Enhancement of aminoglycosides and β-lactams antibiotic activity by essential oil of Lippia sidoides Cham. and the Thymol

1 Introduction

The infections caused by bacteria resistant to the antibiotics constitute one of the largest problems faced by medicine, because they require more complex and expensive treatments, besides they are more difficult to diagnose and to treat (Alanis, 2005). The main mechanisms of bacterial resistance to the aminoglycosides are active efflux, alteration of the objective of the antibiotic to mutation or enzymatic inactivation and alteration of the permeability of the bacterium to the drug (Brooks et al., 2000). The β-lactams (BLA) act by blocking the synthesis of the bacterial cellular wall. The most important mechanisms of resistance to BLA are the cleavage for β-lactamases and changes in the Penicillin Binding Protein (PBP) (Levinson and Jawetz, 2005).

Many researchers have focused on the investigation of natural products as the source of new bioactive molecules because of failure of available antimicrobials to treat infectious diseases. Natural products of vegetable origin can alter the effect of antibiotics, be it increasing or reducing the antibiotic activity (Coutinho et al., 2008). The association between natural products and synthetic drugs has been studied in several works and the results are relevant and promising (Matias et al., 2011; Rodrigues et al., 2010; Coutinho et al., 2008).

The species Lippia sidoides Cham. (Verbenaceae), popularly known as “alecrim-pimenta”, is a bush with odoriferous leaves, typically found in the Northeast of Brazil. In popular medicine, this species has been recommended for external use, prepared by maceration of the leaves in alcohol or used directly for treatment of sore throat and inflammation of the gums, and also for preparation of liquid soap (Matos, 2002; Matos et al., 2004). The same preparations can be made with extracted essential oil of their leaves by hydrodistillation (Matos, 2002).

The chemical composition of the essential oil of the leaves has been described in some studies and the major representatives are thymol and carvacrol (Botelho et al., 2007; Fontenelle et al., 2007). The therapeutic effect of L. sidoides is attributed mainly to the presence of the thymol, however, there are other substances found coming from the vegetable material, possessing certain beneficial influence of the plant, constituting the call phytocomplex (Bush, 2002).

In this work we report the chemical composition of the leaves essential oil from L. sidoides and to determine their potentiation of the antibiotic activity of aminoglycosides and β-lactams.

2 Materials and methods

3 Results and discussion

The essential oil obtained by hydrodistillation presented an income of 1.06% (w/w). Regarding the chemical composition of EOLS, we could identify 97.82% of its chemical constitution, corresponding to 7 compounds (Table 1). The major constituents of the essential oil of L. sidoides were thymol (84.9%), Etil-methyl-carvacrol (5.33%) and p-cymene (3.01%).

In other studies, the majority representative, thymol, came in concentrations of 66.67% (Monteiro et al., 2007) and 59.65% (Camurça-Vasconcelos et al., 2008). The variations in the chemical composition of essential oils are mainly due to the environmental conditions, as soil type, time of the year and schedule of collection of the plants (Taylor et al., 2001).

The results show that there were no differences in the antimicrobial effect between EOLS and certain thymols for the method of microdiluition against to the bacteria in the study (Table 2). EOLS and thymol presented a MIC of 128 μg/mL for S. aureus, 256 μg/mL for S. mutans, K. pneumoniae, P. rettigeri, and E. cloacae and 512 μg/mL for E. faecalis, P. aeruginosa and E. coli.

The Gram-positive bacteria S. aureus was the most sensitive compared to the other stumps. On the other hand, the Gram-positive E. faecalis and the Gram-negatives E. coli and P. aeruginosa were more resistant presenting a larger MIC than the others. These results agree with the ones found in the literature which demonstrate that the bacteria Gram-negatives are more resistant to the action of natural products, as extracts and essential oils, because the membrane present in those bacteria form a complex envelope, protecting them against the action of those agent’s antimicrobials (Oladimeji et al., 2004; Holley and Patel, 2005). Commonly found in channel ridiculers, E. faecalis presents low sensibility to microbial agents and high ability to inactive them (Portenier et al., 2002), explaining larger MIC than the others Gram-positive.

Gochev and Girova (2009) analyzed the essential oil of Thymus vulgaris (containing 43.4% of thymol) and they accomplished a comparative evaluation between the antimicrobial activity of the essential oil of their leaves and the majority composition, thymol, against P. rettigeri, P. aeruginosa, S. aureus and E. coli isolated from nutritious (meats) products for the microdilution method in broth and diffusion in disk. The authors demonstrated that the antimicrobial activity of the oil is dependent on the thymol , because as the essential oil presented only 43.4% of thymol, it was less effective than the pure composition. Other plants from the genus Lippia demonstrated lower levels than that demonstrated by T. vulgaris, indicating a clear difference not only in secondary metabolism, but also in geographic, climatic and seasonal aspects (Veras et al., 2011, 2012; Verma et al., 2011; Soliman et al., 2009).

The antimicrobial activity of the isolated compositions of essential oils depends on their chemical structure (Dorman and Deans, 2000; Inouye et al., 2001; Jirovetz et al., 2007). The representatives that possess a phenolic or alcoholic grouping, such as the thymol and carvacrol, increase the number of double connections allowing larger inhibitory effects on the microbial growth, followed by the aldehydes and ketones (Griffi et al., 1999; Dorman and Deans, 2000). Some studies show that the thymol and carvacrol (isomeric constitutional of the thymol) demonstrate activity antimicrobial against the Gram-positive and Gram-negative bacteria in equality, what means that the position of the hydroxyl group does not have effect on the activity of the compositions (Botelho et al., 2007; Gochev and Girova, 2009).

EOLS contains in their chemical composition, besides the thymol, other compounds with proven antimicrobial activity as carvacrol, p-cimene, β-cariofilene and 1,8-cineole (Kalpoutzakis et al., 2001; Delgado et al., 2004; Sabulal et al., 2006; Kordali et al., 2008). Those components can act sinergically by increasing the potential antimicrobian activities (Burt, 2004). However, our results demonstrated that the thymol is the responsible component for the antibacterial activity presented by EOLS (Table 2).

Due to the great number of chemical components, the essential oils do not possess specific target in the cell. The lipophilic structure facilities the penetration of essential oil across the membranes and destabilizing the transport of the ions and solutes. This process resulting in ion loss and the reduction of the membrane potential, with consequent collapse of the proton bomb, depletion of ATP leads to the cellular death by apoptosis and necrosis (Sikkema et al., 1994; Yoon et al., 2000; Turina et al., 2006).

Tables 3 and 4 show the interference of the essential oil and thymol, in subinhibitory concentrations (MIC/8) on the aminoglycosides activity and ß-lactamases, respectively, demonstrating an interference in the antibiotic activity. EOLS and thymol were not capable to modify the activity of Gentamicin and Neomycin against S. mutans, E. facalis, E. coli, E. cloacae and P. rettigeri. On the other hand, EOLS and thymol reduced CIM of the gentamicin against K. pneumoniae and P. aeruginosa by 32 times (1 μg/mL) and 4 times (8 μg/mL), respectively. There was also reduction of CIM of the gentamicin against S. aureus by 4 times (8 μg/mL) when associated to EOLS and by 16 times (2 μg/mL) when associated to the thymol (Table 3).

The association between OELS and thymol with Neomycin reduced MIC by 4 times (32 μg/mL) and 32 times (4 μg/mL) against S. aureus, respectively. There was also an increase in the activity against P. aeruginosa, reducing MIC of neomycin 128 to 64 μg/mL for both tested samples. In the other analyzed stumps there was no synergism or antagonism between the association of EOLS and thymol with the foregoing aminoglycoside.

In Table 4 are shown MIC of Penicillin G and Ceftriaxone. The results demonstrated against S. mutans a reduction in MIC of penicillin antibiotic 128 for 32 and 2 μg/mL when associated to EOLS and thymol, respectively. Other synergistic effects were observed in the antibiotic activity of Ceftriaxona in association with EOLS and thymol against the E. faecalis with reduction of MIC to 64 for 4 μg/mL (See Fig. 1).

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Figure 1 Chemical structure of the main component of the essential oil of L. sidoides, Thymol.

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In the last years, the resistance to the antimicrobian used at the clinic has been increasing considerably, representing a world problem. About 90–95% of the stumps of S. aureus are resistant to penicillin (Coutinho et al., 2008). Because of the increase of the resistance to the antibiotics, the selection of the new therapeutic agents present in natural product represents one alternative solution of the problem. Many drugs used in the treatment of infectious diseases were isolated and purified starting from medicinal plants (Buttler and Buss, 2006). The synergic action of natural products and clinically relevant antimicrobian is known. Different authors report the decrease of the MIC of antimicrobial in associations with natural product (Matias et al., 2011; Sousa et al., 2011).

An employed strategy to revert the mechanisms of resistance of the bacteria is to use the combination among drugs, as the association between β-lactamases and inhibitors of the β-lactamase. Miranda-Novales et al. (2006) demonstrated that the combination between amikacin and cephalotin or dicloxacline present synergic effect against most of the resistant stumps of S. aureus and coagulase-negative Staphylococcus.

Some classes of secondary metabolites present in natural products were also characterized as modifiers of the antibiotic activity, as terpenes (Nicolson et al., 1999), tannins, alkaloids (Matias et al., 2011), and flavonoids (Sato et al., 2004). The synergic association among natural products and antimicrobian has also been demonstrating in as the essential oils of the leaves of Thymus vulgaris and Pimpinella anisum, by direct contact methods against Gram-positive and Gram-negative bacteria (Al-Bayati, 2008).

The mechanisms for the essential oils to modify the antibiotic actions involve different interactions among the present chemical compositions in the oil and the bacterial lipid membrane. The thymol and carvacrol can be effective against the microorganisms through a lipophilic character action on the cellular membrane, causing the dispersion of the chain of polypeptides of the cellular membrane and destabilizing of the permeability of the cell membrane (Nostro et al., 2004; Cowan, 1999). Studies using the essential oil of the Oreganum vulgare showed the thymol and carvacrol were accumulated in the plasmatic membrane of P. aeruginosa and S. aureus, resulting in a 90% increase of the cellular permeability of those microorganisms (Knowles et al., 2005; Lambert et al., 2001).

Our results demonstrated that synergic effect present in the association of EOLS and thymol with the aminoglycosides reduces MIC of the antibiotic considerably, reducing the necessary dose so that there is therapeutic success. EOLS and thymol presented a synergic effect when associated with Penicillin, reducing MIC considerably for S. mutans and when associated with Ceftriaxone against E. faecalis. Such facts can be justified due to capacity of the thymol in provoking distortions in the physical structure of the cell, causing expansion and consequent desestabilization of the membrane, denaturing essential enzymes and altering the proton pump driving force through variations in the pH and in the electric potential (Helander et al., 1998; Burt, 2004).

This study shows that the thymol is the responsible component for the antimicrobial activity and as a modifier of antibiotics of the essential oil of the leaves of L. sidoides. More studies are necessary in order to evaluate the behavior of that association alive into verify the biodisponibility in “in vivo” test and the possible toxic effects.

Conflict of interest statement

The authors have not any conflict of interest to disclose.