Antonie van Leeuwenhoek (2007) 92:95–100 DOI 10.1007/s10482-006-9139-x
ORIGINAL PAPER
Synergistic bactericidal activity of Eremanthus erythropappus oil or b-bisabolene with ampicillin against Staphylococcus aureus Andre´a M. A. Nascimento Æ Maria G. L. Branda˜o Æ Gabriel B. Oliveira Æ Isabel C. P. Fortes Æ Edmar Chartone-Souza
Received: 14 September 2006 / Accepted: 21 December 2006 / Published online: 18 January 2007 Springer Science+Business Media B.V. 2007
Abstract The activity of Eremanthus erythropappus oil (EO) and some of its compounds and their potential synergistic interaction with ampicillin against different strains of Staphylococcus aureus were investigated. Determination of chemical composition of EO by gas chromatographymass spectrometry (GC/MS) and bioguided chemical fractionation led to the identification of b-bisabolene as the main active compound. A synergistic bactericidal activity of EO or bbisabolene with ampicillin against Staphylococcus aureus was observed in a time-kill assay. EO and b- bisabolene have the potential to restore the effectiveness of ampicillin against resistant S. aureus. A. M. A. Nascimento (&) G. B. Oliveira E. Chartone-Souza Departamento de Biologia Geral, Instituto de Cieˆncias Biolo´gicas – Universidade Federal de Minas Gerais, Avenida Antoˆnio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil e-mail:
[email protected] M. G. L. Branda˜o Departamento de Produtos Farmaceˆuticos, Faculdade de Farma´cia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil I. C. P. Fortes Departamento de Quı´mica, Instituto de Cieˆncias Exatas. Universidade Federal de Minas Gerais, Avenida Antoˆnio Carlos, 6627, Belo Horizonte 31270-901, Brazil
Keywords Eremanthus erythropappus Eremanthus erythropappus oil b-bisabolene Staphylococcus aureus Ampicillin Synergism Abbreviations EO Eremanthus erythropappus oil MHB Mueller-Hinton broth
Introduction Staphylococcus aureus is a versatile pathogen causing a wide variety of community and hospital acquired infections, associated with high morbidity and mortality rates (O’Connell et al. 2003). It also causes economically important diseases, such as mastitis, in cows and sheep. Recently, S. aureus has acquired resistance to most of the antibiotics developed in the past 50 years (Kuroda et al. 2002). Eremanthus erytropappus (D.C.) Mc Leish (Vanillosmopsis erytropappus Schultz-Bip.), Asteraceae, is a small tree found in the savannah areas of the Brazilian Central Plateau. Its popular name, ‘‘candeia’’, which means candlestick, is due to its ability to burn with a luminous flame. In Brazil, its stems have been used as fencing in farms and as roof trusses because of their long life. E. erytropappus oil (EO) is obtained from the
123
96
steam distillation of the wood. EO has achieved a high economic value in the last 20 years due its high content of the anti-inflammatory sesquiterpene a-(-)-bisabolol, also found in small amounts in chamomile oil. EO is currently the main source of commercial a-bisabolol, which is found in several products worldwide (Ammon et al. 1996; Jakolev et al. 1979). Previous studies showed that EO also contains other compounds like isovaleric acid, b-bisabolene, bisabolene diols, bisabolol oxide B, eremanthine and lychnopholide (Braun et al. 2003; Canalle et al. 2001; Lopes et al. 1991; Vichnewski et al. 1989; Lima et al. 1985). In a continuing effort to identify natural products from Brazilian plants that inhibit the growth of S. aureus or reverse its resistance to current antibiotics, we evaluated the potential of EO or some of its compounds to enhance the activity of antibiotics against strains of methicillin-resistant (MRSA) or methicillin-susceptible (MSSA) S. aureus. The bacteriostatic and bactericidal concentrations of EO against S. aureus were also determined.
Materials and methods Oil sample EO was obtained by steam distillation and kindly supplied by Citroleo (Torrinha, Sa˜o Paulo, Brazil). Authentic samples of a-(-)-bisabolol and b-bisabolene were kindly supplied by GivaudanRoure (Brazil). Bacterial strains and growth conditions Eleven S. aureus strains belonging to our private collection, eight b-lactamase-producing strains (six MRSA and two MSSA) and three non-blactamase-producing strains (MSSA), were used. In addition, S. aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATTC 27853) (American Type Culture Collection, Manassas, VA, USA) were used. The bacteria were cultured in Mueller-Hinton broth (MHB; Difco Laboratories, Baltimore, Md., USA).
123
Antonie van Leeuwenhoek (2007) 92:95–100
Antibiotics Ampicillin (Amp), gentamicin, tetracycline, chloramphenicol and nalidixic acid were used. All antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Determination of the minimum inhibitory concentrations (MIC) Susceptibility tests were carried out by the broth dilution method in MHB with an inoculum of approximately 105-colony forming units (CFU)/ ml. The MHB was supplemented with antibiotic concentrations ranging from 0.125 to 1024 lg/ml, EO concentrations ranging from 5 to 1000 lg/ml and its fractions at concentrations ranging from 20 to 100 lg/ml. EO was firstly solubilized in 10% (v/v) ethanol, a vehicle that did not affect the growth of the bacteria. To evaluate the effect of EO in combination with antibiotics, increasing antibiotic concentrations (ranging from 0.125 to 1024 lg/ml) were added to MHB containing crude EO or its fractions at 1/5 or 1/2 · MIC, respectively. Cultures that contained neither EO nor antibiotics served as controls. The data were reported as MIC, e.g. the lowest concentrations of antibiotics, crude EO, EO fractions or their combinations at which no visible growth was detected after 24 h of incubation at 37C. The bactericidal concentration was the lowest concentration at which bacteria failed to grow in MHB or after plating onto Muller-Hinton agar (MHA) (Smith-Palmer et al. 1998). The fractional inhibitory concentration (FIC) was determined by the broth dilution method (Mackay et al. 2000). Synergism between antibiotic and EO was indicated by an FIC index £ 0.5. Determination of b-lactamase activity b-lactamase activity was determined using nitrocefin (Calbiochem, San Diego, Calif., USA) as substrate, as described by Braga et al. (2005). For data analysis, at least three separate experiments were carried in duplicate.
Antonie van Leeuwenhoek (2007) 92:95–100
Survival curves For time-kill assays, a standard inoculum of approximately 104 CFU/ml of an overnight culture was used. Amp (1/3 · MIC), EO (1/5, 1 and 2 · MIC) and b-bisabolene (1/2, 1 and 2 · MIC) were used. Combinations of Amp (1/3 · MIC) plus EO (1/5 · MIC) or Amp (1/3 · MIC) plus b-bisabolene (1/2 · MIC) were also evaluated. A tube containing only MHB was inoculated and served as control. Viable cell counts were carried out at different intervals up to 24 h at 37C. The rate and extent of killing was determined by plotting viable colony counts (CFU/ml) against time. Chemical analysis Sample analysis was performed on a GC/MS system, model HP5890 series II, interfaced to a quadrupole mass spectrometer HP5989A, using an electron impact (EI) mode and the ionization voltage at 70 EV. The GC column was a fused silica capillary column HP-5MS (5% (phenyl) methylpolysiloxane stationary phase; 30 m · 0.25 lm · 0.25 lm). The GC oven was programmed as follows: 50C for 5 min and then up to 280C, at 3C/min and finally kept at 280C for 10 min. Injector and detector temperatures were maintained at 280C. The carrier gas was helium at a flow rate of 1 ml/min. The sample was diluted in CH2Cl2 and 1 ll was injected using a split mode, with a ratio of 1:5. Preliminary identification was done using a Wiley 138 library search and further identification was achieved by comparison of recorded mass spectra with literature data on MS and retention times (Adams 2001; Swigar and Silverstein 1981; von Sydow 1963, 1964). Co-injection was made in cases where reference samples were available. The quantitative composition of the sample was calculated from percentage area of TIC. Chemical fractionation bioguided by antimicrobial assays EO (10 g) was successively submitted to column chromatography in silica gel eluted with toluol: acetone (8:2). The fractions were submitted to
97
antimicrobial assays until the identification of the active components.
Results Chemical analysis GC/MS analysis showed that the major components of EO were a-(-)-bisabolol (63.0%), b-bisabolene (2.7%), a-humulene (1.8%) and bisabolol oxide (6.6%). Identification of the active compound Silica gel fractionation of EO led to several fractions that were further screened for antibacterial activity. One active fraction demonstrated the presence of a main compound. This fraction was successively submitted to column chromatography in silica gel bioguided by the antimicrobial essays. The active compound was identified as b-bisabolene by co-injection of an authentic sample and its mass spectra: m/z (%) 204 (3), 189 (2), 161 (5), 147(3), 133(5), 121(3), 119 (3), 107 (10), 93 (28), 79 (22), 69 (31), 53 (28), 41 (100), 39 (41), 27 (38). One fraction was exclusively composed of a-(-)-bisabolol and showed no inhibitory effect on bacterial growth at the concentrations tested (up to 4 mg/ml). Bacterial susceptibility Table 1 shows that the MICs of EO and b-bisabolene ranged from 50 to 200 lg/ml and from 8 to 40 lg/ml, respectively. The lowest MIC was observed for b-bisabolene (8 lg/ml) against strain ATCC 25922 of S. aureus. MICs of EO and b-bisabolene had a bacteriostatic effect on S. aureus, preventing any growth for at least 24 h (Fig. 1). The minimal bactericidal concentrations had effect on bacteria after 3 h (Fig. 1). Of the five antibiotic combinations tested with EO or b-bisabolene at sub-inhibitory concentrations against S. aureus, only the combinations Amp plus EO or Amp plus b-bisabolene showed a synergistic activity (Table 1 and data not shown). On the other hand, in all strains of S. aureus that were sensitive to EO at the
123
98
Antonie van Leeuwenhoek (2007) 92:95–100
Table 1 MICs of ampicillin, EO and b-bisabolene, alone or in combination, against S. aureus Strain no.
MIC (lg/ml) EO
b-bisabolene
Amp
Ampicillin in combination with a
b
64 128 64 32 32 64 64 64 1 1 1 0.0125
8 16 32 16 32 32 32 8 1 1 1 0.0125
EO
6*+ 13+ 14*+ 17+ 37*+ 40*+ 43*+ 44*+ 30 34 35 ATCC 25922
50 100 100 50 100 200 100 50 50 50 50 50
40 40 40 40 40 40 40 40 40 40 40 8
64 128 256 32 128 256 512 64 1 1 1 0.0125
b-bisabolene
* MRSA; +b-lactamase a
EO (1/5 · MIC);
b
b-bisabolene (1/2 · MIC)
concentration of 50 lg/ml, no change in the Amp MIC was observed. EO also showed no activity against P. aeruginosa and E. coli at concentrations up to 1000 lg/ml (data not shown). Synergism by time-kill curves The time-kill kinetics was evaluated for the eight S. aureus strains for which a synergistic activity was observed. The same synergistic bactericidal profile was observed for these strains (data not shown). Figure 2 shows the synergistic activity of EO or b-bisabolene with Amp against strain 37 of S. aureus. Following the addition of sub-inhibitory concentrations of either EO (1/5 · MIC), 1,00E+08
Survivors (CFU/mL)
Fig. 1 Survival curves of S. aureus strain 37 (MRSA) in the presence of EO 100 lg/ml (MIC) and 200 lg/ml (MBC) alone and b-bisabolene 40 lg/ml (MIC) and 60 lg/ml (MBC). The control tube contained neither EO nor b-bisabolene
b-bisabolene (1/2 · MIC) or Amp (1/3 · MIC) alone, no killing was observed up to 24 h. The growth of S. aureus in Amp, b-bisabolene or EO alone was slightly retarded with an extended lag phase of up to 3 h. However, these populations reached cell densities similar to that observed for the 24 h culture control. In contrast, the effect of the combinations revealed synergistic activity, which resulted in a bactericidal effect after 4 h of incubation. The bacterial population remained at inoculum density for 4 h of incubation and then decreased by more than 3 log of CFU/ml when compared to the growth of bacteria cultured with EO, b-bisabolene or Amp alone.
1,00E+07 1,00E+06 Control EO MIC EO MBC beta-bisabolene MIC beta-bisabolene MBC
1,00E+05 1,00E+04 1,00E+03 1,00E+02 1,00E+01 1,00E+00 0
6
12
Time (h)
123
18
24
Antonie van Leeuwenhoek (2007) 92:95–100 1,00E+08 1,00E+07
Survivors (CFU/mL)
Fig. 2 Time kill-curves of S. aureus strain 37 grown in the presence of ampicillin (1/3 · MIC) with and without EO (1/5 · MIC) or b-bisabolene (1/2 · MIC)
99
1,00E+06
Control ampicillin 1/3 x MIC
1,00E+05
EO 1/5 x MIC 1,00E+04
EO 1/5 x MIC plus ampicillin 1/3 x MIC beta-bisabolene x 1/2 MIC
1,00E+03
beta-bisabolene x 1/2 MIC plus ampicillin 1/3 x MIC
1,00E+02 1,00E+01 1,00E+00 0
6
12
18
24
Time (h)
Discussion Although a-(-)-bisabolol is the main component of EO and displays several biological activities (Mendes et al. 1999; Menezes et al. 1990), it clearly does not present activity against S. aureus. The active compound was identified as b-bisabolene, present in a concentration of 2.7% in EO. It is noteworthy that the antibacterial activity of b-bisabolene described in the present study is in agreement with results reported by other groups showing that the activity of essential oils of other Asteraceae species against S. aureus is also due to this compound (Gouvinden-Soulange et al. 2004; Simic et al. 2005). To overcome the emerging problem of bacterial antibiotic resistance, studies investigating combinations of plant extracts with antibiotics against clinical strains have been reported (Braga et al. 2005; Darwish et al. 2002; Nascimento et al. 2000; Yam et al. 1998). In the present study, we found that combination of EO or b-bisabolene, at sub-MIC concentrations, with Amp significantly improved the activity of the antibiotic (Table 1 and Fig. 2). Synergistic activity of the combination of ampicillin with EO or b-bisabolene was confirmed by time-kill assays. In contrast, three out of eight b-lactamase producing S. aureus strains were sensitive to EO at the concentration of 50 lg/ml and did not demonstrate any synergistic activity of Amp in combination with EO.
However, the synergistic activity was observed in the three b-lactamase-producing S. aureus strains when ampicillin was associated with b-bisabolene. A possible explanation would be that the low b-bisabolene concentration present in EO might not be sufficient to produce synergistic effects. The synergistic interaction appears to be specific for EO and Amp, because no changes were observed in the MICs of other antibiotics evaluated in this study. Colorimetric assays of b-lactamase-producing S. aureus strains with EO or b-bisabolene at sub-MICs concentrations showed that synthesis of this enzyme remained unaffected (data not shown). Thus, it seems that EO and b-bisabolene may have an effect on b-lactamase activity.
Conclusions The in vitro activity of EO and b-bisabolene against S. aureus and their synergistic interaction with Amp have been demonstrated for the first time. EO and b-bisabolene have the potential to restore the effectiveness of Amp against resistant S. aureus and could be useful in the development of valuable clinical treatments. Acknowledgements The authors are indebted to Givaudan Roure do Basil (Sa˜o Paulo, Brazil) for supplying the authentic volatile compounds. This work was supported by a FAPEMIG grant CBB-1106/03.
123
100
References Adams RP (2001) Identification of essential oil components by gas chromatography mass spectrometry. Allured Pub. Corp., Carol Stream, Illinois Ammon HPT, Sabieraj J, Kamille KR (1996) Mechanismus derantiphlogistischen wirkung von Kamillenextract und Inhaltsstoffen. Dtsch Apoth Ztg 36: 1821–1834 Braga LC, Leite AAM, Xavier KGS, Takahashi JA, Bemquerer MP, Chartone-Souza E, Nascimento AMA (2005) Synergic interaction between pomegranate extract and antibiotics against Staphylococcus aureus. Can J Microbiol 51:541–547 Braun NA, Meier M, Kohlenberg B, Hammeschmidt FJ (2003) Two new bisabolene diols from the stem wood essential oil of Vanillosmopsis erytropappa Schultz-Bi (Asteraceae). J Essent Oil Res 15:139–142 Canalle R, Burim RV, Callegari JLL, Takahashi CS (2001) Assessment of the cytotoxic and clastogenic activities of the sesquiterpene lactone lynchnopholide in mammalian cells in vitro and in vivo. Cancer Detect Prev 25:93–101 Darwish RM, Aburjai T, Al-Khalil S, Mahafzah A (2002) Screening of antibiotic resistant inhibitors from local plant materials against two different strains of Staphylococcus aureus. J Ethnopharmacol 79:359–364 Gouvinden-Soulange J, Magan N, Gurib-Fakin A, Gauvin A, Smadja J, Kodja H (2004) Chemical composition and in vitro antimicrobial activities of the essential oils from endemic Psidia species growing in Mauritius. Biol Pharm Bull 27:1814–1818 Jakolev O, Isaac I, Thiemer K, Kunde R (1979) Pharmacological investigations with compounds of chamomile – new investigations on the antiphlogistic effects of a- bisabolol and bisabolol oxides. Planta Med 35:125–140 Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki K-I, Nagai Y, Lian J, Ito T, Kanamori M, Matsumaru H, Maruyama A, Murakami H, Hosoyama A, Mizutani-Ui Y, Takahashi NK, Sawano T, Inoue R-I, Kaito C, Sekimizu K, Hirakawa H, Kuhara S, Goto S, Yabuzaki J, Kanehisa M, Yamashita A, Oshima K, Furuya K, Yoshino C, Shiba T, Hattori M (2002) Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225–1240 Lima PDDB, Garcia M, Rabi JA (1985) Selective extraction of a-methylene-c-lactones Reinvestigation
123
Antonie van Leeuwenhoek (2007) 92:95–100 of Vanillosmopsis erytropappus. J Nat Prod 48:986– 988 Lopes JNC, Lopes JLC, Vichnewsky WR, Dioneia C, Gottlieb OR (1991) Chemical variability of Vanillosmopsis erytropappa. Anais da Academia Brasileira de Cieˆncias 63:21–22 Mackay ML, Milne K, Gould IM (2000) Comparison of methods for assessing synergic antibiotic interactions. Int J Antimicrob Agents 15:125–129 Mendes MN, Queiroz RO, Grandi TSM, Anjos AMG, Oliveira AB, Zani CL (1999) Screening of Asteraceae (Compositae) plant extracts for molluscicidal activity. Mem I Oswaldo Cruz 94:411–412 Menezes AMS, Almeida FRC, Rao VSN (1990). Antiinflammatory activity of the essential oil of Vanillosmopsis arborea. Fitoterapia 61:252–254 Nascimento GGF, Locatelli J, Freitas PC, Silva GL (2000) Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistance bacteria. Braz J Microbiol 31:247–256 O’Connell NH, Humphreys H., Pidgeon C, Smyth EG (2003) Absence of risk factors for community MRSA. Clin Microbiol Infec 9:578–579 Simic N, Palic R, Randjelovc V (2005) Composition and antibacterial activity of Achillea clypeolata essential oil. Flavour Frag J 20:127–130 Smith-Palmer A, Stewart J, Fyfe L (1998) Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett Food Microbiol 26:118–122 Swigar AA, Silverstein RM (1981) Monoterpenes. Aldrich Chemical Co. Inc., Milwaukee, WI Vichnewski W, Takahashi AM, Nasi AMT, Rodrigues DC, Goncalves G, Dias DA, Lopes JNC, Goedken VL, Gutie´rrez AB Herz W (1989) Sesquiterpene lactones and other constituents from Eremanthus seidelii, E. goyazensis and Vanillosmopsis erythropappa. Phytochemistry 28:1441–1451 Von Sydow E. (1963) Mass spectrometry of terpenes monoterpenes alcohol. Acta Chem Scand 17:2504– 2512 Von Sydow E. (1964) Mass spectrometry of terpenes monoterpenes aldehydes and ketones. Acta Chem Scand 18:1099–1101 Yam TS, Hamilton-Mu¨eller JMT, Shah S (1998) The effect of a component of tea (Camellia sinensis) on methicillin resistance, PBP2’ Synthesis, and b-lactamase production in Staphylococcus aureus. J Antimicrob Chemoth 42:211–216
