Skip to main content

Advertisement

Herbal therapy associated with antibiotic therapy: potentiation of the antibiotic activity against methicillin – resistant Staphylococcus aureus by Turnera ulmifolia L

Article metrics

Abstract

Background

Staphylococcus genus is widely spread in nature being part of the indigenous microbiota of skin and mucosa of animal and birds. Some Staphylococcus species are frequently recognized as etiological agents of many animal and human opportunistic infections This is the first report testing the antibiotic resistance-modifying activity of Turnera ulmifolia against methicillin-resistant Staphylococcus aureus – MRSA strain.

Methods

In this study an ethanol extract of Turnera ulmifolia L. and chlorpromazine were tested for their antimicrobial activity alone or in combination with aminoglycosides against an MRSA strain.

Results

The synergism of the ethanol extract and aminoglycosides were verified using microdillution method. A synergistic effect of this extract on gentamicin and kanamycin was demonstrated. Similarly, a potentiating effect of chlorpromazine on kanamycin, gentamicin and neomycin, indicating the involvement of an efflux system in the resistance to these aminoglycosides.

Conclusion

It is therefore suggested that extracts from Turnera ulmifolia could be used as a source of plant-derived natural products with resistance-modifying activity, constituting a new weapon against the problem of bacterial resistance to antibiotics demonstrated in MRSA strains.

Background

Staphylococcus genus is widely spread in nature being part of the indigenous microbiota of skin and mucosa of animal and birds. Some Staphylococcus species are frequently recognized as etiological agents of many animal and human opportunistic infections [1]. S. aureus, S. epidermidis, S. saprophyticus and S. haemolyticus are the most important species as community and nosocomial human infection causing agents. In addition of causing different kinds of intoxications, S. aureus has been the most common etiological agent of festering infections that attack different tissues and/or organs (e.g. furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, bacterial arthritis) [2, 3]. Capsule, peptidoglican, teicoic acids, adesins and synthesis of enzymes and extracelullar toxins are some virulence attributes present in/on S. aureus cell [1].

Clinicians write millions prescriptions for antimicrobials every year [4]. Clinicians need to apply sound concepts when prescribing antimicrobials to both achieve a good outcome and avoid encouraging resistance. There is convincing evidence that inappropriate use of antibiotics directly leads to the development of resistant organisms [5]. To prevent this, is necessary educate all health worker regarding healthy drug use and regarding the natural history of the infection, emphasizing palliative therapies and infection control measures [6]. Infectious diseases are the major cause of morbidity and mortality and experience has shown that an approach that seeks to 'defeat' infectious diseases will not work. Long-term solutions must acknowledge this, and nurses and other health care professionals must take a proactive part in finding alternative solutions [7].

With increased incidence of resistance to antibiotics, natural products from plants could be interesting alternatives [8, 9]. Some plant extracts and phytochemicals are known to have antimicrobial properties, and can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to demonstrate such efficacy [1012].

Many plants have been evaluated not only for direct antimicrobial activity, but also as a resistance-modifying agent [13]. Several chemical compounds, synthetic or from natural sources, such as the phenothiazines, and natural products, have direct activity against many species of bacteria, enhancing the activity of a specific antibiotic, reversing the natural resistance of specific bacteria to given antibiotics, promoting the elimination of plasmids from bacteria and inhibiting transport functions of the plasma membrane in regard to given antibiotics. The inhibition of plasma membrane-based efflux pumps has been observed as well [14, 15]. The enhancement of antibiotic activity or the reversal of antibiotic resistance by natural or synthetic non-conventional antibiotics affords the classification of these compounds as modifiers of antibiotic activity.

Turnera ulmifolia L. (Turneraceae), a small annual herb, can be found in the north and northeast brazilian regions, where it is considered a weed [16]. It grows preferentially in sandy soils and on hill slopes. T. ulmifolia L. is already known to be of medicinal value, being used popularly as an anti-inflammatory, as an expectorant, and in the treatment of several problems [1618]. Authors detected flavonoids, alkaloids, tannins and phenolic compounds in preparations from this plant [1921].

Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome, and the increase in cases of bacterial resistance to aminoglycosides is widely recognized as a serious health threat [22]. The main mechanisms of resistance to aminoglycosides are active efflux and enzymatic inactivation [23].

In this work, we tested an ethanol extract of Turnera ulmifolia as a resistance modifying agent in an aminoglycoside-resistant strain of S. aureus.

Methods

Strains

The strain used was the clinical isolate Staphylococcus aureus 358 (SA358), resistant to several aminoglycosides [24]. The SA-ATCC25923 strain of Staphylococcus aureus was used as a positive control. The strains were maintained in heart infusion agar slants (HIA, Difco), and prior to assay, the cells were grown overnight at 37°C in brain heart infusion (BHI, Difco).

Plant material

Leaves of Turnera ulmifolia were collected in the county of Crato, Ceará State, Brazil. The plant material was identified and a voucher specimen was deposited with the number 1618 at the Herbarium "Dárdano de Andrade Lima" of Universidade Regional do Cariri – URCA.

Preparation of ethanol extract of Turnera ulmifolia (EETU)

A quantity of 200 g of leaves were dried at room temperature and powdered. The powdered material was extracted by maceration using 1 L of 95% ethanol as solvent at room temperature, and the homogenate was allowed to stand for 72 h at room temperature. The extracts were then filtered and concentrated under vacuum in a rotary evaporator [25]. For the tests, the dry extract material was dissolved in DMSO (dimethyl sulfoxide) 0,5%. The DMSO was chosen due its less toxicity than ethanol.

Drugs

Chlorpromazine, gentamicin, tobramycin, kanamycin, amikacin and neomycin were obtained from Sigma Chemical Co. All drugs were dissolved in sterile water.

Drug susceptibility test

The minimum inhibitory concentration (MIC) of EETU, antibiotics and chlorpromazine (CPZ) were determined in BHI by the microdilution assay using suspensions of 105 CFU/ml and a drug concentration range of 1024 to 1 μg/ml (twofold serial dilutions) [26]. MIC was defined as the lowest concentration at which no growth was observed. For the evaluation of EETU as a modulator of antibiotic resistance, MICs of the antibiotics were determined in the presence of EETU (32 μg/ml) and CPZ (16 μg/ml) at sub-inhibitory concentrations, and the plates were incubated for 24 h at 37°C. CPZ was used as positive control for efflux pump inhibition [27]. Each experiment was undertaken in duplicate.

Results

The EETU did not show a substantial antibacterial activity at 1024 μg/ml against the strains assayed (MIC ≥ 1024 μg/ml). None effect was observed when EETU was combined with the aminoglycosides in the strain SA-ATCC25923.

The addiction of EETU to the growth medium at 32 μg/ml produced a dramatic reduction in the MIC for gentamicin and kanamycin in the strain Staphylococcus aureus 358, demonstrating a potentiating effect of EETU on aminoglycoside activity (Table 1).

Table 1 MIC‡ values (μg/ml) of aminoglycosides in the absence and presence of EETU# and CPZ * in Staphylococcus aureus 358.

A MIC reduction for gentamicin and kanamycin was also observed when CPZ was added to the growth medium at 16 μg/ml, which indicates the involvement of an efflux pump in the resistance to these antibiotics (Table 1).

A potentiating effect of CPZ on amikacin and tobramycin was not observed, which suggests the occurrence of other resistance mechanisms (Table 1).

Discussion

Only few articles were published focusing pharmacological activities of the genus Turnera. Some species of Turnera are widely used in folk medicine for different types of inflammatory diseases. Fresh leaves of T. guaianensis Aubl. is used to treat inflammatory diseases in general and as an immunomodulator while the decoction of it's dried leaves is employed to treat furunculosis [17]. T. diffusa Willd., in a decoction of the whole plant, is used to treat otitis and nephritis [28, 29], while the leaf infusion is used in diseases related to the gastrointestinal and respiratory systems [30], reproductive organs in general or specifically for gonorrhoea treatment [17]. T. ulmifolia is used popularly as an anti-inflammatory and as an expectorant [1618], but as far as we know, natural products of Turnera ulmifolia or of any plants from the genus Turnera having a potentiating effect on any drug, mainly antibiotics (as aminoglycosides) have not been previously reported in human or animals.

Phenothiazines, such as chlorpromazine, probably act on the plasma membrane of bacteria affecting the efflux pumps [31]. This modification of permeability could enhance the activity of antibiotics that act within the cell, such as the aminoglycosides.

Efflux pumps have been associated with resistance mechanisms since the 1980s32, representing one of the main mechanism of multidrug resistance (MDR) evolved in the antibiotic resistance to aminoglycosides [32]. Several studies have been performed to identify drugs interfering with these pumps, called resistance modifying agents [33]. Other plant products, as ethanol extract of Mentha arvensis, affected the efflux system of an Escherichia coli multiresistant to aminoglycosides, inhibitinh these resistance mechanism [27].

This strategy is named "herbal shotgun" or "Synergistic multi-target effects" and refers to the use of herbals and drugs in a multitargeted approach, due the fact of mono or multi-extract combinations affect not only a single target, but several ones, cooperating in an agonistic-synergistic way. This approach are not exclusive for extract combinations, but combinations between single natural products or extracts with chemosynthetics or antibiotics are possible too [34, 35].

Conclusion

The results obtained indicate that Turnera ulmifolia (and broadly Turneraceae) could serve as a source of plant-derived natural products with antibiotic resistance-modifying activity to be used against multiresistant bacteria as MRSA strains acquired from hospital and community.

References

  1. 1.

    Nostro A, Blanco AR, Cannatelli MA, Enea V, Flamini G, Morelli I: Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol. FEMS Microbiol Lett. 2004, 230: 191-195. 10.1016/S0378-1097(03)00890-5.

  2. 2.

    Pereira MSV, Siqueira Júnior JP, Takaki GMC: Elimination of resistance to drugs by fluoroquinolones in bovine strains of Staphylococcus aureus. Pesquisa Vet Brasil. 2004, 24: 11-14.

  3. 3.

    Verhoeff J, Beaujean D, Vlok H, Baars A, Meyler A, Werkwn Van Der C: A dutch approach to methicillin-resistance Staphylococcus aureus. Eur J Clin Microbiol Infect Dis. 1999, 18: 461-466. 10.1007/s100960050324.

  4. 4.

    Karras D: Antibiotic misuse in the emergency department. Acad Emerg Med. 2006, 13: 331-333. 10.1111/j.1553-2712.2006.tb01701.x.

  5. 5.

    Malhutra-Kumar S, Lammens C, Coenen S, Van Herk K, Goossens H: Effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in health volunteers: A randomized, double-blind, placebo-controlled study. Lancet. 2007, 369: 482-490. 10.1016/S0140-6736(07)60235-9.

  6. 6.

    Roe VA: Antibiotic resistance: A guide for effective prescribing in women's health. J Midwif Womens Health. 2008, 53: 216-226. 10.1016/j.jmwh.2008.01.003.

  7. 7.

    Purssel E: Evolutionary nursing: the case of infectious disease. J Adv Nurs. 2005, 49: 164-172. 10.1111/j.1365-2648.2004.03276.x.

  8. 8.

    Lu Y, Zhao YP, Wang ZC, Chen SY, Fu CX: Composition and antimicrobial activity of the essential oil of Actinidia macrosperma from China. Nat Prod Res. 2007, 21: 227-233. 10.1080/14786410500463395.

  9. 9.

    Mbwambo ZH, Moshi MJ, Masimba PJ, Kapingu MC, Nondo RS: Antimicrobial activity and brine shrimp toxicity of extracts of Terminalia brownii roots and stem. BMC Complement Altern Med. 2007, 7: 9-10.1186/1472-6882-7-9.

  10. 10.

    Benoit-Vical F, Grellier P, Abdoulaye A, Moussa I, Ousmane A, Berry A: In vitro and in vivo Antiplasmodial Activity of Momordica balsamina Alone or in a Traditional Mixture. Chemotherapy. 2006, 52: 288-292. 10.1159/000095960.

  11. 11.

    Senatore F, Rigano D, Formisano C, Grassia A, Basile A, Sorbo S: Phytogrowth-inhibitory and antibacterial activity of Verbascum sinuatum. Fitoterapia. 2007, 78: 244-247. 10.1016/j.fitote.2006.11.010.

  12. 12.

    Singh G, Maurya S, Delampasona MP, Catalan CA: A comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. Food Chem Toxico. 2007, 45: 1650-1661. 10.1016/j.fct.2007.02.031.

  13. 13.

    Gibbons S: Anti-staphylococcal plant natural products. Nat Prod Rep. 2004, 21: 263-277. 10.1039/b212695h.

  14. 14.

    Gunics G, Farkas S, Motohashi N, Shah A, Harsukh G, Kawase M, Molnár J: Interaction between 3,5-diacetyl-1,4-dihydropyridines and ampicillin, and erythromycin on different E. coli strains. In Vivo. 2006, 20 (3): 367-372.

  15. 15.

    Molnar J, Molnar A, Spengler G, Mandi Y: Infectious plasmid resistance and efflux pump mediated resistance. Acta Microbiol Immunol Hung. 2004, 51 (3): 333-349. 10.1556/AMicr.51.2004.3.11.

  16. 16.

    Braga R: Plantas do nordeste, especialmente do Ceará. 1976, ESAM (Coleção Mossoroense), Fortaleza, 3

  17. 17.

    Pio Corrêa M: Dicionário das plantas úteis do Brasil e das exóticas cultivadas. 1984, Imprensa Nacional, Rio de Janeiro, 3

  18. 18.

    Hosamani KM: Fatty acids in seed oil from Turnera ulmifolia. Phytochemistry. 1993, 34 (5): 1363-1365. 10.1016/0031-9422(91)80030-5.

  19. 19.

    Antonio MA, Souza Brito AR: Oral anti-inflammatory and anti-ulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae). J Ethnopharmacol. 1998, 6 (3): 215-228. 10.1016/S0378-8741(98)00049-X.

  20. 20.

    Gracioso JS, Vilegas W, Hiruma-Lima CA, Souza Brito AR: Effects of tea from Turnera ulmifolia L. on mouse gastric mucosa support the Turneraceae as a new source of antiulcerogenic drugs. Biol Pharmac Bull. 2002, 25 (4): 487-491. 10.1248/bpb.25.487.

  21. 21.

    Nascimento MA, Silva AK, Franca LC, Quignard EL, Lopez JA, Almeida MG: Turnera ulmifolia L. (Turneraceae): Preliminary study of its antioxidant activity. Biores Technol. 2006, 97 (12): 1387-1391. 10.1016/j.biortech.2005.07.009.

  22. 22.

    Jana S, Deb JK: Molecular understanding of aminoglycoside action and resistance. Appl Microbiol Biotechnol. 2006, 70: 140-150. 10.1007/s00253-005-0279-0.

  23. 23.

    Smith E, Williamson M, Wareham N, Kaatz G, Gibbons S: Antibacterial and modulators of bacterial resistance from the immature cones of Chamaecyparis lawsoniana. Phytochemistry. 2007, 68: 210-217. 10.1016/j.phytochem.2006.10.001.

  24. 24.

    Freitas FI, Guedes-Stehling E, Siqueira Júnior JP: Resistance to gentamicin and related aminoglycosides in Staphylococcus aureus isolated in Brazil. Lett Appl Microbiol. 1999, 9 (3): 197-201. 10.1046/j.1365-2672.1999.00617.x.

  25. 25.

    Brasileiro BG, Pizziolo VR, Raslan DS, Jamal CM, Silveira D: Antimicrobial and cytotoxic activities screening of some Brazilian medicinal plants used in Governador Valadares district. Rev Bras Cienc Farm. 2006, 42: 195-202. 10.1590/S1516-93322006000200004.

  26. 26.

    Javadpour MM, Juban MM, Lo WC, Bishop SM, Alberty JB, Cowell SM: De novo antimicrobial peptides with low mammalian cell toxicity. J Med Chem. 1996, 39: 3107-3113. 10.1021/jm9509410.

  27. 27.

    Coutinho HDM, Costa JGM, Falcão-Silva VS, Lima EO, Siqueira Júnior JP: Enhancement of the antibiotic activity against a multiresistant Escherichia coli by Mentha arvensis L. and chlorpromazine. Chemotherapy. 2008, 54: 328-330. 10.1159/000151267.

  28. 28.

    Fryer FA: A chemical investigation of Damiana (Turnera diffusa). Specialites. 1965, 112: 21-25.

  29. 29.

    Perez RM, Ocegueda GA, Munoz JL, Ávila JG, Morrow WW: A study of the hypoglycemic effect of some Mexican plants. J Ethnopharmacol. 1984, 123: 253-262. 10.1016/0378-8741(84)90054-0.

  30. 30.

    Cáceres A: Plantas de Uso Medicinal en Guatemala. 1996, Editorial Universitaria, San Carlos de Guatemala, 1

  31. 31.

    Kristiansen JE, Amaral L: The potential management of resistant infections with non-antibiotics. J Antimicrob Chemother. 1997, 40: 319-327. 10.1093/jac/40.3.319.

  32. 32.

    Van Bambeke F, Glupczynski Y, Plesiat P, Pechère JC, Tulkens PM: Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy. J Antimicrob Chemoth. 2003, 51: 1055-1065. 10.1093/jac/dkg224.

  33. 33.

    Gunics G, Farkas S, Motohashi N, Shah A, Harsukh G, Kawase M, Molnár J: Interaction between 3,5-diacetyl-1,4-dihydropyridines and ampicillin, and erythromycin on different E. coli strains. Int J Antimicrob Agents. 2002, 20: 227-229. 10.1016/S0924-8579(02)00159-0.

  34. 34.

    Hemaiswarya Sh, Kruthiventi AK, Doble M: Synergism between natural products and antibiotics against diseases. Phytomedicine. 2008, 15: 639-652. 10.1016/j.phymed.2008.06.008.

  35. 35.

    Wagner H, Ulrich-Merzenich G: Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009, 16: 97-110. 10.1016/j.phymed.2008.12.018.

Pre-publication history

  1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/9/13/prepub

Download references

Acknowledgements

This work was supported by the following Brazilian agencies CNPQ and FAPESQ/PB.

Author information

Correspondence to Henrique DM Coutinho.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

HDMC was the principal investigator, participated in the planning and execution of the study, performed data entry and data analysis, and was the main responsible author. JGMC, EOL, VSFS and JPSJ participated in the planning of the study and contributed to the writing process.

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

Coutinho, H.D., Costa, J.G., Lima, E.O. et al. Herbal therapy associated with antibiotic therapy: potentiation of the antibiotic activity against methicillin – resistant Staphylococcus aureus by Turnera ulmifolia L. BMC Complement Altern Med 9, 13 (2009) doi:10.1186/1472-6882-9-13

Download citation

Keywords

  • Minimum Inhibitory Concentration
  • Aminoglycosides
  • Ethanol Extract
  • Chlorpromazine
  • Efflux Pump