- Email: [email protected]
Treatment with Hypericum perforatum L. does not trigger decreased resistance in Staphylococcus aureus against antibiotics and Hyperforin
Phytomedicine 10: 206–208, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
SHORT COMMUNICATION
Treatment with Hypericum perforatum L. does not trigger decreased resistance in Staphylococcus aureus against antibiotics and Hyperforin A. T. Hübner Summary Most scientific investigations of Hypericum perforatum L. (Saint John’s wort) concentrated on its antidepressant activity. Only recently, its antibacterial activity against multiresistant Staphylococcus aureus led to speculations regarding the use of hyperforin, an antibacterial principle of hypericum, as an antibiotic. In the present investigation, we show that Staphylococcus aureus is able to acquire a resistance against hyperforin which did not lead to a cross resistance against clinically used antibiotics. Resistance development does not take place, however, at concentrations as low as they are found in human blood plasma during antidepressant treatment with 900 mg Hypericum extract/day. Key words: Hyperforin, Staphylococcus aureus, antibiotics, resistance, phytotherapy, Hypericum perforatum
j Introduction Most scientific investigations of Hypericum perforatum L. (Saint John’s wort), that has traditionally been used for the treatment of burns, insomnia and myalgia, concentrated on its antidepressant activity. One key compound for this activity was named hyperforin (Chatterjee et al., 1998) which also exhibits antibacterial activity. Hypericum perforatum L. contains several antimicrobial compounds that exhibit a broad spectrum of antibacterial and antiviral activity. Besides flavonoids and hypericin especially hyperforin has been characterized as a major antibacterial compound active against even multiresistant gram positive bacteria. (Gurevich et al., 1971; Schempp et al., 1999). This activity against multiresistant Staphylococcus aureus led to speculations regarding the systemic use of hyperforin as an antibiotic (Schempp et al., 1999).
Reports on the bioavailability support this potential use (Biber et al., 1998): in their investigation Biber et al. report hyperforin plasma levels of up to > 400 ng/ml. After administration of the normal therapeutic dose used against depression, which is 3 × 300 mg extract/day, steady state plasma levels of approximately 100 ng/ml hyperforin were found (Biber et al., 1998). In the present report, we investigated the development of resistance against hyperforin in S. aureus strains. This question is especially important because of the large number of people treated for depression with hypericum extracts. The development of resistant strains in these patients may interfere with a wide use of hyperforin as a new antibiotic. Recently concerns regarding therapy with Hypericum perforatum L. have been raised because of interactions with other prescribed medication. Evidence has 0944-7113/03/10/02-03-206 $ 15.00/0
Treatment with Hypericum perforatum L. been provided that these interactions result from the inductions of cytochrome P450 drug metabolizing liver enzymes and intestinal p-glycoprotein (Johne et al., 1999; Maurer et al., 1999; Moore et al., 2000; Obach 2000; Roby et al., 2000). To rule out that the selected S. aureus strains develop a resistance against commonly used antibiotics, agar diffusion tests were carried out to check for cross resistance.
207
of 3 ml. The positive control (PRSA) for the selection system contained penicillin G (Grünenthal GmbH, Aachen, Germany) at concentrations from 0.03 I.U./ml broth to 0.2 I.U./ml. The S. aureus strain used in this investigation was isolated from clinical material in the Department of Microbiology, Clinical Center Ludwigsburg. S. aureus ATCC 29213 (American Type Culture Collection, Rockville, MD, USA) served as the reference strain for the susceptibility testing. The tubes were inoculated with 50 µl of a bacterial j Materials and Methods suspension containing 5* 107 colony-forming-units, CFU/ml (McFarland 0.5). From the second day on each Hyperforin (Dr. Willmar Schwabe GmbH & Co., Karls- set of tubes was inoculated with 100 µl from the tube in ruhe, Germany) was dissolved in normal saline and which bacterial growth occurred at the highest hyperfurther diluted with Mueller-Hinton broth (Difco Labo- forin concentration after incubation. This process was ratories, Detroit, USA) to obtain twofold concentra- repeated five times. tions ranging from 0.025 µg/ml to 4 µg/ml for the fluid The susceptibility of all strains to hyperforin and selection (FS). The concentration used for the plasma penicillin G was determined in 96-well microtiter level selection (PLS) was 0.1 µg/ml at a final volume plates containing twofold dilutions of hyperforin in Mueller-Hinton broth ranging from 4 µg/ml to 0.03 µg/ml and Table 1. Susceptibility of selected S. aureus strains against hyperforin/penicillin G for the positive control, peniin terms of MIC values. cillin G at concentrations ranging from 0.01 I.U./ml broth to µg hyperforin/ml I.U. penicillin G/ml 1.6 I.U./ml. Mueller Hinton broth Mueller Hinton broth The plates were inoculated with a 1:200 dilution of a bacteriS. aureus parent strain 0.5 0.05 al suspension containing approxS. aureus PRSA 0.5 0.4 imately 5* 10 7 CFU/ml (McFarS. aureus PLS 0.5 nd land 0.5) of the respective strain S. aureus FS 1 nd in Mueller-Hinton broth. To rule nd – not determined out contamination by bacteria Results of three independent determinations other than S. aureus each strain was subcultured on agar plates without inhibitory supplement Table 2. Results of the susceptibility testing. before susceptibility testing. The tests were carried out in tripliinhibiton zone diameter in mm cate. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– The susceptibility testing Antibiotic concen- S. aureus S.aureus FS S. aureus S. aureus against antibiotics was carried out tration parent strain PRSA ATCC 29213 on Mueller-Hinton agar plates Amoxicillin 25 µg 29 32 19 19 (BBL, Cockeysville, USA) acOfloxacin 10 µg 27 25 24 24 cording to the procedures deDoxycycline 30 µg 28 24 27 25 scribed by Bauer et al. using comGentamicin 10 µg 20 18 21 20 mercially available antibiotic Cotrimoxazole 25 µg 28 24 27 24 discs (Mast Diagnostica, ReinAmikacin 30 µg 21 20 21 19 feld, Germany). The antibiotics Cefazolin 30 µg 30 35 25 29 tested are listed in Table 1. Erythromycin 15 µg 23 22 28 25 Microtiter plates were covered Lincomycin 15 µg 25 21 27 22 with a lid and incubated in a wet Mezlocillin 30 µg 27 29 19 17 chamber. Tetracycline 30 µg 28 25 28 25 All tubes and susceptibility Oxacillin 5 µg 28 29 24 26 tests were incubated overnight in Penicillin G 10 I.U. 29 28 17 17 the dark at 37 °C.
208
An. T. Hübner
j Results Compared to the other strains, twice the amount of hyperforin was necessary to inhibit growth (Table 1) of the strain that was exposed to the fluid selection. The MIC values for S. aureus FS were 1 µg hyperforin/ml in contrast to a MIC of 0.5 µg/ml hyperforin for the parent strain and the strain exposed to the plasma level selection. Because of these results no antibiotic susceptibility testing was carried out for S. aureus PLS. The positive control showed that the system used to select for resistant bacteria was capable of selecting for resistant mutants; the MIC value for penicillin G increased from 0.05 I.U./ml for the original strain to 0.4 I.U/ml for the selected strain, S. aureus PRSA. All bacterial strains that were exposed to hyperforin and the original strain were susceptible to the tested antibiotics (Table 2). S. aureus PRSA developed a resistance against amoxicillin and penicillin.
j Discussion In this paper we show that exposure of S. aureus to hyperforin, an antibiotic agent from Hypericum perforatum L., leads to a reduced sensitivity towards hyperforin. A concentration of hyperforin, such as found in patients treated with Hypericum perforatum L. against depressions, was not sufficient to elicit decreased sensitivity towards hyperforin. Therefore, the development of hyperforin resistance in patients treated for depression with hypericum extracts does not seem likely. However, further tests need to be done with clinical isolates from patients consuming hypericum extracts to exclude the possibility of resistance development, for example due to hyperforin metabolites. Hyperforin’s potential use as an antibiotic is supported by our investigation which showed that no resistance against hyperforin was developed at low hyperforin concentrations and that even in strains with a reduced susceptibility to hyperforin no cross resistance to clinically used antibiotics could be detected. Acknowledgments
The culture media were a generous gift from Becton & Dickinson. The author is grateful to the Department of Microbiol-
ogy, Clinical Center Ludwigsburg, as well as to Dr. Willmar Schwabe GmbH & Co. for providing hyperforin and support and to Mr. Spiel, M.D. for support. The author thanks Karin Wertz for providing helpful comments on the manuscript.
j References Biber A, Fischer H, Romer A, Chatterjee SS (1998) Oral bioavailability of hyperforin from hypericum extracts in rats and human volunteers. Pharmacopsychiatry 31 Suppl 1: 36–43 Chatterjee SS, Nöldner M, Koch E, Erdelmeier C (1998) Antidepressant Activity of Hypericum perforatum and Hyperforin: the Neglected Possibility. Pharmacopsychiatry 31 Suppl 1: 44–53 Gurevich AI, Dobrynin VN, Kolosov MN, Popravko SA, Ryabova ID, Chernov BK, Derbentzeva NA, Aizeman BE, Garagulya AD (1971) Hyperforin, an antibiotic from Hypericum perforatum. Antibiotiki 16: 510–13 Johne A, Brockmöller J, Bauer S, Maurer A, Langheinrich M, Roots I (1999) Pharmacokinetic interaction of digoxin with an herbal extract from St John’s wort (Hypericum perforatum). Clin Pharmacol Ther 66: 338–345 Maurer A, Johne A, Bauer S, Brockmöller J, Donath F, Roots I, Langheinrich M, Hübner W-D (1999) Interaction of St John’s wort extract with phenprocoumon [abstract]. Eur J Pharmacol 55: A22 Moore LB, Goodwin B, Jones SA, Wisely GB, SerabjitSingh CJ, Willson TM, Collins J L, Kliewer S A (2000) St John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci USA 97: 7500–7502 Obach R S (2000) Inhibition of human cytochrome P450 enzymes by constituents of St John’s wort, an herbal preparation used in the treatment of depression. J Pharmacol Exp Ther 294: 88–95 Roby C A, Anderson G D, Kantor E, Dryer D A, Burstein A H (2000) St John’s wort: effect on CYP3A4 activity. Clin Pharmacol Ther 67: 451–457 Schempp CM, Pelz K, Wittmer A, Schopf E, Simon JC (1999) Antibacterial activity of hyperforin from St John’s wort against multiresistant Staphylococcus aureus and gram-positive bacteria. Lancet 353: 2129
j Address A. T. Hübner, Birkenweg 7, D-71711 Murr, Germany Tel.: ++49-7144-29 032; Fax: ++49-1212-513 603 929; e-mail: [email protected]
Phytomedicine
SHORT COMMUNICATION
Treatment with Hypericum perforatum L. does not trigger decreased resistance in Staphylococcus aureus against antibiotics and Hyperforin A. T. Hübner Summary Most scientific investigations of Hypericum perforatum L. (Saint John’s wort) concentrated on its antidepressant activity. Only recently, its antibacterial activity against multiresistant Staphylococcus aureus led to speculations regarding the use of hyperforin, an antibacterial principle of hypericum, as an antibiotic. In the present investigation, we show that Staphylococcus aureus is able to acquire a resistance against hyperforin which did not lead to a cross resistance against clinically used antibiotics. Resistance development does not take place, however, at concentrations as low as they are found in human blood plasma during antidepressant treatment with 900 mg Hypericum extract/day. Key words: Hyperforin, Staphylococcus aureus, antibiotics, resistance, phytotherapy, Hypericum perforatum
j Introduction Most scientific investigations of Hypericum perforatum L. (Saint John’s wort), that has traditionally been used for the treatment of burns, insomnia and myalgia, concentrated on its antidepressant activity. One key compound for this activity was named hyperforin (Chatterjee et al., 1998) which also exhibits antibacterial activity. Hypericum perforatum L. contains several antimicrobial compounds that exhibit a broad spectrum of antibacterial and antiviral activity. Besides flavonoids and hypericin especially hyperforin has been characterized as a major antibacterial compound active against even multiresistant gram positive bacteria. (Gurevich et al., 1971; Schempp et al., 1999). This activity against multiresistant Staphylococcus aureus led to speculations regarding the systemic use of hyperforin as an antibiotic (Schempp et al., 1999).
Reports on the bioavailability support this potential use (Biber et al., 1998): in their investigation Biber et al. report hyperforin plasma levels of up to > 400 ng/ml. After administration of the normal therapeutic dose used against depression, which is 3 × 300 mg extract/day, steady state plasma levels of approximately 100 ng/ml hyperforin were found (Biber et al., 1998). In the present report, we investigated the development of resistance against hyperforin in S. aureus strains. This question is especially important because of the large number of people treated for depression with hypericum extracts. The development of resistant strains in these patients may interfere with a wide use of hyperforin as a new antibiotic. Recently concerns regarding therapy with Hypericum perforatum L. have been raised because of interactions with other prescribed medication. Evidence has 0944-7113/03/10/02-03-206 $ 15.00/0
Treatment with Hypericum perforatum L. been provided that these interactions result from the inductions of cytochrome P450 drug metabolizing liver enzymes and intestinal p-glycoprotein (Johne et al., 1999; Maurer et al., 1999; Moore et al., 2000; Obach 2000; Roby et al., 2000). To rule out that the selected S. aureus strains develop a resistance against commonly used antibiotics, agar diffusion tests were carried out to check for cross resistance.
207
of 3 ml. The positive control (PRSA) for the selection system contained penicillin G (Grünenthal GmbH, Aachen, Germany) at concentrations from 0.03 I.U./ml broth to 0.2 I.U./ml. The S. aureus strain used in this investigation was isolated from clinical material in the Department of Microbiology, Clinical Center Ludwigsburg. S. aureus ATCC 29213 (American Type Culture Collection, Rockville, MD, USA) served as the reference strain for the susceptibility testing. The tubes were inoculated with 50 µl of a bacterial j Materials and Methods suspension containing 5* 107 colony-forming-units, CFU/ml (McFarland 0.5). From the second day on each Hyperforin (Dr. Willmar Schwabe GmbH & Co., Karls- set of tubes was inoculated with 100 µl from the tube in ruhe, Germany) was dissolved in normal saline and which bacterial growth occurred at the highest hyperfurther diluted with Mueller-Hinton broth (Difco Labo- forin concentration after incubation. This process was ratories, Detroit, USA) to obtain twofold concentra- repeated five times. tions ranging from 0.025 µg/ml to 4 µg/ml for the fluid The susceptibility of all strains to hyperforin and selection (FS). The concentration used for the plasma penicillin G was determined in 96-well microtiter level selection (PLS) was 0.1 µg/ml at a final volume plates containing twofold dilutions of hyperforin in Mueller-Hinton broth ranging from 4 µg/ml to 0.03 µg/ml and Table 1. Susceptibility of selected S. aureus strains against hyperforin/penicillin G for the positive control, peniin terms of MIC values. cillin G at concentrations ranging from 0.01 I.U./ml broth to µg hyperforin/ml I.U. penicillin G/ml 1.6 I.U./ml. Mueller Hinton broth Mueller Hinton broth The plates were inoculated with a 1:200 dilution of a bacteriS. aureus parent strain 0.5 0.05 al suspension containing approxS. aureus PRSA 0.5 0.4 imately 5* 10 7 CFU/ml (McFarS. aureus PLS 0.5 nd land 0.5) of the respective strain S. aureus FS 1 nd in Mueller-Hinton broth. To rule nd – not determined out contamination by bacteria Results of three independent determinations other than S. aureus each strain was subcultured on agar plates without inhibitory supplement Table 2. Results of the susceptibility testing. before susceptibility testing. The tests were carried out in tripliinhibiton zone diameter in mm cate. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– The susceptibility testing Antibiotic concen- S. aureus S.aureus FS S. aureus S. aureus against antibiotics was carried out tration parent strain PRSA ATCC 29213 on Mueller-Hinton agar plates Amoxicillin 25 µg 29 32 19 19 (BBL, Cockeysville, USA) acOfloxacin 10 µg 27 25 24 24 cording to the procedures deDoxycycline 30 µg 28 24 27 25 scribed by Bauer et al. using comGentamicin 10 µg 20 18 21 20 mercially available antibiotic Cotrimoxazole 25 µg 28 24 27 24 discs (Mast Diagnostica, ReinAmikacin 30 µg 21 20 21 19 feld, Germany). The antibiotics Cefazolin 30 µg 30 35 25 29 tested are listed in Table 1. Erythromycin 15 µg 23 22 28 25 Microtiter plates were covered Lincomycin 15 µg 25 21 27 22 with a lid and incubated in a wet Mezlocillin 30 µg 27 29 19 17 chamber. Tetracycline 30 µg 28 25 28 25 All tubes and susceptibility Oxacillin 5 µg 28 29 24 26 tests were incubated overnight in Penicillin G 10 I.U. 29 28 17 17 the dark at 37 °C.
208
An. T. Hübner
j Results Compared to the other strains, twice the amount of hyperforin was necessary to inhibit growth (Table 1) of the strain that was exposed to the fluid selection. The MIC values for S. aureus FS were 1 µg hyperforin/ml in contrast to a MIC of 0.5 µg/ml hyperforin for the parent strain and the strain exposed to the plasma level selection. Because of these results no antibiotic susceptibility testing was carried out for S. aureus PLS. The positive control showed that the system used to select for resistant bacteria was capable of selecting for resistant mutants; the MIC value for penicillin G increased from 0.05 I.U./ml for the original strain to 0.4 I.U/ml for the selected strain, S. aureus PRSA. All bacterial strains that were exposed to hyperforin and the original strain were susceptible to the tested antibiotics (Table 2). S. aureus PRSA developed a resistance against amoxicillin and penicillin.
j Discussion In this paper we show that exposure of S. aureus to hyperforin, an antibiotic agent from Hypericum perforatum L., leads to a reduced sensitivity towards hyperforin. A concentration of hyperforin, such as found in patients treated with Hypericum perforatum L. against depressions, was not sufficient to elicit decreased sensitivity towards hyperforin. Therefore, the development of hyperforin resistance in patients treated for depression with hypericum extracts does not seem likely. However, further tests need to be done with clinical isolates from patients consuming hypericum extracts to exclude the possibility of resistance development, for example due to hyperforin metabolites. Hyperforin’s potential use as an antibiotic is supported by our investigation which showed that no resistance against hyperforin was developed at low hyperforin concentrations and that even in strains with a reduced susceptibility to hyperforin no cross resistance to clinically used antibiotics could be detected. Acknowledgments
The culture media were a generous gift from Becton & Dickinson. The author is grateful to the Department of Microbiol-
ogy, Clinical Center Ludwigsburg, as well as to Dr. Willmar Schwabe GmbH & Co. for providing hyperforin and support and to Mr. Spiel, M.D. for support. The author thanks Karin Wertz for providing helpful comments on the manuscript.
j References Biber A, Fischer H, Romer A, Chatterjee SS (1998) Oral bioavailability of hyperforin from hypericum extracts in rats and human volunteers. Pharmacopsychiatry 31 Suppl 1: 36–43 Chatterjee SS, Nöldner M, Koch E, Erdelmeier C (1998) Antidepressant Activity of Hypericum perforatum and Hyperforin: the Neglected Possibility. Pharmacopsychiatry 31 Suppl 1: 44–53 Gurevich AI, Dobrynin VN, Kolosov MN, Popravko SA, Ryabova ID, Chernov BK, Derbentzeva NA, Aizeman BE, Garagulya AD (1971) Hyperforin, an antibiotic from Hypericum perforatum. Antibiotiki 16: 510–13 Johne A, Brockmöller J, Bauer S, Maurer A, Langheinrich M, Roots I (1999) Pharmacokinetic interaction of digoxin with an herbal extract from St John’s wort (Hypericum perforatum). Clin Pharmacol Ther 66: 338–345 Maurer A, Johne A, Bauer S, Brockmöller J, Donath F, Roots I, Langheinrich M, Hübner W-D (1999) Interaction of St John’s wort extract with phenprocoumon [abstract]. Eur J Pharmacol 55: A22 Moore LB, Goodwin B, Jones SA, Wisely GB, SerabjitSingh CJ, Willson TM, Collins J L, Kliewer S A (2000) St John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci USA 97: 7500–7502 Obach R S (2000) Inhibition of human cytochrome P450 enzymes by constituents of St John’s wort, an herbal preparation used in the treatment of depression. J Pharmacol Exp Ther 294: 88–95 Roby C A, Anderson G D, Kantor E, Dryer D A, Burstein A H (2000) St John’s wort: effect on CYP3A4 activity. Clin Pharmacol Ther 67: 451–457 Schempp CM, Pelz K, Wittmer A, Schopf E, Simon JC (1999) Antibacterial activity of hyperforin from St John’s wort against multiresistant Staphylococcus aureus and gram-positive bacteria. Lancet 353: 2129
j Address A. T. Hübner, Birkenweg 7, D-71711 Murr, Germany Tel.: ++49-7144-29 032; Fax: ++49-1212-513 603 929; e-mail: [email protected]