Antibacterial effect of some essential oils administered alone or in combination with Norfloxacin.

Abstract

The objective of the present study was that of verifying a possible synergistic antibacterial effect between Pelargonium graveolens [Lis-Balchin, M., Deans, S.G., Hart, S., 1996. Bioactive Geranium oils from different commercial sources. J. Essential Oil Res. 8, 281-290.] essential oil (and its main components) and Norfloxacin antibiotic.

As a first step growth inhibition by some types of essential oils was assessed in five microbial species. The antimicrobial effects of P. graveolens oil, as well as those of its components, were evaluated by means of the agar dilution method (ADM) against Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538 and S. aureus ATCC 29213.

The results obtained highlighted the occurrence of a pronounced synergism between P. graveolens essential oil and Norfloxacin against three of the five bacterial species under study with a FIC index in the 0.37-0.50 range. Such antibacterial effects were also shown to increase, although to a lesser extent, when Norfloxacin was given with the main components of P. graveolens essential oil.

Significance and impact of the study: The combination of Norfloxacin with either P. graveolens essential oil, or with some of the main components of this latter, in the treatment of infections caused by some bacterial species is likely to reduce the minimum effective dose of Norfloxacin thus minimizing the side effects of the antibiotic.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Citronellol; Geraniol; Norfloxacin; Pelargonium essential oil; Synergism; Triacetin

Introduction

Higher plant products with an evidence-based action against fungi and pathogenic bacteria are currently playing a growing role as present antibiotic therapies are frequently accompanied by a large number of toxic side effects (Gundidza, 1993). In addition, such therapies can sometimes turn out to be ineffective due to the rapid development of bacterial resistance. Therefore, the search for new antimicrobial natural products continues to draw the attention of many researchers. Plant oils account for a source of very promising natural ingredients for producing new antimicrobial drugs in spite of the fact that their antibacterial and antifungal action has been proven to be remarkably milder than that of commercial synthetic drugs (Bidlack et al., 2000; De Laurentis et al., 2004, 2005; Giamperi et al., 2002).

Some studies have explored the combination of synthetic drugs with essential oils in a view to evaluating and enhancing antimicrobial efficacy. One such studies has, for example, focused on the synergistic effect of Euphorbia characias latex and ketoconazole whose antifungal activity has been proven to be substantially enhanced (Giordani et al., 2001). But no study to date has focused on the synergistic effect of combined administration of Norfloxacin and Pelargonium graveolens essential oil.

The antibiotic under study (Norfloxacin) is one of the antibiotics commonly used to treat either genitourinary tract or prostate infections, and also gonorrhea. Unfortunately though Norfloxacin has frequent use-limiting side effects including headache, abdominal pain, vomiting and systemic toxicity. For this reason, our research effort turned to the study of the antimicrobial activity of essential oils in a view to developing safer drugs.

It is worthwhile remembering that the clinical use of essential oils against systemic infections has been strongly hindered by the fact that they are poorly absorbed by the human intestine and exhibit a "weak" action compared to commercial or synthetic antibiotics. Nevertheless, in spite of these negative aspects, many essential oils have been produced for treating localized bacterial infections (Hollman, 2001). What is more, the activity of essential oils both in vitro and in vivo has been the subject of a number of scientific papers. Many of the essential oils studied have been proven to be efficient only at elevated concentrations (Laredo et al., 1995; Giamperi et al., 2002) when their microbicidal action turns out to be more pronounced. For this reason, some synergistic combinations among different essential oils have been recently studied in a view to increasing their antibacterial action without additionally increasing their concentration (Cassella et al., 2002; Bidlack et al., 2000). As a first step in our research, which was intended to develop a broad-spectrum antibacterial agent, some essential oils were studied (by agar dilution test) that are used (in both aroma therapy and complementary medicine) to treat bacterial infections against five species of bacteria. The combination of P. graveolens essential oil (an essential oil with an elevated antibacterial activity, which contains also some alcohols including Citronellol and Geraniol) with Norfloxacin (an antibiotic commonly administered to treat urinary tract infections) was explored in a second phase.

Materials and methods

Plant-derived materials

Stems and leaves of Rosmarinus officinalis, Salvia officinalis, Origanum vulgare, Thymus serphyllum, P. graveolens and Myrtus communis (Pintore et al., 2002), collected in different areas of the region Apulia in July 2004, were sampled and identified. The relevant specimens were kept at the Department of Botany of the University of Bari. Once dried the aerial parts of these plants underwent hydrodistillation by means of a Clevenger apparatus (Buchi). The essential oils obtained were dehydrated over anhydrous sodium sulfate and after filtration stored at 4 [degrees]C until tested. Two [micro]l was used for gas chromatographer and mass spectrometer (GCMS) measurements.

Preparation of samples for testing antibacterial activity

The essential oils of R. officinalis, S. officinalis, O. vulgare, T. serphyllum, P. graveolens and M. communis were obtained by steam extraction. The composition of essential oils was analyzed by GCMS, an apparatus made by a HP 6890 and a HP 5973 equipped with a HP-5 capillary column. The following components of essential oils were studied in terms of their antimicrobial activity: Carvacrol, Thymol, Eucaliptol, Citronellol, Geraniol, Triacetin. The components in question had been purchased from Sigma Chemical Co. (St. Louis, MO, USA). (Adams 1989).

Antimicrobial activity

Bacterial strains

Some control bacterial strains from American Type Culture Collection (ATCC, Rockville, MD, USA) were used to test the antibacterial properties of essential oils and of some of their components. The bacterial strains used were aerobic, Gram positive and negative. Moreover all the strains used were aerobic and accounted for the major group of pathogens.

Antibacterial activity was assessed against the following bacterial strains: Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538, S. aureus ATCC 29213. Test conditions were in full agreement with CLSI Protocol M7A6 requirements.

Assessment of the minimal inhibitory concentration of essential oils by means of modified ADM

The minimum inhibitory concentration (MIC) values of the bacterial strains under study were determined by the agar dilution method (ADM) as envisaged by CLSI Protocol M7A6, but with some modifications: a final concentration of 0.5% Tween 20 (Sigma)(v/v) was incorporated into the agar medium before autoclaving to enhance oil solubility.

MIC was defined as the lowest concentration that did not result in any visible growth of the microorganism compared with the growth in the control plate; the presence of one or two colonies was not taken into account for final assessment of MIC (Santos et al., 1997; Hammer, 1999). Table 1 shows the results of the antimicrobial activity of the six essential oils and of some of their components against the five bacterial strains expressed as MIC (mg/ml); in addition the total activity of the oils is expressed as mg/ml (Eloff, 2004).

Agar dilution checkerboard method used to evaluate the synergic action of Pelargonium graveolens essential oil

Twelve serial two-fold dilutions of Geraniol, Citronellol, Triacetin and P. graveolens essential oil were prepared following the same ADM adopted to assess MIC. Some dilutions of the oil and of its components were prepared (0.09-0.72 mg/ml), together with a series of two-fold serial dilutions of Norfloxacin in the 0.06-1 [micro]g/ml range. In this way, all Norfloxacin dilutions were mixed with the appropriate concentration of oil thus obtaining a series of combinations of Norfloxacin and P. graveolens essential oil and its components (White et al., 1996). The concentrations prepared corresponded to 1/2, 1/4, 1/8 and 1/16 of the MIC value. The analysis of the combination of the substances was obtained by calculating the FIC index (FICI) as follows: FIC = ([MIC.sub.a] of the combination/[MIC.sub.a] alone) + ([MIC.sub.b] of the combination/[MIC.sub.b] alone), where a is either Pelargonium oil or some of its components and b is Norfloxacin (critical agent) in the combination. The FICI was interpreted as: (i) a synergistic effect when [less than or equal to] 0.5; (ii) an additive or indifferent effect when > 0.5 and < 1 and (iii) an antagonistic effect when > 1 (Eucast, 2000). The combination of the two components is shown graphically by a Cartesian diagram by applying the isobole method. The non-interaction of the two components results in a straight line, whereas the occurrence of an interaction is shown by a concave isobole. (Williamson, 2001; Shin and Kang, 2003; Shin and Lim, 2004).

Results and discussion

Combined effects of Pelargomium graveolens essential oil, and its main components, with Norfloxacin.

Among the essential oils analyzed, the oil of P. graveolens was used against the five bacterial strains under consideration. What is more, P. graveolens essential oil was shown to contain relatively stable monoterpenic primary alcohols (Citronellol and Geraniol) as its main components. Norfloxacin MIC and FIC indices in combination with Pelargonium essential oil are given in Table 2. A synergistic effect was obtained with three strains B. cereus ATCC 11778, S. aureus ATCC 6538 and S. aureus ATCC 29213 with a FICI of 0.50, 0.37 and 0.38, respectively. No synergistic effect was instead observed in the experiments with B. subtilis ATCC 6633 (FICI = 0.51) and E. coli ATCC 35218 (FICI = 0.57). Other results were obtained by combining Norfloxacin with either Gerianol, Triacetin or Citronellol with a FICI of 0.50 for B. cereus ATCC 11778, S. aureus ATCC 6538 and S. aureus ATCC 29213. Figs. 1a and b show an isobole curve against S. aureus ATCC 6538 and S. aureus ATCC 29213, thus suggesting a good synergism between the compounds used in the different combinations and Norfloxacin as highlighted by the pronounced leftward shift of the curve. B. cereus ATCC 11778 was less sensitive to the combination Norfloxacin and Pelargonium essential oil with a FICI of 0.50 (Fig. 1c). Several authors have been able to demonstrate the activity of essential oils and of plant extracts including Rosemary, Origan, Basil, Melaleuca (Tea Tree) and Myrtle (Lis-Balchin et al., 1996; Pintore et al., 2002; Laredo et al., 1995; Hethelyi et al., 1989) against the bacterial strains under consideration in the present study. These results were confirmed by the present study, thus highlighting the efficacy of essential oils and of their main components when combined to Norfloxacin.

P. graveolens essential oil was however taken into consideration, together with its main components, essentially because alcohols (which are present in P. graveolens essential oil) may act as powerful antibacterial agents, thanks to their stability, especially in terms of pH, whereas the antimicrobial activity of phenols (which are present in the other essential oils currently under our investigation), including timol and carvacrol, is likely to be affected by an increase in pH level (Shin and Lim, 2004). Unfortunately we have not been able to explain the rationale of the diversified inhibition of the different bacterial species under study also because the mechanism underlying essential oil-induced inhibition is still obscure. Some authors argue that this inhibition mechanism is likely to be associated to both composition and construction of the bacterial cell wall (Russell and Chopra, 1996; Godwin and Michniak, 1999; Scholar and Prat, 2000).

In conclusion, we suggest that the combination Norfloxacin--P. graveolens administered against the bacterial strains under consideration is likely to reduce Norfloxacin minimum efficient dose thus minimizing potential undesirable side effect. In fact, as far as the strains under study are concerned, the concentrations used to assess the synergic effect of the combinations were decidedly (from 4 to 8 times) lower than those of the MIC of each and any drug used. What is more, the use of therapeutic doses could also be a fix to counter microbial resistance and avoid those drug-drug interactions likely to be induced by the administration of currently available antimicrobial drugs. (Shin and Lim, 2004).

[FIGURE 1 OMITTED]

Our results cannot be compared to the data concerning the MIC obtained by other authors because oil concentration is variable and depends on many factors including plant chemotype and plant harvest and storage conditions. The results of the various independent studies carried out in this field could differ because of these uncontrollable variables and not really in terms of actual antibacterial activity (Lis-Balchin et al., 1996; Hammer et al., 1999).

As a result additional in vitro experiments are required to: (i) perform synergy tests on new bacterial strains and to (ii) evaluate antimicrobial potentials in terms of new therapeutic applications.

References

Adams, R.P., 1989. Identification of Essential Oils by Trap Mass Spectrometry. Academic Press, San Diego, pp. 34-177.

Bidlack, W.R., Omaye, S.T., Meskin, M.S., Topham, D., 2000. Phytochemicals as Bioactive Agents. Technomic Publishing Company, Lancaster, UK, pp. 106-110.

Cassella, S., Cassella, J., Smith, I., 2002. Synergistic antifungal activity of tea tree (Melaleuca alternifolia) and lavender (Lavandula angustifolia) essential oil against dermatophyte infection. Int. J. Aromather. 12, 2-15.

De Laurentis, N., Rosato, A., Milillo, M.A., Leone, L., 2004. Chemical composition and antifungal activity of Pistacia terebinthus. 1. Aerial part essential oil. Riv. Ital. Eppos 38, 11-18.

De Laurentis, N., Rosato, A., Gallo, L., Leone, L., Milillo, M.A., 2005. Chemical composition and antimicrobial activity of Myrtus communis. Riv. Ital. Eppos 39, 3-8.

Eloff, J.N., 2004. Quantification the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine 11 (4), 370-371.

Eucast definitive document E.Def 1.2, 2000. Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

Giamperi, L., Fraternale, D., Ricci, D., 2002. The in vitro action of essential oils on different organisms. J. Essential Oil Res. 14, 312-318.

Giordani, R., Trebaux, J., Masi, M., Regli, P., 2001. Enhanced antifungal activity of ketoconazole by Euphorbia characias latex against Candida albicans. J. Ethnopharmacol. 78, 1-5.

Godwin, D.A., Michniak, B.B., 1999. Influence of drug lipophilicity on terpenes as transdermal pentetration enhancers. Drug Dev. Ind. Pharm. 25, 905-915.

Gundidza, M., 1993. Antimicrobial activity of essential oil from Shimus molle. Cent. Afr. J. Med. 39, 231-234.

Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oils and other plant extract. J. Appl. Microbiol. 86, 985-990.

Hethelyi, E., Koczka, I., Tetenyi, P., 1989. Phytochemical and antimicrobial analysis of essential oils. Herba Hung. 28, 1-2.

Hollman, P.C.H., 2001. Evidence for health benefits of plant phenols: local or systemic effects? J. Sci. Food Agric. 81 (9), 842-852.

Laredo, J.V., Abut, M., Calvo-Torras, M.A., 1995. Antimicrobial activity of essences from Labiatae. Microbios 82, 171-762.

Lis-Balchin, M., Deans, S.G., Hart, S., 1996. Bioactive geranium oils from different commercial sources. J. Essential Oil Res. 8, 281-290.

Pintore, G., Usai, M., Bradesi, P., Juliano, C., Boatto, G., et al., 2002. Chemical composition and antimicrobial activity of Rosmarinus officinalis L. oils from Sardinia and Corsica. Flavour Fragrances J. 17, 15-19.

Russell, A.D., Chopra, I., 1996. Antiseptics, disinfectans and preservatives: their properties, mechanisms of action and uptake into bacteria. In: Russell, A.D., Chopra, I. (Eds.), Understanding Antibacterial Action and Resistance. Ellis Horwood, London, pp. 96-149.

Santos, F.A., Cunha, G.M.A., Viana, G.S.B., et al., 1997. Antibacterial activity of essential oils from Psidium and Pilocarpus species of plants. Phytother. Res. 11, 67-69.

Scholar, S.K., Prat, W.B., 2000. The Antimicrobial Drugs. Oxford University Press, Oxford, UK, 348pp.

Shin, S., Kang, C.A., 2003. Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole. Lett. Appl. Microbiol. 36, 111-115.

Shin, S.W., Lim, S., 2004. Antifungal effects of herbal essential oils and in combination with ketoconazolo against Trichopyton spp. J. Appl. Microbiol. 97, 1289-1296.

White, R.L., Burgess, D.S., Manduru, M., Bosso, J.A., 1996. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob. Agents Chemother. 40, 1914-1918.

Williamson, E.M., 2001. Synergy and other interactions in phytomedicines. Phytomedicine 8 (5), 401-409.

Antonio Rosato (a,*), Cesare Vitali (a), Nicolino De Laurentis (a), Domenico Armenise (a), Maria Antonietta Milillo (b)

(a) Department Farmaco Chimico, Section of Microbiology, Faculty of Pharmacy, University of Bari, Via Orabona 4, Bari, Italy

(b) Department of Animal Health and Welfare, Faculty of Veterinary Medicine, University of Bari, S.P. Casamassima Km. 3, 70010 Valenzano (Ba), Italy

*Corresponding author. Tel.: + 39 80 5442768; fax: + 39 80 5442230.

Table 1. Minimum inhibitory concentrations (MICs) (a), total
activity (b) of selected essential oils and some active compounds
against five different microorganisms

Essential oil and active  Yield mg for 1 g  B. subtilis ATCC 6633
compounds                 of dried leaves   MIC    Total activity

Myrtus communis           15.00              1.40   10.71
Origanum vulgare          39.00              0.35  111.43
Pelargonium graveolens     9.80              0.72   27.22
Rosmarinus officinalis    57.00              5.60   10.18
Salvia officinalis        40.00              5.60    7.14
Thymus serphyllum         45.00              0.70   64.29
Citronellol                                  0.35
Eucalyptol                                   5.60
Geraniol                                     0.08
Thymol                                       0.70
Carvacrol                                    0.35
Triacetin                                   22.40

Essential oil and active  B. cereus ATCC 11778   S. aureus ATCC 6538
compounds                 MIC    Total activity  MIC    Total activity

Myrtus communis            1.40  10.71            2.80    5.36
Origanum vulgare           0.70  55.71            0.70   55.71
Pelargonium graveolens     0.36  13.61            0.72   13.61
Rosmarinus officinalis     1.40  40.71            5.60   10.18
Salvia officinalis         1.40  28.57           11.20    3.57
Thymus serphyllum          1.40  32.14            0.28  160.71
Citronellol                0.70                   0.70
Eucalyptol                 2.80                   2.80
Geraniol                   0.70                   0.70
Thymol                     0.70                   0.70
Carvacrol                  2.80                   0.70
Triacetin                 22.40                  22.40

Essential oil and active  S. aureus ATCC 29213   E. coli ATCC 35218
compounds                 MIC    Total activity  MIC     Total activity

Myrtus communis            2.80   5.36            11.20   1.34
Origanum vulgare           0.70  55.71             0.70  55.71
Pelargonium graveolens     0.72  13.61             5.60   1.75
Rosmarinus officinalis     5.60  10.18           -11.20   5.09
Salvia officinalis         5.60   7.14            11.20   3.57
Thymus serphyllum          0.70  64.29             0.70  64.29
Citronellol                0.70                    1.40
Eucalyptol                 2.80                    2.80
Geraniol                   0.70                    1.40
Thymol                     0.70                    1.40
Carvacrol                  0.70                    1.40
Triacetin                 22.40                   22.40

(a) MIC, minimum inhibitory concentration, values given as mg/ml from
the triplicate experiments.
(b) Total activity, the units are expressed in mg/ml and indicate the
degree to which the active compounds in 1 g of plant material can be
diluted while still inhibiting the growth of the microorganisms under
study.

Table 2. Fractional inhibitory concentration (FIC) and FIC indices

                                     B. subtilis ATCC 6633
                             [MIC.sub.o]  [MIC.sub.c]  FIC   FICI

P. graveolens-Norfloxacin
  P. graveolens (mg/ml)      0.72         0.09         0.13  0.51
  Norfloxacin ([micro]g/ml)  0.13         0.05         0.40
Citronellol-Norfloxacin
  Citronellol (mg/ml)
  Norfloxacin ([micro]g/ml)
Geraniol-Norfloxacin
  Geraniol (mg/ml)
  Norfloxacin ([micro]g/ml)
Triacetin-Norfloxacin
  Triacetin (mg/ml)
  Norfloxacin ([micro]g/ml)

                                     B. cereus ATCC 11778
                             [MIC.sub.o]  [MIC.sub.c]  FIC   FICI

P. graveolens-Norfloxacin
  P. graveolens (mg/ml)      0.36         0.09         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Citronellol-Norfloxacin
  Citronellol (mg/ml)        0.64         0.16         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Geraniol-Norfloxacin
  Geraniol (mg/ml)           0.16         0.04         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Triacetin-Norfloxacin
  Triacetin (mg/ml)          2.30         0.57         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25

                                     S. aureus ATCC 6538
                             [MIC.sub.o]  [MIC.sub.c]  FIC    FICI

P. graveolens-Norfloxacin
  P. graveolens (mg/ml)      0.72         0.18         0.25   0.37
  Norfloxacin ([micro]g/ml)  0.50         0.06         0.12
Citronellol-Norfloxacin
  Citronellol (mg/ml)        0.64         0.16         0.25   0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Geraniol-Norfloxacin
  Geraniol (mg/ml)           0.68         0.17         0.25   0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Triacetin-Norfloxacin
  Triacetin (mg/ml)          2.30         0.57         0.256  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25

                                     S. aureus ATCC 29213
                             [MIC.sub.o]  [MIC.sub.c]  FIC   FICI

P. graveolens-Norfloxacin
  P. graveolens (mg/ml)      0.72         0.09         0.13  0.38
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Citronellol-Norfloxacin
  Citronellol (mg/ml)        0.64         0.16         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Geraniol-Norfloxacin
  Geraniol (mg/ml)           0.68         0.17         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25
Triacetin-Norfloxacin
  Triacetin (mg/ml)          2.30         0.57         0.25  0.50
  Norfloxacin ([micro]g/ml)  0.50         0.13         0.25

                                     E. coli ATCC 35218
                             [MIC.sub.o]  [MIC.sub.c]  FIC   FICI

P. graveolens-Norfloxacin
  P. graveolens (mg/ml)      5.76         2.32         0.40  0.57
  Norfloxacin ([micro]g/ml)  0.06         0.01         0.17
Citronellol-Norfloxacin
  Citronellol (mg/ml)
  Norfloxacin ([micro]g/ml)
Geraniol-Norfloxacin
  Geraniol (mg/ml)
  Norfloxacin ([micro]g/ml)
Triacetin-Norfloxacin
  Triacetin (mg/ml)
  Norfloxacin ([micro]g/ml)

[MIC.sub.o], MIC of one sample alone; [MIC.sub.c], MIC of one sample of
the most effective combination.
FIC of oil = MIC of oil in combination with Norfloxacin/MIC of oil
alone.
FIC of Norfloxacin = MIC of Norfloxacin in combination with oil/MIC of
Norfloxacin alone.
FIC index = FIC of oil + FIC of Norfloxacin.