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Synergism and postantibiotic effect of tobramycin and Melaleuca alternifolia (tea tree) oil against Staphylococcus aureus and Escherichia coli.

ABSTRACT

The application of antimicrobial combinations may address the rising resistance to established classes of both systemic and topical agents and their clinical relevance is related to the presence of a significant postantibiotic effect (PAE). We investigated the effectiveness in vitro of the association between tobramycin and tea tree oil (TTO) against Gram-positive and Gram-negative bacteria. The minimal inhibitory concentrations, the bacterial killing and the PAE of tobramycin and TTO were determined both singly and in combination against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. A synergistic interaction was observed against both strains tested: the mean PAEs were 1.3 and 1.7 h for tobramycin against E. coli and S. aureus respectively, 10.8 h for tobramycin and TTO (0.05%) against E. coli, 10.4 h and 17.4 h against S. aureus for tobramycin and TTO (0.25 and 0.50%, respectively). Longer PASMEs were observed with S. aureus after TTO/tobramycin exposure. In vitro interactions can improve the antimicrobial effectiveness of the antibiotic and may contribute for the development of novel topical agents for the treatment of skin lesions including conjunctiva and respiratory infections by inhalation.

[c] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Post antibiotic effect

Tea tree oil

Melaleuca alternifolia

Tobramycin

Synergism

Introduction

Plant essential oils are well known for their pharmacological activities, including antibacterial activity, and may represent a promising source for new natural drugs (Dorman and Deans 2000). Melaleuca alternifolia essential oil, also known as tea tree oil (TTO), is incorporated in topical formulations for the treatment of cutaneous infections (Carson et al. 2006; Hammer et al. 2006). The broad-spectrum antimicrobial activity of TTO is mainly attributed to terpinen-4-ol and 1,8- cineole, major components of the oil, and includes antibacterial, antifungal, antiviral, antiprotozoal and antimycoplasmal activities, all promoting TTO as therapeutic agent (Furneri et al. 2006).

The emergence and spread of antibiotic resistance at elevated concentrations of the antimicrobial has significantly affected the use of established antibiotics and poses a serious threat to public health worldwide. Between aminoglycosides, tobramycin (TOB) shows excellent in vitro and clinical activities against susceptible strains of E. coli and S. aureus, both inhibiting plasma membrane functions and specifically targeting the ribosomal A site. However, a growing number of reports document resistance to this antibacterial agent (Oteo et al. 2006; Cuevas et al. 2002). As a result, non traditional agents may be used in combination with antibiotics in order to provide better efficacy for combating various infections and drug resistance. The pharmacodynamic drug interaction is generally described by qualitative terms, such as synergy, indifference, and antagonism. The postantibiotic effect (PAE) represents the delay before regrowth of bacteria following a short antibiotic exposure. Other basic pharmacodynamic properties include the rate of bacterial killing at different concentrations and different inocula, as well as the postantibiotic sub-MIC effect (PASME), which describes the effect of subihibitory concentrations after exposure to suprainhibitory concentrations (Odenholt 1993). Such properties are useful for the evaluation of the pharmacokinetic/pharmacodynamic indices correlated with the efficacy of antimicrobial agents. With the goal of developing a new highly active antibacterial therapeutic combination of essential oils and synthetic antibacterial agents, we investigated the in vitro association properties of tobramycin and TTO against S. aureus and E. coli: checkerboard microdilution assay, timekill methods, PAE and PASMEs were determined. The association tobramycin/TTO could be incorporated into topical formulations for the treatment of staphylococcical and E. coli skin lesions, as well as for respiratory and lung infections by inhalation.

Materials and methods

Drugs used

The tobramycin was dissolved in 1 ml of phosphate-buffered saline (PBS) pH 7.2 and thereafter diluted in Mueller-Hinton broth (MHB). Australian TTO was a high pH distillation of a flush growth (young leaves) sample of Melaleuca alternifolia. The oil was diluted 1:10 in methanol prior to GC injection. For GC-FID analyses a Shimadzu GC-2010 system, equipped with an AOC-20i autosampler and a split/splitless injector was used. The column was a SLB-5MS Supelco (Milan, Italy), 30 m x 0.25 [micro]m i.d. x 0.25 [micro]md, coated with 5% diphenyl-95% polydimethylsiloxane, operated with the following oven program temperature: 45 [degrees]C, held 6 min, at 3 [degrees]C/min to 250[degrees]C. GC-MS analyses were carried out on a GCMS-QP2010 system (Shimadzu). Data handling was supported by the software GCMS solution ver. 2.51 (Shimadzu). For the identification of compounds, a GC-MS database dedicated to flavour and fragrance material (FFNSC ver.1.2, Shimadzu) was used along with comparison of Linear Retention Indices with those reported by specific literature (Shellie et al. 2003). The major component of TTO was terpinen-4-ol (40%), followed by [gamma]-terpinen (13%), p-cimene (13%), and [alpha]-terpinen (4%). Other compounds included eucalyptol (3%), [alpha]-pinene (3%), [alpha]-terpineol (3%), terpinolene (2%), [beta]-phellandrene (2%). Minor indentified compounds were the following: [alpha]-thujene (1%), aromadendrene (1%) [beta]-pinene (0.8%), [delta]-cadinene (0.8%), sabinene (0.6%), myrcene (0.6%), viridiflorene (0.6%), transascaridol glycol (0.5%), zonarene (0.4%), globulol (0.4%), cis-para-mentha-2-en-l-ol (0.3%), transpara- mentha-2-en-1-ol (0.3%), [delta]-elemene (0.3%), [alpha]-gurjunene (0.3%), caryophyllene (0.3%). Pure essential oil was dissolved in MHB.

Fresh solutions of tobramycin and TTO were made on the days of experiments.

Bacterial strains and antimicrobial testing

The bacteria used were S. aureus ATCC 29213 and E. coli ATCC 25922. Both strains were grown in MHB at 37[degrees]C overnight, resulting in approximately 5 x [10.sup.8]CFU/ml. The MICs of TOB and TTO were determined by the broth microdilution method, according to CLSI (Clinical and Laboratory Standards Institute 2008). The MICs were also performed in the Bioscreen C (Labsystems Oy, Helsinki, Finland) for all strains as follows: twofold serial dilutions of drug were added to broth in 400 [micro]1 wells microplate and inoculated with the test strains yielding to a bacterial density of approximately 5 x [l0.sup.5] CFU/ml. After 20 h incubation in the Bioscreen C, the MIC was defined as the lowest concentration which prevented growth, as determined by optical density (Lowdin et al. 1993). The lowest detectable level of OD for the strains was approximately 5 x [10.sup.1] CFU/ml. MIC determinations were done in triplicate in three independent assays.

In the combination assays, the "checkerboard" procedure described by White et al. (1996) was followed. This method allows varying the concentrations of each antimicrobial along the different axes, thus ensuring that each well contained a different combination. The combination assays were performed in a Bioscreen honeycomb 100-well plates containing appropriate media with test compounds represented by TOB serially diluted on the x axis, ranging from 0 to 4 x MIC, and increasing concentrations of TTO ranging from 0 to 4 x MIC on the y axis. Diluted cell cultures were then added and the bacterial growth was monitored using Bioscreen C for 20 h. The [OD.sub.540] was measured at 10 min intervals. Controls grown with equivalent levels of media were included in all assays.

MIC data of TOB and TTO were converted into fractional inhibitory concentration (FIC), defined as ratio of the concentration of the antimicrobial in an inhibitory concentration with a second compound to the concentration of the antimicrobial by itself (Te Dorsthorst et al. 2002).

FICA = MIC of A with B/MIC of A

The FIC index was then calculated as follows: FIC index = FICA+FICB.

Time-kill curves

Tubes containing TOB at concentrations corresponding to 1, 2, 4, 8, 16 and 32 x MIC were inoculated with a suspension of each test strain, yielding to a final bacterial density of 5 x [10.sup.5] CFU/ml and then incubated at 37 [degrees]C in a shaking incubator. A growth control was also performed. Samples for viable counting were withdrawn at 0, 1, 2, 4, 8, 12 and 24 h and, if necessary, diluted in fresh medium. At least four dilutions of each sample were spread on agar plates (MHA), incubated at 37[degrees]C and counted after 24 h. Time-kill studies were also performed using a combination of TOB and TTO: solutions of the two drugs were added to tubes containing a suspension of each test strain giving a final bacterial concentration of 5 x [10.sup.5] CFU/ml. The final concentrations of drugs in each tube for E. coli were the following: (1) control (without drug); (2) TTO (0.05% v/v); (3) TOB (1 x MIC) and TTO (0.05% v/v); (4) TOB (2 x MIC) and TTO (0.05% v/v); (5) TOB (1 x MIC); (6) TOB (2 x MIC). The final concentrations in each tube for S. aureus were the following: (1) control (without drug); (2) TTO (0.50% v/v); (3) TTO (0.25% v/v); (4) TOB (2 x MIC) and TTO (0.50% v/v); (5) TOB (2 x MIC) and TTO (0.25%v/v); (6) TOB (2 x MIC). Samples for viable counting were drawn at 0, 2, 4, 8, 12 and 24 h. All the experiments were performed in triplicate for each strain.

PAEs and PASMEs of tobramycin and TTO

PAEs and PASMEs determinations were performed in the Bioscreen C. An inoculum of approximately 5 x [10.sup.7] CFU/ml was exposed to each drug (alone or in combination) for 1 h at 37 [degrees]C in a shaking incubator. The final concentration of drugs in each tubes were as described above, with the exception of TOB concentration (4 x MIC). After exposure the strains were washes three times, centrifuged each time for 5 min at 1400 g and thereafter diluted in fresh medium. The unexposed control strains were washed similarly but then diluted [10.sup.-1], [10.sup.-2], [10.sup.-3], [10.sup.-4] and [10.sup.-5], in order to obtain inocula close to those of the exposed strains. Viable counts of both controls and exposed strains were performed before drug exposure, after 1 h induction and after washing. Thereafter, a volume of 40 [micro]l of each sample was inoculated into a microtiter well together with 360 [micro]l of fresh medium and incubated in the Bioscreen C at 37 [degrees]C. Growth curves were automatically measured as optical density (OD) at 540 nm every 10 min for 20 h. The PAE was calculated as the difference of time for the exposed curve and the corresponding control to reach a definite point ([A.sub.50]) on the OD curve, corresponding to 50% of the maximal OD of the control (Odenholt 1993).

To determine the PASME, the strains in the postantibiotic phase, as well as the different dilutions of the control, were thereafter exposed to subinhibitory concentrations of MIC, corresponding to 0.1, 0.2 and 0.3 times the MICs of the TOB in MHB and incubated in the Bioscreen C as described above. The PASME was defined as the difference in time required for the cultures exposed to subinhibitory concentrations of MIC and the corresponding control to reach a definite point ([A.sub.50]) on the OD curve, as described above (Odenholt 1993).

All experiments were performed in triplicate.

Results

Minimum inhibitory concentrations

The MICs of TOB and TTO determined by the microdilution method were 0.39 mg/I and 0.25% (v/v) for E. coli and 0.20 mg/1 and 0.50% (v/v) for S. aureus, respectively. Analogue values were obtained in the Bioscreen C. In the combination assays, the FIC indices calculated for TOB and TTO were 0.37 and 0.62 against E. coli and S. aureus, respectively. Although the interpretation of the FIC indices depends on which of the several definitions described in the literature are used, in this study we have considered as synergistic if the FIC index is </ = 0.5, additive or indifferent if > 0.5 but < / = 4 and antagonistic if >4 (Visalli et al. 1998). Indifference to synergism was observed between tobramycin and TTO against S. aureus, whereas a more synergistic effect between the two compounds was obtained against E. coli.

Bacterial killing

Concentration-dependent killing was observed with TOB against E. coli and S. aureus (Fig. la and b). At 1 h there was a >5[1og.sub.10] difference in CFU between 1 x MIC and 32 x MIC with E. coli (Fig. 1a) and complete bacterial killing was achieved within 8 h exposure at 4, 8, 16 and 32 x MIC. Tobramycin gave overall less killing against S. aureus, although at 4h there was a > 4[log.sub.10] difference in CFU between 1 x MIC and 32 x MIC (Fig. lb). Complete bacterial killing was observed only after 24 h exposure at the concentration of 16 x MIC, whereas 32 x MIC caused the most prominent bactericidal effect. The killing curves of TTO against the two strains tested are shown in Fig. 2a and b, E. coli being more susceptible than S. aureus. As TTO concentrations of 1.00% v/v, 0.50% v/v and 0.25% v/v exerted a very rapid bactericidal effect against E. coli after 10 min (results not shown), a concentration of 0.05% v/v, corresponding to 1/5 x MIC, was chosen for killing experiments in association with TOB and PAE determination.

[FIGURE 1 OMITTED]

TTO alone (0.05% v/v) produced only an initial slight reduction of > 1[log.sub.10] with E. coli (Fig. 2a) at 1 h, after which the number of CFU constantly increased towards the control levels. In the combination assays, the association of TOB (1 x MIC) with TTO determined a reduction of > 5 [log.sub.10] for E. coli after 8 h incubation time, whereas a faster reduction was observed incubating with TOB (2 x MIC) and TTO (1/2 x MIC) (Fig. 2a). Concentration dependent killing was observed with TTO against S. aureus with a reduction of > 4[1og.sub.l0] after 24 h incubation time with 0.5% (v/v) TTO, whereas nearly 3 [log.sub.10] was observed with 0.25% (v/v) TTO under the same conditions. In the association killing curve a synergistic effect was observed when treating S. aureus with tobramycin in combination with TTO, and a concentration-dependent killing was exerted (Fig. 2b). A reduction of > [5log.sub.10] was shown after 4 and 8 h with TOB (2 x MIC) and TTO 0.50 (v/v) and 0.25% (v/v), respectively. This data confirmed the combination between TOB and TTO had a synergistic effect against the two stains investigated, the Gram-negative strain being overall more susceptible than the Gram-positive strain.

[FIGURE 2 OMITTED]

Postantibiotic effect and postantibiotic sub-MIC effect--TOB in combination with TTO

Tables 1 and 2 report the PAEs and PASMEs of E. coli and S. aureus, respectively. TOB alone exerted short PAEs against the two strains tested. Significantly longer PASMEs were noted against S. aureus compared with E. coli. TTO alone produced a concentration-dependent PAE (6.0 h at 1 x MIC and 3.8 h at 1/2 x MIC) against S. aureus. TOB and TTO acted synergistically against both S. aureus and E. coli and this trend was confirmed by longer PAEs observed with the association. In order to investigate the increase in the postantibiotic effect of the association antibiotic/TTO, bacterial cells were further incubated with sub-MIC concentrations of TOB: an increase in the PAE and very long PASMEs were recorded when 0.50% (v/v) TTO was used. However, different behavior was observed against E. coli, demonstrating different combination effects against Gram-positive and Gram-negative bacteria.
Table 1
PAE and PASME (mean [+ or -] SD) of tobramycin, tea tree oil and their
combination against E. coli.

E. coli ATCC 25922           PAE (h)       PASME (h) 0.1 x MIC

Tobramycin              1.3 [+ or -] 0.15   2.6 [+ or -] 0.18
TTO 0.05%               9.2 [+ or -] 0.02  -
Tobramycin with 0.05%  10.8 [+ or -] 0.04  13.6 [+ or -] 0.47
TTO

E. coli ATCC 25922     PASME (h) 0.2 x MIC  PASME (h) 0.3 x MIC

Tobramycin              3.3 [+ or -] 0.09    5.3 [+ or -] 0.11
TTO 0.05%              -                    -
Tobramycin with 0.05%  14.5 [+ or -] 0.45   15.0 [+ or -] 0.42
TTO

In vitro values are the means [+ or -] SD, n = 6.
TTO: tea tree oil.
-: not detected.

Table 2
PAE and PASME (mean [+ or -] SD) of tobramycin, tea tree oil at
different concentrations and their combinations against S. aureus.

S. aureus ATCC 29213         PAE (h)       PASME (h) 0.1 x MIC

Tobramycin              1.7 [+ or -] 0.09  19.9 [+ or -] 0.69
TTO 0.50%               6.0 [+ or -] 0.24  -
TTO 0.25%               3.8 [+ or -] 0.25  -
Tobramycin with 0.50%  17.4 [+ or -] 0.85  35.2 [+ or -] 1.52
TTO
Tobramycin with 0.25%  10.4 [+ or -] 0.69  14.1 [+ or -] 0.74
TTO

S. aureus ATCC 29213   PASME (h) 0.2 x MIC  PASME (h) 0.3 x MIC

Tobramycin              22.4 [+ or -] 1.2   24.6 [+ or -] 0.56
TTO 0.50%               -                   -
TTO 0.25%               -                   -
Tobramycin with 0.50%   53.1 [+ or -] 1.74  70.8 [+ or -] 1.96
TTO
Tobramycin with 0.25%   22.1 [+ or -] 1.26  33.9 [+ or -] 1.34
TTO

In vitro values are the means [+ or -]SD, n = 6.
TTO: tea tree oil.
-: not detected.


Discussion

In this study we report the antimicrobial activity of TTO against S. aureus and E. coli, and its synergistic effect when incorporated with tobramycin. As many authors indicate, it is rather difficult to give an unequivocal definition for synergy effect (Loewe 1957; Greco et al. 1995; Barrera et al. 2005) and the "isobole method" of Berenbaum (1989) seems one of the most demonstrative for the proof of synergy effects. The standardization of these techniques for laboratory testing is crucial for the development of combination therapies against the increasing numbers of multiple-drug resistant strains (Hemaiswarya et al. 2008). The mechanism of action of TTO against E. coli and S. aureus has partly been elucidated: leakage of potassium ions and inhibition of respiration occurred with both strains, with relevant changes in their morphology under electron microscopy (Gustafson et al. 1998). Most of the antimicrobial activity of TTO is attributed to terpinen-4-ol and [alpha]-terpineol, the former representing the major component of the oil (Raman et al. 1995). Other components showed some degree of antimicrobial activity, which has been correlated with the presence of functional groups, such as alcohols, and the solubility in biological membranes (Cristani et al. 2007). It has been recently demonstrated that monoterpenes contained in essential oils interact with model membranes and their antimicrobial effect may result from the damage of the microbial lipid membrane fraction, in relation to the lipid composition and the net surface charge of the microbial membranes, the Gram-negative outer membrane presenting a strong negative charge conferred by the lipopolisaccharide (Trombetta et al. 2005). Previous studies investigating the mechanisms involved in the PAE during exposure of E. coli to tobramycin have shown that a global macromolecular synthesis was inhibited, including DNA, RNA and protein synthesis (Gottfredsson et al. 1995). Furthermore, the presence of free tobramycin remaining inside the cells may partly cause growth retardation, as observed at concentration of 5 x the MIC. Tobramycin also induced PAE at sub-MIC levels, suggesting that continuous bacterial growth suppression can be sustained when the antibiotic concentration is below the MIC. The synergistic interaction of TOB and TTO against S. aureus and E. coli could result in structural conformation changes which significantly affect reaction with cell membranes thus enhancing uptake by the bacterial cells. Interactions between antimicrobial compounds can alter effectiveness and synergistic or antagonistic relationships may result in competition for possible primary target sites (Mandalari et al. 2007). If single constituents of a mono-extract or a multi-extract combination are able to affect several targets in an agonistic way, a synergistic multi-target effect occurs, which may involve enzymes, substrates, metabolites and proteins, receptors, ion channels, transport proteins, DNA/RNA, ribosomes, monoclonal antibodies and physicochemical mechanisms (Wagner and Ulrich-Merzenich 2009; Imming et al. 2006). Alternatively, interaction between different compounds may lead to changes in structural conformation, thus resulting in the reduction of inhibitory activity (Lis-Balchim and Deans 1997).

In conclusion we have shown that in vitro association between tobramycin and tea tree oil against E. coli and S. aureus was found to produce a substantial PAE, which may help the formulation of novel topical agents for the treatment of conjunctiva and respiratory infections by inhalation. However, the significance of these in vitro effects has to be evaluated in clinical trials.

Acknowledgments

We would like to thank Prof. Mondello from the University of Messina for providing the tea tree oil.

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Manuela D'Arrigo (a), Giovanna Ginestra (a), Giuseppina Mandalari (a), P.M. Furneri (b), G. Bisignano (a), *

(a) Pharmaco-Biological Department, University of Messina, Vill. SS. Annunziata 98168 Messina, Italy

(b) Department of Microbiological and Gynecological Sciences, University of Catania, Via Androne 81 Catania 95124, Italy

* Corresponding author. Tel: +390906766434; fax: +39090 6766474.

E-mail address: bisignano@pharma.unime.it (G. Bisignano).

doi: 10.1016/j.phymed.2009.07.008
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Author:D'Arrigo, Manuela; Ginestra, Giovanna; Mandalari, Giuseppina; Furneri, P.M.; Bisignano, G.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUIT
Date:Apr 1, 2010
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