella pneumophila
La presente invenzione si riferisce all'uso di terpinene-4-olo come agente antimicrobico contro Legionella pneumophila. In particolare, la presente invenzione riguarda l'uso di terpinene-4-olo, sia in soluzioni acquose che sotto forma di vapore, come agente antimicrobico contro L. pneumophila per la disinfezione, dal suddetto batterio, di impianti per la distribuzione idrica o contenenti acqua come gli impianti idrici negli edifici, le torri di raffreddamento, le terme, le piccole condotte idriche come i riuniti dentali, i dispositivi medici presenti negli ospedali e nelle strutture per l'assistenza sanitaria o i dispositivi medici respiratori. The present invention relates to the use of terpinen-4-ol as an antimicrobial agent against Legionella pneumophila. In particular, the present invention relates to the use of terpinene-4-ol, both in aqueous solutions and in the form of vapor, as an antimicrobial agent against L. pneumophila for the disinfection, from the aforementioned bacterium, of systems for water distribution or containing water such as building water systems, cooling towers, spas, small water pipes such as dental units, medical devices in hospitals and health care facilities, or respiratory medical devices.
L. pneumophila à ̈ 1<1>agente responsabile di due sindromi cliniche nell'uomo: una polmonite grave chiamata "Malattia del Legionario" (6) una malattia simil-influenzale autolimitantesi come la "Febbre di Pontiac" (7). Legionella à ̈ ubiquitaria negli ambienti d'acqua dolce dove sopravvive come parassita intracellulare di protozoi e può essere associata a biofilm (8,9). L'infezione avviene attraverso l'inalazione o l'aspirazione di aerosol contaminati da Legionella, seguita dalla sua replicazione all'interno dei macrofagi degli alveoli polmonari. La malattia insorge per la capacità dei batteri di replicarsi all'interno del fagosoma e di sfuggire alla degradazione lisosomiale (10,11). In Europa, le specie di Legionella causano circa 1,9% di tutti i casi di polmonite acquisita in comunità , il 4,9% di quelle ospedaliere, e nel 7,9% dei ricoveri in terapia intensiva (12).Epidemie di legionellosi sono state in gran parte associate ad ambienti artificiali contaminati, come gli impianti idrici degli edifici, le torri di raffreddamento, le terme e simili. Gli impianti idrici e i dispositivi medici che producono aerosol sono le principali fonti di legionellosi associate a terapie mediche (13,14). L. pneumophila is the 1 <1> agent responsible for two clinical syndromes in humans: a severe pneumonia called "Legionnaire's Disease" (6) a self-limiting flu-like illness such as "Pontiac Fever" (7). Legionella is ubiquitous in freshwater environments where it survives as an intracellular parasite of protozoa and can be associated with biofilms (8,9). Infection occurs through the inhalation or aspiration of aerosols contaminated with Legionella, followed by its replication within the macrophages of the pulmonary alveoli. The disease arises from the ability of bacteria to replicate within the phagosome and escape lysosomal degradation (10,11). In Europe, Legionella species cause approximately 1.9% of all community-acquired pneumonia cases, 4.9% of hospital-acquired pneumonia, and 7.9% of ICU admissions (12). they have been largely associated with contaminated man-made environments, such as building water systems, cooling towers, spas and the like. Water systems and medical devices that produce aerosols are the main sources of legionellosis associated with medical therapies (13,14).
Sebbene siano stati studiati vari metodi di disinfezione chimica e fisica per ridurre la contaminazione da Legionella nelle fonti idriche artificiali (15,16,17,18), il controllo ambientale della Legionella à ̈ ancora una grande sfida, e la strategia ottimale per la riduzione a lungo termine della carica batterica rimane problematica (19, 20). Although various chemical and physical disinfection methods have been studied 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 reduction long-term bacterial load remains problematic (19, 20).
Alla luce di quanto suddetto, à ̈ evidente la necessità di nuovi metodi di disinfezione per prevenire la colonizzazione di Legionella nei sistemi di distribuzione idrica o contenenti acqua che consentano di superare i limiti delle tecniche sino ad ora adottate. In the light of the above, it is clear the need for new disinfection methods to prevent the colonization of Legionella in water distribution systems or systems containing water that make it possible to overcome the limits of the techniques adopted up to now.
Recentemente, gli oli essenziali (OE), i loro componenti e i prodotti del metabolismo secondario delle piante sono stati valutati per la loro attività antimicrobica (1,2,3,4,5) nella prospettiva di utilizzo delle loro proprietà terapeutiche potenziali. Recently, essential oils (EOs), their components and the products of secondary metabolism of plants have been evaluated for their antimicrobial activity (1,2,3,4,5) with a view to using their potential therapeutic properties.
Tra gli OE, il tea tree oil australiano (olio dell'albero del tà ̈: TTO) à ̈ uno dei più intensamente studiati a causa dell'ampio spettro di attività antibatteriche, antifungine, antivirali e altre proprietà biologiche. In particolare, le attività di TTO e di terpinene-4-olo sono state valutate nei confronti di un elevato numero di specie batteriche, utilizzando metodologie standardizzate o modificate. Tuttavia, i dati sull'attività in vitro di terpinene-4-olo, il principale componente del TTO, nei confronti di L. pneumophila sono carenti, probabilmente anche a causa della difficoltà di standardizzare i metodi di valutazione della suscettibilità agli antimicrobici di questo microrganismo difficile da coltivare. Among the EOs, Australian tea tree oil (tea tree oil: TTO) is one of the most intensively studied due to the broad spectrum of antibacterial, antifungal, antiviral and other biological properties. In particular, the activities of TTO and terpinen-4-ol were evaluated against a large number of bacterial species, using standardized or modified methods. However, data on the in vitro activity of terpinen-4-ol, the main component of TTO, against L. pneumophila are lacking, probably also due to the difficulty of standardizing the methods for assessing susceptibility to antimicrobials of this microorganism. difficult to grow.
Gli OE e i loro componenti costituiscono una categoria interessante di composti dotati di ampio spettro di attività antimicrobica (21, 22, 23). Negli ultimi anni, alcuni studi hanno evidenziato l'efficacia di una varietà di OE allo stato di vapore contro gli agenti patogeni del tratto respiratorio (1). Uno dei primi studi sull'attività di OE contro L.pneumophila, à ̈ stato eseguito da Ishimatsu et al. (24).Questi autori hanno dimostrato con il metodo di diffusione in agar con disco, l'attività anti-Legionella di hinokitiol (beta-thujaplicin) , che à ̈ uno dei principali componenti dell'olio essenziale di Chamaecyparis ottuse. In un documento più recente, Chang et al. (25) descrivono l'attività di altri OE estratti dalle foglie di Cinnamomum osmophloeum e da differenti tessuti di Cryptomeria japonica contro L. pneumophila a 42°C. In particolare, questi autori hanno sottolineato il ruolo potenziale degli OE per controllare la contaminazione da Legìonella negli impianti di acqua calda. EOs and their components constitute an interesting category of compounds with a broad spectrum of antimicrobial activity (21, 22, 23). In recent years, studies have highlighted the efficacy of a variety of vaporized EOs against pathogens in the respiratory tract (1). One of the first studies on EO activity against L. pneumophila was performed by Ishimatsu et al. (24) These authors demonstrated with the disk agar diffusion method, the anti-Legionella activity of hinokitiol (beta-thujaplicin), which is one of the main components of Chamaecyparis ottuse essential oil. In a more recent paper, Chang et al. (25) describe the activity of other EOs extracted from Cinnamomum osmophloeum leaves and from different Cryptomeria japonica tissues against L. pneumophila at 42 ° C. In particular, these authors highlighted the potential role of EOs to control Legìonella contamination in hot water systems.
Complessivamente, gli studi riportati finora sono stati condotti con metodi diversi, e sono state impiegate differenti valutazioni dei risultati, con poca possibilità di confronto tra loro. Overall, the studies reported so far have been conducted with different methods, and different evaluations of the results have been employed, with little possibility of comparison between them.
Queste differenze hanno fortemente influenzato la determinazione delle attività anti-Legionella degli OE, costituendo in tal modo una limitazione importante per la comprensione del loro vero potenziale di disinfezione per il controllo della Legìonella. Inoltre, sembra che nei due studi precedenti (24,25) sugli OE, sia stato impiegato un solo ceppo di Legìonella. These differences strongly influenced the determination of the anti-Legionella activities of the EOs, thus constituting an important limitation for the understanding of their true disinfection potential for the control of Legìonella. Furthermore, it appears that in the two previous studies (24,25) on EO, only one strain of Legìonella was used.
E<1>noto l'articolo Mondello et al. (Journal of Microbiological Methods, Elsevier, Amsterdam, NL, voi. 77, no. 2, 1 May 2009, pages 243-248) concernente uno studio sulla efficacia dell'olio di Melaleuca alternìfolia Cheel (tea tree) in forma di vapore contro ceppi di Legìonella pneumophila. Inoltre, à ̈ nota la domanda di brevetto WO88/10122 concernente l'uso di olio di Melaleuca (tea tree oil) disciolto in alcol per la disinfezione di condizionatori d'aria, ad esempio contro la Legìonella pneumophila. The article Mondello et al. Is known. (Journal of Microbiological Methods, Elsevier, Amsterdam, NL, vol. 77, no. 2, 1 May 2009, pages 243-248) concerning a study on the efficacy of Melaleuca alternìfolia Cheel (tea tree) oil in the form of vapor against strains of Legìonella pneumophila. Furthermore, patent application WO88 / 10122 is known concerning the use of Melaleuca oil (tea tree oil) dissolved in alcohol for the disinfection of air conditioners, for example against Legìonella pneumophila.
Barzanò & Zanardo Barzanò & Zanardo
5 5
Gli autori della presente invenzione hanno adesso scoperto che il terpinene-4-olo dimostra un'attività antibatterica contro Legionella. In particolare, questa invenzione riguarda la determinazione dell'attività in vitro del terpinene-4-olo nei confronti di vari sierogruppi (SG) di L. pneumophila, provenienti da diverse fonti (ceppi di riferimento e ceppi di origine ambientale e clinica) utilizzando due metodi diversi: il metodo di microdiluizione in brodo, con lievi modifiche, del Clinical and Laboratory Standards Institute (CLSI))(26), e il metodo di diffusione in micro-atmosfera (27,28). Inoltre, sono state migliorate le condizioni dei saggi sperimentali mediante un'accurata considerazione dell'attività dell'olio in fase acquosa, impedendo l'evaporazione con sigillazione della micropiastra. The authors of the present invention have now discovered that terpinen-4-ol demonstrates antibacterial activity against Legionella. In particular, this invention concerns the determination of the in vitro activity of terpinen-4-ol against various serogroups (SG) of L. pneumophila, coming from different sources (reference strains and strains of environmental and clinical origin) using two different methods: the broth microdilution method, with slight modifications, of the Clinical and Laboratory Standards Institute (CLSI)) (26), and the micro-atmosphere diffusion method (27,28). Furthermore, the conditions of the experimental assays were improved by careful consideration of the activity of the oil in the aqueous phase, preventing evaporation with sealing of the microplate.
L'attività in vitro del terpinene-4-olo, il principale componente del tea tree oil (TTO), à ̈ stata analizzata nei confronti di 18 ceppi di L.pneumophila di differenti sierogruppi e fonti di isolamento. Per questo scopo, sono stati utilizzati un metodo standard validato di micro-diluizione in brodo, con lievi modifiche, e il metodo di diffusione in microatmosfera. Inoltre à ̈ stata valutata la procedura di sigillazione, semplice ma molto efficace, nel metodo di micro-diluizione per determinare l'attività antibatterica del terpinene-4 -olo nei confronti di Legionella nella fase acquosa. I risultati hanno mostrato che L.pneumophila, à ̈ sensibile al terpinene 4-olo, con una concentrazione minima inibitoria (MIC) che varia da 0,06 a 0,125% v/v, a seconda del ceppo, e un'attività battericida a 0,5% v/v. Inoltre, abbiamo dimostrato che i vapori del terpinene-4-olo esercitano un'attività critica e il loro effetto deve essere controllato per una determinazione riproducibile della MIC. Pertanto, questi dati suggeriscono che il terpinene-4 -olo à ̈ attivo come disinfettante per L. pneumophila, come possibile misura di controllo per la contaminazione da Legionella degli impianti idrici, specialmente nelle terme, nelle piccole condotte idriche o in particolari dispositivi medici respiratori . The in vitro activity of terpinene-4-ol, the main component of tea tree oil (TTO), was analyzed against 18 L.pneumophila strains of different serogroups and sources of isolation. For this purpose, a standard validated broth micro-dilution method, with slight modifications, and the micro-atmosphere diffusion method were used. Furthermore, the simple but very effective sealing procedure was evaluated in the micro-dilution method to determine the antibacterial activity of terpinene-4 -ol against Legionella in the aqueous phase. The results showed that L. pneumophila is sensitive to terpinen 4-ol, with a minimum inhibitory concentration (MIC) ranging from 0.06 to 0.125% v / v, depending on the strain, and bactericidal activity at 0.5% v / v. Furthermore, we have shown that terpinene-4-ol vapors exert critical activity and their effect must be controlled for reproducible MIC determination. Therefore, these data suggest that terpinen-4 -ol is active as a disinfectant for L. pneumophila, as a possible control measure for Legionella contamination of water systems, especially in spas, small water pipes or particular respiratory medical devices. .
E'importante evidenziare che prima di questa invenzione, pochi ceppi di L. pneumophila si sono dimostrati sensibili ad altri tipi di oli essenziali (24,25), ma questa à ̈ la prima dimostrazione della suscettibilità di L. pneumophila al terpinene-4-olo, anche in considerazione dei diversi metodi usati per valutare le proprietà antimicrobiche degli OE in vitro. It is important to note that before this invention, few strains of L. pneumophila have been shown to be sensitive to other types of essential oils (24,25), but this is the first demonstration of the susceptibility of L. pneumophila to terpinen-4- ol, also in consideration of the different methods used to evaluate the antimicrobial properties of EOs in vitro.
Al fine di saggiare la suscettibilità di L. pneumophila al terpinene-4-olo, gli inventori hanno per prima cosa migliorato i metodi per rilevare e quantificare la suscettibilità di Legionella al composto suddetto, tenendo conto della difficoltà che comporta la crescita di questi microrganismi esigenti nei saggi standardizzati di inibizione della crescita e la ben nota separazione della miscela antimicrobica attiva del terpinene-4-olo tra la fase acquosa e la fase vapore. In order to test the susceptibility of L. pneumophila to terpinen-4-ol, the inventors first improved the methods for detecting and quantifying the susceptibility of Legionella to the aforementioned compound, taking into account the difficulty involved in the growth of these fastidious microorganisms. in standardized growth inhibition assays and the well-known separation of the active antimicrobial mixture of terpinen-4-ol between the aqueous and vapor phases.
Sono stati usate procedure standardizzate e linee guida validate per valutare l'attività antimicrobica di diversi agenti antimicrobici idrosolubili e non volatili. Ciò nonostante, questi metodi non sono stati idonei per la valutazione dell'attività antimicrobica di sostanze scarsamente solubili e volatili degli oli essenziali, come il terpinene-4 -olo, a causa delle loro peculiari proprietà fisiche (23). Differenti strategie sono state adottate per superare questo problema, come, ad esempio, l'aggiunta di agenti solubilizzanti nel brodo e nell'agar dei terreni utilizzati per i saggi (34). Tuttavia, i tensioattivi potrebbero diminuire l'efficacia degli OE contro ceppi microbici, interferendo nell'interpretazione dei risultati (21,35). Pertanto, sono state proposte varie modificazioni di metodi convenzionali standardizzati, che contribuiscono a dimostrare la forte attività antimicrobica degli OE, sia in fase liquida sia in fase vapore (1,5,34,35,37,38,39). Nonostante ciò, sono ancora da stabilire metodi di suscettibilità adeguati e comunemente accettati per testare l'attività antimicrobica di tali oli, soprattutto per quanto riguarda l'effetto dei vapori dell'olio. Standardized procedures and validated guidelines were used to evaluate the antimicrobial activity of several water-soluble and non-volatile antimicrobial agents. However, these methods have not been suitable for evaluating the antimicrobial activity of poorly soluble and volatile substances in essential oils, such as terpinen-4 -ol, due to their unique physical properties (23). Different strategies have been adopted to overcome this problem, such as, for example, the addition of solubilizing agents in the broth and agar of the media used for the assays (34). However, surfactants may decrease the efficacy of EOs against microbial strains, interfering in the interpretation of the results (21,35). Therefore, various modifications of standardized conventional methods have been proposed, which help to demonstrate the strong antimicrobial activity of EOs, both in the liquid phase and in the vapor phase (1,5,34,35,37,38,39). Despite this, adequate and commonly accepted susceptibility methods for testing the antimicrobial activity of such oils, especially with regard to the effect of oil vapors, are yet to be established.
In questa invenzione, sono stati confrontati due differenti metodi, per la valutazione dell'attività antimicrobica del terpinene-4 -olo contro L.pneumophila, possibilmente applicabili anche agli altri OE, in particolare alla miscela madre TTO. Queste condizioni consideravano particolarmente gli effetti potenziali della diffusione di vapore dell'olio e dei suoi componenti in fase acquosa, prevenendo l'evaporazione dalla piastra con sigillazione di quest 'ultima. In precedenza, gli effetti del vapore degli OE sono stati esaminati in fase acquosa con sigillazione della piastra solo per determinare l'attività antifungina e mai per l'attività antibatterica.Così, per determinare l'attività antibatterica di terpinene-4-olo nei confronti di L. pneumophila in fase acquosa à ̈ stata valutata per la prima volta una procedura di sigillazione semplice, ma molto efficace nel metodo di micro-diluizione. Studi preliminari effettuati con piastre non sigillate avevano dimostrato che durante l'incubazione venivano emessi vapori di TTO, che inibivano la crescita di Staphylococcus aureus (40). L'evaporazione dell'olio provocava notevoli discordanze negli effetti antimicrobici, con valori apparentemente molto bassi delle MIC, rendendo in qualche modo non attendibile e irriproducibile l'esatta determinazione quantitativa degli effetti inibitori. I vapori dell'olio rimanevano intrappolati tra la micropiastra ed il suo coperchio causando una distribuzione non omogenea della concentrazione dell'olio nei pozzetti della micropiastra. In this invention, two different methods have been compared, for the evaluation of the antimicrobial activity of terpinen-4 -ol against L. pneumophila, possibly applicable also to the other EOs, in particular to the TTO parent mixture. These conditions particularly considered the potential effects of vapor diffusion of the oil and its components in the aqueous phase, preventing evaporation from the plate with sealing of the latter. Previously, the vapor effects of EOs were examined in the aqueous phase with plate sealing only to determine antifungal activity and never for antibacterial activity. Thus, to determine the antibacterial activity of terpinen-4-ol in comparisons of L. pneumophila in the aqueous phase A simple but highly effective sealing procedure in the micro-dilution method was evaluated for the first time. Preliminary studies performed with unsealed plates had shown that TTO vapors were emitted during incubation, which inhibited the growth of Staphylococcus aureus (40). The evaporation of the oil caused considerable discrepancies in the antimicrobial effects, with apparently very low MIC values, making the exact quantitative determination of the inhibitory effects in some way unreliable and irreproducible. The oil vapors were trapped between the microplate and its lid causing an uneven distribution of the oil concentration in the microplate wells.
Sulla base di quanto suddetto, Ã ̈ evidente l'importanza di stabilire un metodo che tenga conto della diffusione del vapore anche studiando l'effetto diretto del vapore del terpinene-4-olo contro L.pneumophila con un metodo di diffusione in microatmosfera. Con questo metodo, il vapore originato da 10 pi di terpinene-4-olo causava l'inibizione della crescita totale, misurata dopo 2 giorni di coltura, con l'abbattimento totale dopo 7 giorni di coltura. On the basis of the above, it is evident the importance of establishing a method that takes into account the diffusion of the vapor also by studying the direct effect of the vapor of terpinene-4-ol against L. pneumophila with a method of diffusion in the micro atmosphere. With this method, the vapor originating from 10 µl of terpinen-4-ol caused the inhibition of total growth, measured after 2 days of culture, with total culling after 7 days of culture.
In conclusione, la presente invenzione mostra che L . pneumophila, anche indipendentemente dal sierogruppo e dalla fonte di isolamento, à ̈ estremamente sensibile al terpinene-4-olo, il componente principale della miscela TTO, con un effetto battericida.Abbiamo anche dimostrato che il vapore del terpinene-4-olo esercita un'attività critica e la sua azione deve essere controllata per una determinazione riproducibile della MIC. Poiché il TTO contiene più componenti, ciascuno con proprietà individuali, sarebbe più pratico fare una formulazione contenente un unico principio attivo come potenziale farmaco alternativo per il controllo delle infezioni da Legionella. In conclusion, the present invention shows that L. pneumophila, even regardless of serogroup and source of isolation, is extremely sensitive to terpinen-4-ol, the main component of the TTO blend, with a bactericidal effect. We have also shown that the vapor of terpinen-4-ol exerts a critical activity and its action must be controlled for reproducible MIC determination. Since TTO contains multiple components, each with individual properties, it would be more practical to make a formulation containing a single active ingredient as a potential alternative drug for controlling Legionella infections.
Forma, pertanto, oggetto specifico della presente invenzione l'utilizzo del terpinene-4-olo con una percentuale di purezza maggiore del 97%, in forma di soluzione acquosa o di vapore, come agente antimicrobico contro L.pneumophila, in particolare per la disinfezione degli impianti per la distribuzione idrica o contenenti acqua ad esempio, ma non solo, gli impianti idrici degli edifici, le torri di raffreddamento, le terme, le piccole condotte idriche, quali i riuniti dentali, i dispositivi medici respiratori presenti negli ospedali e nelle strutture per l'assistenza sanitaria o altri dispositivi medici dove può avvenire il contatto con acqua contaminata con L. pneumophila. Nel complesso, molte altre strutture potrebbero essere disinfettate da terpinene-4-olo, come di seguito elencate: Therefore, the specific object of the present invention forms the use of terpinen-4-ol with a percentage of purity greater than 97%, in the form of aqueous or vapor solution, as an antimicrobial agent against L. pneumophila, in particular for disinfection of water distribution systems or systems containing water, for example, but not limited to, building water systems, cooling towers, spas, small water pipes, such as dental units, respiratory medical devices in hospitals and structures for healthcare or other medical devices where contact with water contaminated with L. pneumophila may occur. Overall, many other facilities could be disinfected by terpinen-4-ol, as listed below:
• Gli impianti di condizionamento dell'aria, come umidificatori, nebulizzatori, sistemi a spruzzamento pompe di calore; â € ¢ Air conditioning systems, such as humidifiers, nebulizers, spraying systems, heat pumps;
• i sistemi idrici di emergenza, come le docce di decontaminazione, le stazioni di lavaggio oculare e i sistemi sprinkler anti-incendio; â € ¢ emergency water systems, such as decontamination showers, eye wash stations and fire sprinkler systems;
• le fontane ad intermittenza che possono essere ad alto rischio di contaminazione; â € ¢ intermittent fountains which can be at high risk of contamination;
• i serbatoi di accumulo, normalmente presenti negli impianti solari per la produzione di ACS (acqua calda sanitaria, la cui normale temperatura di funzionamento à ̈ di circa 50°C); â € ¢ storage tanks, normally present in solar plants for the production of DHW (domestic hot water, whose normal operating temperature is around 50 ° C);
• le piscine con getti d'acqua per idromassaggio; • gli impianti di acqua calda ad uso domestico. â € ¢ swimming pools with hydromassage jets; â € ¢ domestic hot water systems.
• soprattutto le piccole condotte idriche, in particolare i dispositivi medici respiratori o le terme rappresentano un rischio importante per l'acquisizione della legionellosi , dovuta all'inalazione di aerosol spesso diretta o molto ravvicinata e, alla presenza in questo caso, di persone più suscettibili, come documentato dalle numerose epidemie di legionellosi in queste strutture (41,42,43,44,45). â € ¢ especially small water pipes, in particular respiratory medical devices or spas, represent an important risk for the acquisition of legionellosis, due to the inhalation of aerosols often direct or very close and, in this case, of people more susceptible, as documented by the numerous outbreaks of Legionnaires' disease in these structures (41,42,43,44,45).
La concentrazione del terpinene-4 -olo in soluzione acquosa varia dal 5% allo 0,125% v/v, preferibilmente dal 2,5% all'1% v/v. The concentration of terpinene-4 -ol in aqueous solution varies from 5% to 0.125% v / v, preferably from 2.5% to 1% v / v.
In particolare, il terpinene-4-olo in forma di soluzione acquosa o vapore può essere utilizzato a temperature comprese da 4°C a 60°C, preferibilmente dalla temperatura ambiente (18° C) a 55°C. In particular, terpinene-4-ol in the form of an aqueous solution or vapor can be used at temperatures ranging from 4 ° C to 60 ° C, preferably from room temperature (18 ° C) to 55 ° C.
La presente invenzione verrà ora descritta in modo illustrativo e non limitativo secondo le sue forme di realizzazione preferite. The present invention will now be described in an illustrative and non-limiting manner according to its preferred embodiments.
Esempio 1: Studio dell'attività antibatterica del terpinene-4-olo Example 1: Study of the antibacterial activity of terpinen-4-ol
Materiali e metodi Materials and methods
Olio essenziale di Melaleuca alternifolia-Cheel (tea tree oil) e suoi componenti Melaleuca alternifolia-Cheel essential oil (tea tree oil) and its components
Anche se conforme alla norma internazionale ISO 4730 (29) l'olio australiano Melaleuca alternifolia (Maiden e Betch) Cheel, fornito da Variati (Milano, Italia), prima di utilizzarlo, Ã ̈ stato analizzato mediante gascromatografia (GC-FID) e gascromatografiaspettrometria di massa (GC-MS), per la determinazione esatta dei singoli costituenti come precedentemente riportato (30). I componenti tipici che caratterizzano la composizione standard del TTO erano terpinene-4-olo e 1,8 cineolo, ad una concentrazione del 42,35% e del 3,57%, rispettivamente, in accordo con la prescrizione della Farmacopea Europea (31) e delle norme internazionali ISO 4730. Terpinene-4-olo, acquistato da Fluka (Buchs, Svizzera) e 1,8-cineolo da Sigma-Aldrich (St. Louis, MO, USA), sono stati utilizzati come marcatori positivi.Entrambi i componenti erano puri>97% . Although it complies with the international standard ISO 4730 (29), the Australian oil Melaleuca alternifolia (Maiden and Betch) Cheel, supplied by Variati (Milan, Italy), was analyzed by gas chromatography (GC-FID) and gas chromatography spectrometry before using it. mass (GC-MS), for the exact determination of the single constituents as previously reported (30). Typical components that characterize the standard composition of TTO were terpinen-4-ol and 1.8 cineole, at a concentration of 42.35% and 3.57%, respectively, in accordance with the European Pharmacopoeia prescription (31) and international standards ISO 4730. Terpinen-4-ol, purchased from Fluka (Buchs, Switzerland) and 1,8-cineole from Sigma-Aldrich (St. Louis, MO, USA), were used as positive markers. components were> 97% pure.
Ceppi batterici e terreni di coltura Bacterial strains and culture media
Sono stati testati diciotto ceppi di L. pneumophila provenienti da colture di riferimento e di origine clinica ed ambientale. Tra questi: L. pneumophila SG 1 (Lp 1) ATCC 33152, L.pneumophila SG 6 (Lp 6) ATCC 33215, 5 isolati clinici Lpl, 5 isolati ambientali Lpl, 5 isolati clinici Lp6 e 1 isolato ambientale Lp6. Tutti i ceppi sono stati conservati a -70°C in latte scremato sterile fino all'uso. Subcolture di batteri congelati sono state coltivate su agar tamponato, con aggiunta di carbone, estratto di lievito, integrato con 0,1% di alfa-chetoglutarato (BCYE-alfa, Oxoid, LTD, Basingstoke, Inghilterra). Le piastre sono state incubate a 36 ± 1°C in aria umidificata con 2.5% di C02per 72 ore. E' stato utilizzato come controllo di qualità E.coli ATCC 25922. Eighteen strains of L. pneumophila from reference and clinical and environmental cultures were tested. These include: L. pneumophila SG 1 (Lp 1) ATCC 33152, L.pneumophila SG 6 (Lp 6) ATCC 33215, 5 clinical isolates Lpl, 5 environmental isolates Lpl, 5 clinical isolates Lp6 and 1 environmental isolate Lp6. All strains were stored at -70 ° C in sterile skim milk until use. Subcultures of frozen bacteria were grown on buffered agar, with added charcoal, yeast extract, supplemented with 0.1% alpha-ketoglutarate (BCYE-alpha, Oxoid, LTD, Basingstoke, England). The plates were incubated at 36 ± 1 ° C in humidified air with 2.5% CO2 for 72 hours. E.coli ATCC 25922 was used as a quality control.
Il metodo di micro-diluizione per L. pneumophila e per il ceppo per il controllo di qualità , à ̈ stato effettuato utilizzando brodo con estratto di lievito tamponato (BYEB) preparato con lievito Bacto (Becton Dickinson and Company Sparks, MD, USA), addizionato con supplemento di crescita Legionella BCYE (Oxoid LTD, Basingstoke,Inghilterra). Tween-80 (Sigraa-Aldrich, St Louis, MO, USA) à ̈ stato aggiunto al BYEB per facilitare la solubilità dell'olio(32). Dopo aver preparato le piastre per miero-diluizione con le soluzioni con gli EO, le micropiastre riempite sono state sigillate in sacchetti di plastica e subito messe in un congelatore a ≤-20°C fino al momento dell'uso. The micro-dilution method for L. pneumophila and for the quality control strain was performed using buffered yeast extract broth (BYEB) prepared with Bacto yeast (Becton Dickinson and Company Sparks, MD, USA), added with Legionella BCYE growth supplement (Oxoid LTD, Basingstoke, England). Tween-80 (Sigraa-Aldrich, St Louis, MO, USA) was added to BYEB to facilitate the solubility of the oil (32). After preparing the micro-dilution plates with the EO solutions, the filled microplates were sealed in plastic bags and immediately placed in a freezer at â ‰ ¤ -20 ° C until use.
Metodo della micro-diluizione per il saggio dell 'attività di terpinene-4-olo contro L.pneumophila Determinazione dell'inoculo di L. pneumophila Micro-dilution method for the assay of the activity of terpinen-4-ol against L. pneumophila Determination of the inoculum of L. pneumophila
Gli esperimenti preliminari effettuati con inoculo standardizzato per testare la suscettibilità agli antibiotici secondo il protocollo CLSI M7-A7 (26) non consentono una realistica valutazione riproducibile dell'attività di terpinene-4-olo (anche della miscela madre TTO) contro L. pneumophila. Per valutare la dimensione dell'inoculo, sono state sospese, in acqua distillata sterile, colture di L.pneumophila di 72 ore su agar fino a densità ottica 0,6 misurata a 600 nm (≥10<9>CFU/ml), mescolate con "vortex" e diluite per dare le seguenti concentrazioni: ≡10<5>, IO<6>, IO<7>, IO<8>CFU/ml. 50 Î1⁄4Î di ciascuna sospensione diluita sono stati aggiunti in duplicato in diluizioni a raddoppio di soluzioni di TTO o di terpinene-4-olo per ottenere concentrazioni di IO<4>, IO<5>, IO<6>, IO<7>CFU/ micropozzetto (volume finale di 100 Î1⁄4Î /micropozzetto) in piastre da 96 pozzetti per microtitolazione. Le concentrazioni finali testate di TTO e di terpinene-4-olo variavano da 0,0078% a 4% v/v. Il ceppo utilizzato in questo studio come controllo di qualità era come descritto nel protocollo standard CLSI. L'inoculo vitale à ̈ stato determinato con la conta delle CFU (in terreno agar BCYE-alfa) . Gli esperimenti sono stati eseguiti con inoculo finale approssimativamente di 5xl0<4>CFU/ml per E. coli ATCC 25922. Preliminary experiments carried out with standardized inoculum to test susceptibility to antibiotics according to the CLSI M7-A7 protocol (26) do not allow a realistic and reproducible evaluation of the activity of terpinen-4-ol (also of the TTO mother mix) against L. pneumophila. To evaluate the inoculum size, 72-hour cultures of L. pneumophila were suspended in sterile distilled water on agar up to optical density 0.6 measured at 600 nm (â ‰ ¥ 10 <9> CFU / ml) , mix with "vortex" and dilute to give the following concentrations: â ‰ ¡10 <5>, IO <6>, IO <7>, IO <8> CFU / ml. 50 Î1⁄4Î of each diluted suspension was added in duplicate in two-fold dilutions of TTO or terpinene-4-ol solutions to obtain concentrations of 10 <4>, 10 <5>, 10 <6>, 10 <7 > CFU / microwell (final volume of 100 Î1⁄4Î / microwell) in 96-well microtiter plates. The final tested concentrations of TTO and terpinen-4-ol ranged from 0.0078% to 4% v / v. The strain used in this study as a quality control was as described in the CLSI standard protocol. Viable inoculum was determined with CFU count (in BCYE-alpha agar medium). The experiments were performed with a final inoculum of approximately 5xl0 <4> CFU / ml for E. coli ATCC 25922.
Solubilizzazione di TTO e di terpinene-4-olo Solubilization of TTO and terpinene-4-ol
Esperimenti preliminari sono stati condotti per valutare la minima concentrazione di Tween 80 necessaria per solubilizzare TTO e in grado di mantenere la vitalità di L. pneumophila. Diluizioni seriali di Tween 80 sono state eseguite in duplicato dalla concentrazione di 1% a 0,0015% v/v in brodo BYE-alfa in piastre a 96 pozzetti da microtitolazione. 10<®>batteri per mi sono state inoculate ad una concentrazione finale di ~2.5xl0<6>cellule/pozzetto .Dopo coltura di 72 ore a 36 ± 1°C in atmosfera di 2,5% C02, la densità ottica à ̈ stata misurata spettrofotometricamente a 600 nra, e il numero di cellule di Legionella in ogni pozzetto à ̈ stato determinato con la conta CFU in terreno agar BCYE-alfa. Preliminary experiments were conducted to evaluate the minimum concentration of Tween 80 necessary to solubilize TTO and able to maintain the viability of L. pneumophila. Serial dilutions of Tween 80 were performed in duplicate from the concentration of 1% to 0.0015% v / v in BYE-alpha broth in 96-well microtiter plates. 10 <®> bacteria per mi were inoculated to a final concentration of ~ 2.5xl0 <6> cells / well. After 72 hour culture at 36 ± 1 ° C in an atmosphere of 2.5% C02, the optical density is ̈ was measured spectrophotometrically at 600 nra, and the number of Legionella cells in each well was determined with the CFU count in BCYE-alpha agar medium.
Determinazione della minima concentrazione inibente e battericida Determination of the minimum inhibitory and bactericidal concentration
Saggi di suscettibilità al TTO e al terpinene-4-olo sono stati eseguiti secondo il metodo di micro-diluizione CLSI in brodo, con le seguenti modifiche: 1) BYEB invece di brodo Muller-Hinton, 2) emulsionante Tween 80, 3) la grandezza dell'inoculo IO<8>invece di IO<5>CFU/ml, 4) uso di una pellicola sigillante sterilizzata trasparente per micropiastre (TMS) durante l'incubazione per 72 ore. TTO e terpinene-4 -olo sono stati diluiti con BYEB in presenza di Tween 80 a 0,001% v/v. Aliquote di 50 Î1⁄4Î di diluizioni a raddoppio di soluzioni di TTO o di terpinene-4-olo sono stati distribuiti in piastre da micro-titolazione a 96 pozzetti. La concentrazione finale dell'OE e del componente varia da 0,0078% a 4% v/v per TTO e terpinene-4-olo. Per l'inoculo, sono state sospese in acqua distillata sterile colture di 72 ore in agar di L.pneumophila alla densità ottica 0,6 misurata a 600 nm (≡10<9>CFU/ml), mescolate con "vortex" e diluite per dare IO<8>CFU/ml. 50 Î1⁄4Î di ciascuna sospensione diluita (IO<8>CFU/ml) à ̈ stata aggiunta in duplicato in diluizioni a raddoppio di soluzioni di TTO o di terpinene-4-olo ad una concentrazione finale di ~2.5 xl0<e>cellule/pozzetto. Le micropiastre sono state poi incubate a 36 ± 1° C con 2,5% di C02per 72 ore coperte con o senza pellicola sigillante sterilizzata trasparente per micropiastre (TMS) (AMP Llseal, Greiner Bio-One, Germania). Il controllo della crescita batterica à ̈ stata effettuata in terreno privo di TTO e di terpinene-4-olo con aggiunta di Tween 80 allo 0,001% v/v. Ogni vaschetta includeva una colonna di sterilità (otto pozzetti) non inoculata. TTO and terpinen-4-ol susceptibility assays were performed according to the CLSI broth micro-dilution method, with the following modifications: 1) BYEB instead of Muller-Hinton broth, 2) Tween 80 emulsifier, 3) la inoculum size IO <8> instead of IO <5> CFU / ml, 4) use of a sterilized transparent microplate sealant (TMS) during incubation for 72 hours. TTO and terpinen-4 -ol were diluted with BYEB in the presence of Tween 80 at 0.001% v / v. Aliquots of 50 Î1⁄4ε doubled dilutions of TTO or terpinen-4-ol solutions were distributed in 96-well micro-titration plates. The final OE and component concentration ranges from 0.0078% to 4% v / v for TTO and terpinene-4-ol. For inoculation, 72-hour agar cultures of L. pneumophila at optical density 0.6 measured at 600 nm (â ‰ ¡10 <9> CFU / ml) were suspended in sterile distilled water, mixed with "vortex" and diluted to give 10 <8> CFU / ml. 50 Î1⁄4Î of each diluted suspension (10 <8> CFU / ml) was added in duplicate in two-fold dilutions of TTO or terpinen-4-ol solutions to a final concentration of ~ 2.5 xl0 <e> cells / cockpit. The microplates were then incubated at 36 ± 1 ° C with 2.5% CO2 for 72 hours covered with or without sterilized transparent microplate sealant (TMS) (AMP Llseal, Greiner Bio-One, Germany). The control of bacterial growth was carried out in a medium without TTO and terpinen-4-ol with the addition of Tween 80 at 0.001% v / v. Each tray included an uninoculated sterility column (eight wells).
I valori della concentrazione minima inibente (MIC) sono stati definiti come la più bassa concentrazione di TTO o di terpinene-4-olo che mostrava un'inibizione della crescita del 100% rispetto al controllo di crescita di L.pneumophila, privo di olio, come risultava spettrofotometricamente o visualmente. I valori della concentrazione minima battericida (MBC) sono stati determinati piastrando 10 Î1⁄4Î di ogni pozzetto, senza crescita apparente, su un terreno agar BCYE-alfa e incubato a 36 1° C con 2,5% di C02. Dopo 72 ore à ̈ stata valutata la vitalità . Ogni esperimento à ̈ stato condotto in doppio. La MBC era definita come la più bassa concentrazione di olio che causa la morte del 99,9% dell'inoculo iniziale. Minimum inhibitory concentration (MIC) values were defined as the lowest concentration of TTO or terpinen-4-ol showing 100% growth inhibition compared to the growth control of L.pneumophila, oil-free, as it turned out spectrophotometrically or visually. Minimum bactericidal concentration (MBC) values were determined by plating 10 Î1⁄4Î of each well, with no apparent growth, on BCYE-alpha agar medium and incubated at 36 1 ° C with 2.5% C02. Viability was assessed after 72 hours. Each experiment was conducted in duplicate. MBC was defined as the lowest oil concentration causing 99.9% death in the initial inoculum.
Metodo di diffusione in agar Method of diffusion in agar
In questo saggio à ̈ stato utilizzato il terreno agar BCYE-alfa con aggiunta di Tween 80 sterile (0,01% v/v) per analizzare la suscettibilità al terpinene-4-olo di ceppi di L. pneumophila di riferimento, clinici e ambientali Lpl e Lp6.Sono stati distribuiti circa 1x10<s>CFU/ mi sul terreno solido.Un disco di carta del diametro di 6 mm (Oxoid, LTD, Basingstoke, Inghilterra) à ̈ stato posto sulla superficie di agar inoculato e, impregnato con 10 Î1⁄4Î di terpinene-4-olo puro. Tutte le piastre sono state sigillate con due fogli di parafilm (Pechiney Plastic Packaging, USA), incubate a 36 ± 1°C con 2,5% di C02e osservate dopo due e sette giorni per verificare se gli effetti erano stati batteriostatici o battericidi. E' stato utilizzato come controllo di crescita positivo agar BCYE-alfa con Tween 80 (0,01%), ma senza olio. Ogni prova à ̈ stata effettuata in duplicato ed i valori medi della zona di inibizione della crescita sono stati determinati. Il diametro di inibizione à ̈ stato misurato mediante un righello e, quando non si verificava crescita su tutta la superficie della piastra, la zona di inibizione veniva registrata come >90 mm, che corrispondeva al diametro interno della piastra. In this assay, BCYE-alpha agar medium with the addition of sterile Tween 80 (0.01% v / v) was used to analyze the susceptibility to terpinen-4-ol of reference, clinical and environmental strains of L. pneumophila Lpl and Lp6. Approximately 1x10 <s> CFU / ml were distributed on the solid medium. A 6 mm diameter paper disc (Oxoid, LTD, Basingstoke, England) was placed on the inoculated agar surface and impregnated with 10 Î1⁄4Î of pure terpinene-4-ol. All plates were sealed with two parafilm sheets (Pechiney Plastic Packaging, USA), incubated at 36 ± 1 ° C with 2.5% C02 and observed after two and seven days to verify whether the effects were bacteriostatic or bactericidal. BCYE-alpha agar with Tween 80 (0.01%) was used as a positive growth control, but without oil. Each test was performed in duplicate and the mean values of the growth inhibition zone were determined. The diameter of inhibition was measured using a ruler and, when no growth occurred over the entire surface of the plate, the zone of inhibition was recorded as> 90 mm, which corresponded to the internal diameter of the plate.
Metodo di diffusione in miero-atmosfera Method of diffusion in the miero-atmosphere
Un metodo di diffusione in agar, leggermente modificato, à ̈ stato utilizzato per stimare l'attività di terpinene-4-olo, in fase di vapore, su ceppi di L. pneumophila di riferimento, clinici ed ambientali Lpl e Lp6 (27,28). Un micro-coprivetrino (Prestige, Italia, 22 mm x 22 mm) à ̈ stato attaccato con una goccia di smalto commerciale sul coperchio superiore di una piastra di Petri. Successivamente un disco di carta (Oxoid, Unipath LTD, Basingstoke, Inghilterra) di 6 mm di diametro, inumidito con 10 Î1⁄4Î di terpinene-4-olo puro, à ̈ stato messo al centro di questo vetrino. La superficie del disco era ad una distanza di circa 4 mm dalla superficie di crescita dell'organismo saggiato. Il coperchio superiore delle piastre à ̈ stato poi invertito, sigillato con parafilm e incubato a 36 ± 1°C con 2,5% di C02e osservato dopo due e sette giorni. Agar BCYE-alfa con Tween 80 (0,01%) senza olio, à ̈ stato utilizzato come controllo di crescita positivo. Lo spazio all'interno della capsula di Petri chiusa era pari a ~60 cm<3>di aria.Ogni saggio à ̈ stato eseguito in duplicato ed i valori medi della zona di inibizione della crescita sono stati determinati dopo due e sei giorni. I risultati sono stati dati come sopra descritto nel metodo di diffusione in agar. A slightly modified agar diffusion method was used to estimate the activity of terpinen-4-ol, in the vapor phase, on reference, clinical and environmental L. pneumophila strains Lpl and Lp6 (27,28 ). A micro-coverslip (Prestige, Italy, 22mm x 22mm) was attached with a drop of commercial glaze to the top lid of a Petri dish. Subsequently a 6 mm diameter paper disc (Oxoid, Unipath LTD, Basingstoke, England), moistened with 10 Î1⁄4Î of pure terpinen-4-ol, was placed in the center of this slide. The disc surface was approximately 4 mm from the growth surface of the test organism. The top lid of the plates was then inverted, sealed with parafilm and incubated at 36 ± 1 ° C with 2.5% C02e observed after two and seven days. BCYE-alpha Agar with Tween 80 (0.01%) without oil was used as a positive growth control. The space inside the closed Petri dish was ~ 60 cm <3> of air. Each assay 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 the agar diffusion method.
Risultati Results
Effetto di Tween 80 sulla crescita di L. pneumophila Effect of Tween 80 on L. pneumophila growth
Al fine di escludere l'interferenza del detergente nel nostro saggio abbiamo effettuato esperimenti preliminari nella nostra condizione di lavoro per stabilire la concentrazione solubilizzante corretta, ma non-inibitoria di Tween 80. In effetti, e come già descritto (33), detergenti vari potrebbero avere un effetto inibitorio nei confronti di alcune specie di Legionella in funzione della concentrazione. L'attività inibitoria di Tween 80 sulla crescita di L. pneumophìla SG1 e SG6 à ̈ stato testato con concentrazioni di Tween 80 variabili dall'1% allo 0,0015% v/v. È stato osservato che concentrazioni superiori allo 0,06% inibivano la crescita di tutti i ceppi testati, a concentrazioni più basse (tra 0,06% e 0,015% v/v) di Tween 80 erano ancora presenti batteri vitali, come determinato dalla conta delle CFU.Poiché nessun effetto tossico à ̈ stato evidenziato con concentrazioni di Tween 80 inferiori, tutti gli ulteriori esperimenti sono stati realizzati con una concentrazione di Tween 80 di 0,001 % v/v. In order to rule out detergent interference in our assay we carried out preliminary experiments in our working condition to establish the correct, but non-inhibitory solubilizing concentration of Tween 80. Indeed, and as already described (33), various detergents could have an inhibitory effect against some Legionella species as a function of concentration. The inhibitory activity of Tween 80 on the growth of L. pneumophìla SG1 and SG6 has been tested with Tween 80 concentrations ranging from 1% to 0.0015% v / v. Concentrations above 0.06% were observed to inhibit 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 count. Since no toxic effect was shown with lower Tween 80 concentrations, all further experiments were performed with a Tween 80 concentration of 0.001% v / v.
Attività di terpinene-4-olo anti-Legionella Anti-Legionella terpinene-4-ol activity
Prima di esaminare l'attività di terpinene-4-olo contro tutti i diversi ceppi di Legionella, gli esperimenti sono stati condotti con il metodo di microdiluizione in saggi ripetuti su ceppi di riferimento di L. pneumophila SG 1 o 6. In questi esperimenti, inizialmente senza TMS, abbiamo notato una variazione inter-sperimentale molto grande, associata a bassissimi valori di MIC. Di conseguenza, abbiamo fatto ricorso all'uso di una TMS, che à ̈ stata messa su ogni singolo pozzetto, impedendo così che la diffusione del vapore si potesse estendere sui pozzetti con differenti concentrazioni di terpinene-4 -olo oltre quelle del TTO. Before examining the activity of terpinen-4-ol against all the different Legionella strains, the experiments were conducted with the microdilution method in repeated assays on reference strains of L. pneumophila SG 1 or 6. In these experiments, initially without TMS, we noticed a very large inter-experimental variation, associated with very low MIC values. Consequently, we resorted to the use of a TMS, which was placed on each individual well, thus preventing the diffusion of vapor from spreading over the wells with different concentrations of terpinene-4 -ol beyond those of the TTO.
Con questa modifica, la MIC ripetuta di L. pneumophila sierotipo 1 e 6 misurava costantemente 0,25% v/v. Analogamente, la MBC altamente variabile in esperimenti ripetuti in pozzetti non sigillati, si conformava allo 0.5% v/v, in condizioni sigillate. Così, tutte le determinazioni ulteriori delle MIC e delle MBC sono state effettuate con tutti i ceppi in micropiastre sigillate. With this modification, the repeated MIC of L. pneumophila serotype 1 and 6 consistently measured 0.25% v / v. Similarly, the highly variable MBC in repeated experiments in unsealed wells conformed to 0.5% v / v under sealed conditions. Thus, all further MIC and MBC determinations were performed with all strains in sealed microplates.
La tabella 1 mostra l'attività di terpinene-4-olo, in confronto con TTO, sia come MIC e MBC, per ogni singolo ceppo di Lp. Table 1 shows the activity of terpinen-4-ol, in comparison with TTO, both as MIC and MBC, for each individual Lp strain.
Tabella 1 Table 1
Attività di tea tree oil e terpinene-4 -olo nei confronti di singoli ceppi di Legionella pneumophila con metodo di micro-diluizione con pellicola sigillante Activity of tea tree oil and terpinene-4 -ol against single strains of Legionella pneumophila with micro-dilution method with sealing film
Tea tree oil Terpinene-4 -olo Microrganismi MIC% MBC% MIC% MBC% Tea tree oil Terpinen-4 -ol Microorganisms MIC% MBC% MIC% MBC%
(v/v) (v/v) {v/v) {v/v) Lpl ATCC 0,25 0,5 0,125 0,5 .Lpl 1/3224* 0,25 1,0 0,125 0,5 .Lpl 2510* 0,25 0,5 0,06 0,5 Lpl 41/2883* 0,25 0,5 0,125 0,5 Lpl 3.260* 0,125 0,5 0,125 0,5 Lpl 4.357* 0,25 0,5 0,125 0,5 Lpl 73** 0,25 0,5 0,125 0,5 Lpl 66** 0,25 0,5 0,125 0,5 Lpl 55/3646 ** 0,25 1,0 0,125 0,5 -Lpl 2258** 0,25 0,5 0,125 0,5 1.Lpl 3.261 ** 0,25 0,5 0,125 0,5 .Lp6 ATCC 33215 0,25 0,5 0,125 0,5 .Lp6 11/2378 * 0,5 0,5 0,125 0,5 .Lp6 51/3380 * 0,25 0,5 0,125 0,5 .Lp6 3265 * 0,25 0,5 0,125 0,5 .Lp6 3374 * 0,25 0,5 0,125 0,5 .Lp6 3303 * 0,25 0,5 0,25 0,5 .Lp6 4405 ** 0,25 0,5 0,125 0,25<♦>isolato clinico; ** isolato ambientale (v / v) (v / v) {v / v) {v / v) Lpl ATCC 0.25 0.5 0.125 0.5 .Lpl 1/3224 * 0.25 1.0 0.125 0.5 .Lpl 2510 * 0.25 0.5 0.06 0.5 Lpl 41/2883 * 0.25 0.5 0.125 0.5 Lpl 3.260 * 0.125 0.5 0.125 0.5 Lpl 4.357 * 0.25 0.5 0.125 0.5 Lpl 73 ** 0.25 0.5 0.125 0.5 Lpl 66 ** 0.25 0.5 0.125 0.5 Lpl 55/3646 ** 0.25 1.0 0.125 0.5 -Lpl 2258 ** 0.25 0.5 0.125 0.5 1.Lpl 3.261 ** 0.25 0.5 0.125 0.5 .Lp6 ATCC 33215 0.25 0.5 0.125 0.5 .Lp6 11/2378 * 0, 5 0.5 0.125 0.5 .Lp6 51/3380 * 0.25 0.5 0.125 0.5 .Lp6 3265 * 0.25 0.5 0.125 0.5 .Lp6 3374 * 0.25 0.5 0.125 0 , 5 .Lp6 3303 * 0.25 0.5 0.25 0.5 .Lp6 4405 ** 0.25 0.5 0.125 0.25 <â ™ ¦> clinical isolate; ** environmental isolated
La tabella 2 confronta la MIC50e la MIC90dei due sierogruppi isolati. In entrambe le tabelle, l'uniformità dei valori di MIC allo 0,25% v/v ed i valori di MBC allo 0,5% v/v sono evidenziati, con rare eccezioni, e non vi à ̈ nessuna differenza tra i due sierogruppi o tra gli isolati ambientali e clinici. Table 2 compares the MIC50 and MIC90 of the two isolated serogroups. In both tables, the uniformity of MIC values at 0.25% v / v and MBC values at 0.5% v / v are highlighted, with rare exceptions, and there is no difference between the two. serogroups or between environmental and clinical isolates.
Tabella 2 Table 2
Attività di terpinene-4-olo nei confronti di Legionella pneumophila con metodo di micro-diluizione con pellicola sigillante Activity of terpinen-4-ol against Legionella pneumophila by micro-dilution method with sealing film
Organismi n MIC50% MIC90% MIC range MBC50% MBC90% MBC v/v v/v % v/v v/v v/v range 3⁄4 v/v Organisms n MIC50% MIC90% MIC range MBC50% MBC90% MBC v / v v / v% v / v v / v v / v range 3⁄4 v / v
Lpl<§>Il 0,125 0,125 0,06-0,125 0,5 0,5 0,5 Lpl <§> The 0.125 0.125 0.06-0.125 0.5 0.5 0.5
Lp6<55>7 0,125 0,125 0,125-0,25 0,5 0,5 0,25-0,5 * (1 ceppo di riferimento ATCC 33152 5 ceppi clinici e 5 ambientali) Lp6 <55> 7 0.125 0.125 0.125-0.25 0.5 0.5 0.25-0.5 * (1 reference strain ATCC 33152 5 clinical and 5 environmental strains)
* * (1 ceppo di riferimento ATCC 33215 5 ceppi clinici e 1 ambientale) * * (1 reference strain ATCC 33215 5 clinical and 1 environmental strains)
n, numero â–¡â–¡ n, number â – ¡â – ¡
Metodo di diffusione in micro-atmosfera Micro-atmosphere diffusion method
Gli esperimenti di cui sopra suggerivano l'importanza dei vapori del terpinene-4-olo nell'attività anti-Legionella.Ad ulteriore conferma di questo dato, abbiamo testato l'attività di terpinene-4-olo e TTO con il metodo di diffusione in microatmosfera, che ha permesso il contatto dei soli vapori di TTO o di terpinene-4-olo con le cellule batteriche (vedi Materiali e Metodi).Come indicato nella tabella 3, dopo due giorni à ̈ stata osservata un'inibizione totale della crescita (il diametro della zona di inibizione à ̈ uguale al diametro della capsula di Petri) . Dopo 7 giorni, l'inibizione era ancora totale per tutti i ceppi, tranne che per Lp6 ATCC, suggerendo che il vapore rilasciato da una soluzione pura di terpinene-4-olo à ̈ sufficiente da solo a uccidere i batteri nelle condizioni del saggio di micro-atmosfera. I dati ottenuti con il metodo standard di diffusione con disco, ha mostrato l'inibizione totale della crescita di L. pneumophila dopo due giorni, ma una crescita parziale dopo 7 giorni (dati non riportati). The above experiments suggested the importance of terpinen-4-ol vapors in the anti-Legionella activity. To further confirm this data, we tested the activity of terpinen-4-ol and TTO with the diffusion method in microatmosphere, which allowed only TTO or terpinene-4-ol vapors to come into contact with bacterial cells (see Materials and Methods) .As indicated in Table 3, a total growth inhibition was observed after two days ( the diameter of the inhibition zone is equal to the diameter of the Petri dish). After 7 days, the inhibition was still total for all strains except Lp6 ATCC, suggesting that the vapor released by a pure solution of terpinen-4-ol is sufficient alone to kill the bacteria under the conditions of the test. micro-atmosphere. Data obtained with the standard disc diffusion method showed total growth inhibition of L. pneumophila after two days, but partial growth after 7 days (data not shown).
Tabella 3 Table 3
Metodo di diffusione in micro-atmosfera: effetto dei vapori di terpinene-4-olo sulla crescita di sierogruppi di Legionella pneumophila Micro-atmosphere diffusion method: effect of terpinen-4-ol vapors on the growth of Legionella pneumophila serogroups
Terpinene-4-olo Microrganismi Controllo Due giorni di Sette contatto<5>giorni di contatto<®>Lpl ATCC 33152 0 90 90 Lpl 2510* 0 90 90 Lpl 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 Terpinen-4-ol Microorganisms Control Two days of Seven contact <5> days of contact <®> Lpl ATCC 33152 0 90 90 Lpl 2510 * 0 90 90 Lpl 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
* zona di inibizione in rara * zone of inhibition in rare
* isolato clinico; isolato ambientale ** â–¡ * clinical isolate; environmental isolated ** â – ¡
Bibliografia Bibliography
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| PCT/IT2011/000267 WO2012014244A1 (en) | 2010-07-26 | 2011-07-25 | USE OF TERPINEN-4-ol AS ANTIMICROBIAL AGENT AGAINST BACTERIA OF LEGIONELLA GENUS |
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| WO1988010122A1 (en) * | 1987-06-22 | 1988-12-29 | The Commonwealth Industrial Gases Limited | Treatment of air conditioning system |
| US20040071757A1 (en) * | 2001-11-20 | 2004-04-15 | David Rolf | Inhalation antiviral patch |
| FR2918571A1 (en) * | 2007-07-10 | 2009-01-16 | Aroma Technologies Soc Par Act | ANTIBIOTIC COMPOSITIONS BASED ON ESSENTIAL OILS PROPHYLAXIS AND TREATMENT OF NOSOCOMIAL INFECTIONS |
| KR20090029431A (en) * | 2007-09-18 | 2009-03-23 | (주)지앤지 | A composition using antibacterial containing chamaecyparis obtusa oil |
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| WO1988010122A1 (en) * | 1987-06-22 | 1988-12-29 | The Commonwealth Industrial Gases Limited | Treatment of air conditioning system |
| US20040071757A1 (en) * | 2001-11-20 | 2004-04-15 | David Rolf | Inhalation antiviral patch |
| FR2918571A1 (en) * | 2007-07-10 | 2009-01-16 | Aroma Technologies Soc Par Act | ANTIBIOTIC COMPOSITIONS BASED ON ESSENTIAL OILS PROPHYLAXIS AND TREATMENT OF NOSOCOMIAL INFECTIONS |
| KR20090029431A (en) * | 2007-09-18 | 2009-03-23 | (주)지앤지 | A composition using antibacterial containing chamaecyparis obtusa oil |
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| DATABASE WPI Week 200927, Derwent World Patents Index; AN 2009-H18012, XP002624390 * |
| MONDELLO F ET AL: "Determination of Legionella pneumophila susceptibility to Melaleuca alternifolia Cheel (tea tree) oil by an improved broth micro-dilution method under vapour controlled conditions", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 77, no. 2, 1 May 2009 (2009-05-01), pages 243 - 248, XP026048408, ISSN: 0167-7012, [retrieved on 20090303], DOI: DOI:10.1016/J.MIMET.2009.02.012 * |
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