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In vitro growth-inhibitory effect of plant-derived extracts and compounds against Paenibacillus larvae and their acute oral toxicity to adult honey bees
Veterinary Microbiology 145 (2010) 129–133
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
In vitro growth-inhibitory effect of plant-derived extracts and compounds against Paenibacillus larvae and their acute oral toxicity to adult honey bees Jaroslav Flesar a, Jaroslav Havlik a,*, Pavel Kloucek b, Vojtech Rada a, Dalibor Titera c,d, Michal Bednar d, Michal Stropnicky c,d, Ladislav Kokoska c,e a Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamycka 129, Prague 165 21, Czech Republic b Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Crop Production, Kamycka 129, Prague 165 21, Czech Republic c Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Zoology and Fisheries, Kamycka 129, Prague 165 21, Czech Republic d Bee Research Institute at Dol, Libcice nad Vltavou 252 66, Czech Republic e Czech University of Life Sciences Prague, Institute of Tropics and Subtropics, Department of Crop Sciences and Agroforestry, Kamycka 129, Prague 165 21, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2010 Received in revised form 12 March 2010 Accepted 19 March 2010
In total, 26 natural compounds of various chemical classes (flavonoids, alkaloids, terpenoids) and 19 crude extracts from selected plants were tested in vitro for antibacterial activity against three strains of P. larvae, the causal agent of American Foulbrood Disease of honey bees (AFB) by the broth microdilution method. Among the individual substances, sanguinarine (MIC 4 mg/ml), followed by thymoquinone, capsaicin, trans-2-hexenal and nordihydroguaiaretic acid (MIC 4–32 mg/ml) possessed the strongest antibacterial effect. In case of extracts, common hop (Humulus lupulus L.) and myrtle (Myrtus communis L.) methanolic-dichloromethane extracts exhibited the highest growth-inhibitory effect with MICs ranging from 2 to 8 mg/ml. Acute oral toxicity of the most active natural products was determined on adult honey bees, showing them as non-toxic at concentrations as high as 100 mg peer bee. Our study leads to identification of highly potent natural products effective against AFB in vitro with very low MICs compared to those reported in literature, low toxicity to adult honey bees and commercial availability suggesting them as perspective, low cost and consumer-acceptable agents for control of AFB. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Paenibacillus larvae Apis mellifera Honey bee Antimicrobial activity Myrtus communis Humulus lupulus Thymoquinone Toxicity
1. Introduction American foulbrood (AFB) is a serious worldwidespreading disease of honey bee (Apis mellifera L.) caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. A popular approach in treatment of AFB in some countries, such as US, is suppressing of the clinical phase of
* Corresponding author. Tel.: +420 224 382 679; fax: +420 224 382 760. E-mail address: [email protected] (J. Havlik). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.03.018
the disease with supplemented antibiotics, however, this practice has been shown to lead to bacterial resistance (Eguaras et al., 2005) or contamination of apiary products (Mutinelli, 2003). In contrary, antibiotics in bee keeping are legally banned in European Union and affected colonies are often destroyed by burning the hives (European Commission, 2009a). This situation calls for an alternative and effective control of the disease with either therapeutics or prophylactic feed additives that do not contribute to the phenomenon of bacterial resistance and comply with strict
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J. Flesar et al. / Veterinary Microbiology 145 (2010) 129–133
EU policies and current trends of green consumerism. Higher plants are considered as important sources of bioactive molecules, some of them exhibiting antibacterial effect with future prospects in pharmaco-chemistry or food control (Lewis and Ausubel, 2006). Based on this presumption, various natural products e.g. allicin (Aronstein and Hayes, 2004), thymol (Fuselli et al., 2006b), citral, geraniol (Gochnauer et al., 1979), fatty acids (Feldlaufer et al., 1993), essential oils (Alippi et al., 1996; Floris and Carta, 1990; Fuselli et al., 2006a) or honey bee products (Antunez et al., 2008) were previously investigated in a number of studies, exhibiting growth-inhibitory effect against several diseases of honey bee brood, including AFB. Although some of these molecules or plantmixtures were found to be active against AFB, according to our knowledge, there is no systemic screening study evaluating their effect on P. larvae isolates promoting further research in this field. Thus, the aim of this study is to evaluate effect of natural products against three strains of P. larvae together with oral acute toxicity tests to adult bees for most potent compounds to provide the initial assessment of their possible oral administration to the colony. 2. Materials and methods 2.1. Extracts and natural compounds In our study, selected pure compounds of plant origin and plant extracts were tested in vitro on activity against P. larvae. Some of the plants and natural compounds (flavonoids, alkaloids and terpenoids) were selected based on their previously reported antimicrobial properties, ethno-pharmacological use or growth-inhibitory activity against Bacillus sp. Commonly available herbs and commercially available compounds were selected due to the presumption, that much is known about their biological activities, and their commercial availability often refers to known synthesis protocols and therefore presumably lower market price. Following natural compounds of high commercially available purities were tested: berberine chloride (98%), 1,8-cineol (93%), caffeic acid (98%), epigallocatechin gallate from green tea (95%), harmane (98%), chinin-hydrochlorid dihydrate (96%), naringenin (98%), quercetin dehydrate (98%), resveratrol (99%), sanguinarine chlorid hydrate (98%), thymoquinone (99%), trans-cinnamic acid (99%), trans-2-hexenal (98%), eugenol (99%), nordihydroguaiaretic acid (97%), piperin (98%), pyrogalol (98%), terpinen-4-ol (98%), all obtained from Sigma–Aldrich (CZ). Catechin (100%), chelidonic acid (98%), ellagic acid (96%), hesperetin (99%), thymol (100%), tomatin (100%) were obtained from Carl Roth (DE). Two compounds were available only in lower purities: natural capsaicin (65% capsaicin, 35% dihydrocapsaicin), and curcumin from Curcuma longa in purity 70% from Sigma–Aldrich (CZ). Reference antibiotics tylosin tartrate (100%) and oxytetracyclin (95%) were obtained from Sigma–Aldrich (CZ). Plant material was collected, dried and subjected to the extraction using Soxhlet apparatus (methanol-dichloromethane, 1:1). List of species used in the study and part used are shown in Table 2. After extraction, samples were
dried in a rotary evaporator at 40 8C, diluted in DMSO to concentration 25.6 mg/ml and stored at 20 8C until used. 2.2. Microorganisms and media Two strains of P. larvae used in this study were isolated in 2007–2008 from the winter wax debris collected from hives exhibiting clinical symptoms of AFB. Strain 17/08;h3 was isolated from a colony in Sudomerice–Zamosti (488510 5100 N, 178150 1400 E) and strain 1153/07;15 was sampled in Valtice–Polesi (488440 5100 N, 168470 1900 E). Both strains were identified at Bee Research Institute at Dol, according to the method described by The World Organisation for Animal Health (2008). The third was the type strain P. larvae ATCC 9545 (LGC Promochem, DE). Cultures were grown on MYPGP broth (Dingman and Stahly, 1983) consisting of 1.5% yeast extract (Oxoid, CZ), 1.0% Mueller–Hinton broth (Oxoid, CZ), 0.2% glucose, 0.3% K2HPO4, 0.1% sodium pyruvate. 2.3. Antimicrobial assay In vitro antibacterial activity was determined by the broth microdilution method (Jorgensen et al., 1999). Twofold dilutions (eight) of each compound or extract tested were carried out, starting from a concentration of 128 mg/ ml and 256 mg/ml, respectively. The wells were inoculated with a bacterial suspension (10 ml) at a density of 107 CFU/ ml, incubated at 37 8C for 48 h, and then observed for the minimum inhibitory concentration (MIC). The growth of microorganisms was determined spectrophotometrically as turbidity using Multiscan Ascent Microplate reader (Thermo Fisher Scientific, Waltham, USA) at 405 nm. The MIC was determined as the lowest dilution that resulted in 80% reduction in growth compared with the growth control (Jorgensen et al., 1999). The solution of DMSO (1%, v/v) was simultaneously assayed as the negative control. All samples were tested in triplicate. For the controls, MYPGP broth with antibiotics (tylosin, oxytetracycline) was assayed. 2.4. The acute oral toxicity to worker honey bees Acute oral toxicity of most active (MIC below 2–32 mg/ ml) natural compounds (capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, trans-2-hexenal) and extracts (Humulus lupulus, Myrtus communis) to adult honey bees (A. mellifera subsp. carnica) was evaluated by ICPBR technique (1993) and expressed as LD50. Young adult worker bees collected in the morning from frames without brood were used in the toxicity test. Groups of ten bees in plastic cup covered by permeable fabric were kept in an incubator (25 8C) under artificial light. Known amounts of natural products were dissolved in a feeding solution (50% w/v sucrose in distilled water) to obtain the desired concentrations. The test compounds were solubilised in DMSO (final concentration 5%. Bees were left starving for 2 h and afterwards 200 ml of test solution was offered in glass capillary feeding tubes put on the cover of the test cup. After consuming all of the solutions tested, bees were fed ad libitum with 50% toxicant-free sucrose
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solution. Dimethoate was used as a toxic standard (Gough et al., 1994). DMSO was assayed as a negative control. The LD50 at 24 h and its fiducial limits were calculated by probit regression analysis. 3. Results and discussion 3.1. Antimicrobial assay In total, 26 natural compounds of different chemical classes and 19 crude extracts were investigated. Determination of the MICs by means of the broth microdilution method showed that 13 compounds (Table 1) and 16 extracts tested (Table 2) exhibited an antimicrobial effect against P. larvae with MICs values ranging from 2 to 256 mg/ml. Out of all plant-derived products tested, the greatest antibacterial action against P. larvae was observed for sanguinarine with MIC values 4 mg/ml, followed by thymoquinone with MIC values ranging from 8 to 16 mg/ ml (depending upon the strain tested), capsaicin (MIC 32 mg/ml), nordihydroguaiaretic acid (MIC 32 mg/ml), trans-2-hexenal (32 mg/ml) and extracts of H. lupulus (MIC 2–4 mg/ml) and M. communis (MIC 2–8 mg/ml). MIC values of reference antibiotics (Table 1) ranged from 0.016 to 0.031 mg/ml. Both isolated strains were more susceptible to natural products than the type strain but not to antibiotics. The data published (Miyagi et al., 2000) show that some strains of P. larvae are resistant to oxytetracycline and MIC values in some cases exceed 32 mg/ml, which is considerably higher than those of our most potent natural products. Sanguinarine, the most active out of the compounds is considered as relatively non-toxic for humans, is often used as an antibacterial in toothpastes or mouthwashes and is commercially available as feed additive, registered as Macleaya extract by European Commission (2009b) for Table 1 Antibacterial activity of natural compounds against P. larvae. Compound Sanguinarine Thymoquinone Capsaicin Nordihydroguaiaretic acid trans-2-Hexenal Curcumin Harmane Quininin hydrochloride Naringenin Resveratrol Eugenol Thymol Hesperetin Catechin 1,8-Cineol Berberin chloride
MIC (mg/ml) 4 8–16 32 32 32 32–64 32–64 64 64 64
Compound
MIC (mg/ml)
Caffeic acid Ellagic acid Epigallocatechin Chelidonic acid
>128 >128 >128 >128
Piperine Pyrogallol Quercetin dihydrate Terpinen-4-ol Tomatin trans-Cinnamic acid
>128 >128 >128 >128 >128 >128
64–128 64–128 128 >128 >128 >128
Antibiotics Tylosin tartrate Oxytetracycline
0.016–0.031 0.016–0.031
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Table 2 Antibacterial activity of selected plant extracts against P. larvae. Extract
Part tested
MIC (mg/ml)
Humulus lupulus L.—hop flour Humulus lupulus L. Myrtus communis L. Eucalyptus gunnii Hook.f. Laurus nobilis L. Rosmarinus officinalis L. Capparis spinosa L. Catha edulis Forssk. Eucalyptus citriodora Hook. Zingiber officinale Roscoe Bixa orellana L. Curcuma longa L. Mangifera indica L. Camellia sinensis Kuntze Olea europaea L. Punica granatum L. Carica papaya L. Croton sp. Psidium quajava L.
Lupulin glands Cone Leaves Leaves Leaves Aerial parts Aerial parts Leaves Leaves Roots Leaves Roots Leaves Leaves Leaves Rind Leaves Leaves Leaves
2 2–4 2–8 16–32 16–32 16–32 32 32–64 32–64 32–256 64–256 64–256 128–256 256 256 256 >256 >256 >256
various species of animals (yet except bees); crude extracts from this plant were found to contain from 0.7 to 2.5% of sanguinarine (Zhang et al., 2005). Therefore, commercial application in beehives remains feasible regarding effect, toxicity, and availability. Similarly, both hop and myrtle extracts and herbs are already among approved additives in nutrition of various classes of animals according to the regulation of European Commission and therefore considered as safe. Hop and hop-derived compounds were previously reported to inhibit wide spectra of microorganisms, including Grampositive bacteria such as different species of Bacillus, Micrococcus, Staphylococcus and their effect may be attributed to the content of bitter resins (humulone, lupulone) (Schmalreck et al., 1975). Several studies have found a strong antibacterial activity of various extracts of M. communis. This plant contains acylphloroglucinols that are responsible for antibacterial activity against some Gram-positive and Gram-negative bacteria (Shaheen et al., 2006). Effect of feeding of selected extracts to honey bees is not new and was previously indicated in the literature by Pohorecka (2004) who studied the influence of selected plant extracts to general physiological conditions of honey bees, concluding that some of the extracts, e.g. nettle extract (Urtica dioica) may have potential in bee disease prevention. In our study, pure plant-derived compounds (capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, trans-2-hexenal), extracts from hop cone and myrtle leaves showed much lower MIC values in vitro against P. larvae than any other products reported by other researchers in previous studies. Among others, Floris and Carta (1990) tested essential oils of Citrus sinensis, Cinnamomum spp., Cuminum cyminum, Eugenia spp., Thymus vulgaris or Verbena sp. (MIC 100–200 mg/ml). Alippi et al. (1996) described activity of essential oils from Cymbopogon citratus, Satureja hortensis, Lavandula intermedia, Mentha piperita, Origanum vulgare, T. vulgaris, Rosmarius officinalis (MIC 50–700 mg/ml). Essential oils from Acantholippia seriphioides, Schinus molle, Minthosta-
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Table 3 Acute oral toxicity of tested natural products to worker honey bees. Compound/extract
24 h LD50 (mg/bee)
Capsaicin Nordihydroguaiaretic acid Sanguinarine Thymoquinone trans-2-Hexenal Humulus lupulus—cone extract Myrtus communis—leaves extract Dimethoate
>100 >200 153 >100 >200 >100 >100 0.12*
* Accepted range for dimethoate according to the OECD (1998) guideline for the oral toxicity test: 0.10 to 0.35 mg/bee.
chys mollis, Tagetes minuta, Lippia turbinata showed MIC 216–800 mg/ml (Fuselli et al., 2006a), or that from Cinnamomum zeylanicum exhibited MIC 25–100 mg/ml (Gende et al., 2008). Out of pure compounds, thymol (Fuselli et al., 2006b) or allicin (Aronstein and Hayes, 2004) were tested with MICs 100–130 mg/ml and 350 mg/ml, respectively.
growth of P. larvae in vitro at applicable concentrations. The use of perspective non-toxic compounds could represent a natural alternative to synthetic antibiotics in the control of American foulbrood, lessening considerably the controversies of antibiotic residues and resistance. Because of their significant growth-inhibitory action on P. larvae and their low toxicity to bees, these plant-derived products are potentially useful alternatives in suppressing American foulbrood, while being commercially available. The subsequent work will focus on field efficacy, methods of dosage (sugar solution, candy or aerosol) and delivery. Acknowledgements This research was supported by grants GACR No. 525/ 08/H060 (Czech Science Foundation), NAZV QH72144 (Grant Agency of the Ministry of Agriculture of the Czech Republic), and MSM No. 6046070901 (Ministry of Education, Youth, and Sports of the Czech Republic). References
3.2. The acute toxicity to worker honey bees The drugs could be administered to honey bees either orally, in vapour phase or topically on the thorax, therefore determination of toxicity represents an essential step for the follow up studies. In our opinion, one of the most promising methods is administration via feed, because compounds contained in the feeds are subsequently passed to bee products including royal jelly, as young larvae are fed royal jelly, later nectar or diluted honey and pollen. Therefore the oral toxicity to adult bees is of crucial importance, as they will be affected with the most concentrated product. Toxicity of products tested expressed as LD50 could not be precisely determined, as most of them showed lethal effect to bees at very high concentrations close to the solubility limits (LD50 values of capsaicin, nordihydroguaiaretic acid, thymoquinone, trans-2-hexenal, H. lupulus, and M. communis were >100, >200, >100, >200, >100, and >100 mg per bee, respectively) and were therefore virtually non-toxic. The LD50 could have been determined by probit analysis only for sanguinarine and was 153 mg peer bee, referring to slight toxicity at much higher concentrations than the potential therapeutic dose. Results of the oral toxicity tests are summarised in Table 3. Negative control consisting of DMSO solution showed no mortality at two-fold concentration as used (10%) and LD50 of DMSO was >6000 mg/bee. The LD50 value with 95% confidence for reference compound dimethoate was 0.12 mg/bee after 24 h, which corresponds to range of dimethoate according to the OECD (1998) guideline for the oral toxicity test: 0.10–0.35 mg/bee. Tests of oral toxicity indicated slight toxicity only in case of sanguinarine (LD50 = 153 mg peer bee), other compounds were virtually non-toxic. 4. Conclusion Our study indicates that some plant products (H. lupulus and M. communis) as well as plant-derived compounds capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, and trans-2-hexenal have potential to control
Alippi, A.M., Ringuelet, J.A., Cerimele, E.L., Re, M.S., Henning, C.P., 1996. Antimicrobial activity of some essential oils against Paenibacillus larvae, the causal agent of American foulbrood disease. J. Herbs Spices Med. Plants 4, 9–16. Antunez, K., Harriet, J., Gende, L., Maggi, M., Eguaras, M., Zunino, P., 2008. Efficacy of natural propolis extract in the control of American Foulbrood. Vet. Microbiol. 131, 324–331. Aronstein, K.A., Hayes, G.W., 2004. Antimicrobial activity of allicin against honey bee pathogens. J. Apicult. Res. 43, 57–59. Dingman, D.W., Stahly, D.P., 1983. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microb. 46, 860–869. Eguaras, M.J., Fuselli, S., Gende, L., Fritz, R., Ruffinengo, S.R., Clement, G., Gonzalez, A., Bailac, P.N., Ponzi, M.I., 2005. An in vitro evaluation of Tagetes minuta essential oil for the control of the honeybee pathogens Paenibacillus larvae and Ascosphaera apis, and the parasitic mite Varroa destructor. J. Essent. Oil Res. 17, 336–340. European Commission, 2009a. Report of the Meeting of the Terrestrial Animal Health Standards Commission Paris, 2–6 March 2009. Available at http://ec.europa.eu/food/international/organisations/ oie_en.htm (accessed 10.3.2010). European Commission, 2009b. Community Register of Feed Additives pursuant to Regulation (EC) No. 1831/2003. Available at http://ec.europa.eu/food/food/animalnutrition/feedadditives/comm_register_feed_additives_1831-03.pdf (accessed 10.12.2009). Feldlaufer, M.F., Knox, D.A., Lusby, W.R., Shimanuki, H., 1993. Antimicrobial activity of fatty acids against Bacillus larvae, the causative agent of American foulbrood disease. Apidologie 24, 95–99. Floris, I., Carta, C., 1990. In vivo activity of Cinnamomum zeylanicum Nees essential oil against Bacillus larvae White. Apicoltura 6, 57–61. Fuselli, S.R., de la Rosa, S.B.G., Gende, L.B., Eguaras, M.J., Fritz, R., 2006a. Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American foulbrood (AFB). J. Apicult. Res. 45, 2–7. Fuselli, S.R., de la Rosa, S.B.G., Gende, L.B., Eguaras, M.J., Fritz, R., 2006b. Inhibition of Paenibacillus larvae employing a mixture of essential oils and thymol. Rev. Argent. Microbiol. 38, 89–92. Gende, L.B., Floris, I., Fritz, R., Eguaras, M.J., 2008. Antimicrobial activity of cinnamon (Cinnamomum zeylanicum) essential oil and its main components against Paenibacillus larvae from Argentine. Bull. Insectol. 61, 1–4. Gochnauer, T.A., Boch, R., Margetts, V.J., 1979. Inhibition of Ascosphaera apis by citral and geraniol. J. Invertebr. Pathol. 34, 57–61. Gough, H.J., McIndoe, E.C., Lewis, G.B., 1994. The use of dimethoate as a reference compound in laboratory acute toxicity tests on honey bees (Apis mellifera L.) 1981–1992. J. Apicult. Res. 33, 119–125. International Commission for Plant-Bee Relationships (ICPBR), 1993. Hazard of Pesticides to Bees. Guideline on test methods for evaluating the side-effects of plant protection products on honeybees. Wageningen, The Netherlands, 1993 ver. 24/10/93 Appendix 22: EPPO G 170. European and Mediterranean Plant Protection Organization.
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Pohorecka, K., 2004. Effect of standardized plant herb extracts on general condition of the honeybee (Apis mellifera L.). Bull. Vet. Inst. Pulawy 48, 415–419. Shaheen, F., Ahmad, M., Khan, S.N., Hussain, S.S., Anjum, S., Tashkhodjaev, B., Turgunov, K., Sultankhodzhaev, M.N., Choudhary, M.I., Atta-UrRahman, 2006. New alpha-glucosidase inhibitors and antibacterial compounds from Myrtus communis L. Eur. J. Org. Chem. 10, 2371–2377. Schmalreck, A.F., Teuber, M., Reininger, W., Hartl, A., 1975. Structural features determining antibiotic potencies of natural and synthetic hop bitter resins, their precursors and derivatives. Can. J. Microbiol. 21, 205–212. The World Organisation for Animal Health (OIE), 2008. Manual of diagnostic tests and vaccines for terrestrial animals: American Foulbrood of Honey bees. Available at http://www.oie.int/eng/normes/mmanual/A_summry.htm Accessed 17.7.2008. Zhang, F., Chen, B., Xiao, S., Yao, S., 2005. Optimization and comparison of different extraction techniques for sanguinarine and chelerythrine in fruits of Macleaya cordata (Willd). R. Br. Sep. Purif. Technol. 42, 283– 290.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
In vitro growth-inhibitory effect of plant-derived extracts and compounds against Paenibacillus larvae and their acute oral toxicity to adult honey bees Jaroslav Flesar a, Jaroslav Havlik a,*, Pavel Kloucek b, Vojtech Rada a, Dalibor Titera c,d, Michal Bednar d, Michal Stropnicky c,d, Ladislav Kokoska c,e a Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamycka 129, Prague 165 21, Czech Republic b Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Crop Production, Kamycka 129, Prague 165 21, Czech Republic c Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Zoology and Fisheries, Kamycka 129, Prague 165 21, Czech Republic d Bee Research Institute at Dol, Libcice nad Vltavou 252 66, Czech Republic e Czech University of Life Sciences Prague, Institute of Tropics and Subtropics, Department of Crop Sciences and Agroforestry, Kamycka 129, Prague 165 21, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2010 Received in revised form 12 March 2010 Accepted 19 March 2010
In total, 26 natural compounds of various chemical classes (flavonoids, alkaloids, terpenoids) and 19 crude extracts from selected plants were tested in vitro for antibacterial activity against three strains of P. larvae, the causal agent of American Foulbrood Disease of honey bees (AFB) by the broth microdilution method. Among the individual substances, sanguinarine (MIC 4 mg/ml), followed by thymoquinone, capsaicin, trans-2-hexenal and nordihydroguaiaretic acid (MIC 4–32 mg/ml) possessed the strongest antibacterial effect. In case of extracts, common hop (Humulus lupulus L.) and myrtle (Myrtus communis L.) methanolic-dichloromethane extracts exhibited the highest growth-inhibitory effect with MICs ranging from 2 to 8 mg/ml. Acute oral toxicity of the most active natural products was determined on adult honey bees, showing them as non-toxic at concentrations as high as 100 mg peer bee. Our study leads to identification of highly potent natural products effective against AFB in vitro with very low MICs compared to those reported in literature, low toxicity to adult honey bees and commercial availability suggesting them as perspective, low cost and consumer-acceptable agents for control of AFB. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Paenibacillus larvae Apis mellifera Honey bee Antimicrobial activity Myrtus communis Humulus lupulus Thymoquinone Toxicity
1. Introduction American foulbrood (AFB) is a serious worldwidespreading disease of honey bee (Apis mellifera L.) caused by the spore-forming, Gram-positive bacterium Paenibacillus larvae. A popular approach in treatment of AFB in some countries, such as US, is suppressing of the clinical phase of
* Corresponding author. Tel.: +420 224 382 679; fax: +420 224 382 760. E-mail address: [email protected] (J. Havlik). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.03.018
the disease with supplemented antibiotics, however, this practice has been shown to lead to bacterial resistance (Eguaras et al., 2005) or contamination of apiary products (Mutinelli, 2003). In contrary, antibiotics in bee keeping are legally banned in European Union and affected colonies are often destroyed by burning the hives (European Commission, 2009a). This situation calls for an alternative and effective control of the disease with either therapeutics or prophylactic feed additives that do not contribute to the phenomenon of bacterial resistance and comply with strict
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EU policies and current trends of green consumerism. Higher plants are considered as important sources of bioactive molecules, some of them exhibiting antibacterial effect with future prospects in pharmaco-chemistry or food control (Lewis and Ausubel, 2006). Based on this presumption, various natural products e.g. allicin (Aronstein and Hayes, 2004), thymol (Fuselli et al., 2006b), citral, geraniol (Gochnauer et al., 1979), fatty acids (Feldlaufer et al., 1993), essential oils (Alippi et al., 1996; Floris and Carta, 1990; Fuselli et al., 2006a) or honey bee products (Antunez et al., 2008) were previously investigated in a number of studies, exhibiting growth-inhibitory effect against several diseases of honey bee brood, including AFB. Although some of these molecules or plantmixtures were found to be active against AFB, according to our knowledge, there is no systemic screening study evaluating their effect on P. larvae isolates promoting further research in this field. Thus, the aim of this study is to evaluate effect of natural products against three strains of P. larvae together with oral acute toxicity tests to adult bees for most potent compounds to provide the initial assessment of their possible oral administration to the colony. 2. Materials and methods 2.1. Extracts and natural compounds In our study, selected pure compounds of plant origin and plant extracts were tested in vitro on activity against P. larvae. Some of the plants and natural compounds (flavonoids, alkaloids and terpenoids) were selected based on their previously reported antimicrobial properties, ethno-pharmacological use or growth-inhibitory activity against Bacillus sp. Commonly available herbs and commercially available compounds were selected due to the presumption, that much is known about their biological activities, and their commercial availability often refers to known synthesis protocols and therefore presumably lower market price. Following natural compounds of high commercially available purities were tested: berberine chloride (98%), 1,8-cineol (93%), caffeic acid (98%), epigallocatechin gallate from green tea (95%), harmane (98%), chinin-hydrochlorid dihydrate (96%), naringenin (98%), quercetin dehydrate (98%), resveratrol (99%), sanguinarine chlorid hydrate (98%), thymoquinone (99%), trans-cinnamic acid (99%), trans-2-hexenal (98%), eugenol (99%), nordihydroguaiaretic acid (97%), piperin (98%), pyrogalol (98%), terpinen-4-ol (98%), all obtained from Sigma–Aldrich (CZ). Catechin (100%), chelidonic acid (98%), ellagic acid (96%), hesperetin (99%), thymol (100%), tomatin (100%) were obtained from Carl Roth (DE). Two compounds were available only in lower purities: natural capsaicin (65% capsaicin, 35% dihydrocapsaicin), and curcumin from Curcuma longa in purity 70% from Sigma–Aldrich (CZ). Reference antibiotics tylosin tartrate (100%) and oxytetracyclin (95%) were obtained from Sigma–Aldrich (CZ). Plant material was collected, dried and subjected to the extraction using Soxhlet apparatus (methanol-dichloromethane, 1:1). List of species used in the study and part used are shown in Table 2. After extraction, samples were
dried in a rotary evaporator at 40 8C, diluted in DMSO to concentration 25.6 mg/ml and stored at 20 8C until used. 2.2. Microorganisms and media Two strains of P. larvae used in this study were isolated in 2007–2008 from the winter wax debris collected from hives exhibiting clinical symptoms of AFB. Strain 17/08;h3 was isolated from a colony in Sudomerice–Zamosti (488510 5100 N, 178150 1400 E) and strain 1153/07;15 was sampled in Valtice–Polesi (488440 5100 N, 168470 1900 E). Both strains were identified at Bee Research Institute at Dol, according to the method described by The World Organisation for Animal Health (2008). The third was the type strain P. larvae ATCC 9545 (LGC Promochem, DE). Cultures were grown on MYPGP broth (Dingman and Stahly, 1983) consisting of 1.5% yeast extract (Oxoid, CZ), 1.0% Mueller–Hinton broth (Oxoid, CZ), 0.2% glucose, 0.3% K2HPO4, 0.1% sodium pyruvate. 2.3. Antimicrobial assay In vitro antibacterial activity was determined by the broth microdilution method (Jorgensen et al., 1999). Twofold dilutions (eight) of each compound or extract tested were carried out, starting from a concentration of 128 mg/ ml and 256 mg/ml, respectively. The wells were inoculated with a bacterial suspension (10 ml) at a density of 107 CFU/ ml, incubated at 37 8C for 48 h, and then observed for the minimum inhibitory concentration (MIC). The growth of microorganisms was determined spectrophotometrically as turbidity using Multiscan Ascent Microplate reader (Thermo Fisher Scientific, Waltham, USA) at 405 nm. The MIC was determined as the lowest dilution that resulted in 80% reduction in growth compared with the growth control (Jorgensen et al., 1999). The solution of DMSO (1%, v/v) was simultaneously assayed as the negative control. All samples were tested in triplicate. For the controls, MYPGP broth with antibiotics (tylosin, oxytetracycline) was assayed. 2.4. The acute oral toxicity to worker honey bees Acute oral toxicity of most active (MIC below 2–32 mg/ ml) natural compounds (capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, trans-2-hexenal) and extracts (Humulus lupulus, Myrtus communis) to adult honey bees (A. mellifera subsp. carnica) was evaluated by ICPBR technique (1993) and expressed as LD50. Young adult worker bees collected in the morning from frames without brood were used in the toxicity test. Groups of ten bees in plastic cup covered by permeable fabric were kept in an incubator (25 8C) under artificial light. Known amounts of natural products were dissolved in a feeding solution (50% w/v sucrose in distilled water) to obtain the desired concentrations. The test compounds were solubilised in DMSO (final concentration 5%. Bees were left starving for 2 h and afterwards 200 ml of test solution was offered in glass capillary feeding tubes put on the cover of the test cup. After consuming all of the solutions tested, bees were fed ad libitum with 50% toxicant-free sucrose
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solution. Dimethoate was used as a toxic standard (Gough et al., 1994). DMSO was assayed as a negative control. The LD50 at 24 h and its fiducial limits were calculated by probit regression analysis. 3. Results and discussion 3.1. Antimicrobial assay In total, 26 natural compounds of different chemical classes and 19 crude extracts were investigated. Determination of the MICs by means of the broth microdilution method showed that 13 compounds (Table 1) and 16 extracts tested (Table 2) exhibited an antimicrobial effect against P. larvae with MICs values ranging from 2 to 256 mg/ml. Out of all plant-derived products tested, the greatest antibacterial action against P. larvae was observed for sanguinarine with MIC values 4 mg/ml, followed by thymoquinone with MIC values ranging from 8 to 16 mg/ ml (depending upon the strain tested), capsaicin (MIC 32 mg/ml), nordihydroguaiaretic acid (MIC 32 mg/ml), trans-2-hexenal (32 mg/ml) and extracts of H. lupulus (MIC 2–4 mg/ml) and M. communis (MIC 2–8 mg/ml). MIC values of reference antibiotics (Table 1) ranged from 0.016 to 0.031 mg/ml. Both isolated strains were more susceptible to natural products than the type strain but not to antibiotics. The data published (Miyagi et al., 2000) show that some strains of P. larvae are resistant to oxytetracycline and MIC values in some cases exceed 32 mg/ml, which is considerably higher than those of our most potent natural products. Sanguinarine, the most active out of the compounds is considered as relatively non-toxic for humans, is often used as an antibacterial in toothpastes or mouthwashes and is commercially available as feed additive, registered as Macleaya extract by European Commission (2009b) for Table 1 Antibacterial activity of natural compounds against P. larvae. Compound Sanguinarine Thymoquinone Capsaicin Nordihydroguaiaretic acid trans-2-Hexenal Curcumin Harmane Quininin hydrochloride Naringenin Resveratrol Eugenol Thymol Hesperetin Catechin 1,8-Cineol Berberin chloride
MIC (mg/ml) 4 8–16 32 32 32 32–64 32–64 64 64 64
Compound
MIC (mg/ml)
Caffeic acid Ellagic acid Epigallocatechin Chelidonic acid
>128 >128 >128 >128
Piperine Pyrogallol Quercetin dihydrate Terpinen-4-ol Tomatin trans-Cinnamic acid
>128 >128 >128 >128 >128 >128
64–128 64–128 128 >128 >128 >128
Antibiotics Tylosin tartrate Oxytetracycline
0.016–0.031 0.016–0.031
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Table 2 Antibacterial activity of selected plant extracts against P. larvae. Extract
Part tested
MIC (mg/ml)
Humulus lupulus L.—hop flour Humulus lupulus L. Myrtus communis L. Eucalyptus gunnii Hook.f. Laurus nobilis L. Rosmarinus officinalis L. Capparis spinosa L. Catha edulis Forssk. Eucalyptus citriodora Hook. Zingiber officinale Roscoe Bixa orellana L. Curcuma longa L. Mangifera indica L. Camellia sinensis Kuntze Olea europaea L. Punica granatum L. Carica papaya L. Croton sp. Psidium quajava L.
Lupulin glands Cone Leaves Leaves Leaves Aerial parts Aerial parts Leaves Leaves Roots Leaves Roots Leaves Leaves Leaves Rind Leaves Leaves Leaves
2 2–4 2–8 16–32 16–32 16–32 32 32–64 32–64 32–256 64–256 64–256 128–256 256 256 256 >256 >256 >256
various species of animals (yet except bees); crude extracts from this plant were found to contain from 0.7 to 2.5% of sanguinarine (Zhang et al., 2005). Therefore, commercial application in beehives remains feasible regarding effect, toxicity, and availability. Similarly, both hop and myrtle extracts and herbs are already among approved additives in nutrition of various classes of animals according to the regulation of European Commission and therefore considered as safe. Hop and hop-derived compounds were previously reported to inhibit wide spectra of microorganisms, including Grampositive bacteria such as different species of Bacillus, Micrococcus, Staphylococcus and their effect may be attributed to the content of bitter resins (humulone, lupulone) (Schmalreck et al., 1975). Several studies have found a strong antibacterial activity of various extracts of M. communis. This plant contains acylphloroglucinols that are responsible for antibacterial activity against some Gram-positive and Gram-negative bacteria (Shaheen et al., 2006). Effect of feeding of selected extracts to honey bees is not new and was previously indicated in the literature by Pohorecka (2004) who studied the influence of selected plant extracts to general physiological conditions of honey bees, concluding that some of the extracts, e.g. nettle extract (Urtica dioica) may have potential in bee disease prevention. In our study, pure plant-derived compounds (capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, trans-2-hexenal), extracts from hop cone and myrtle leaves showed much lower MIC values in vitro against P. larvae than any other products reported by other researchers in previous studies. Among others, Floris and Carta (1990) tested essential oils of Citrus sinensis, Cinnamomum spp., Cuminum cyminum, Eugenia spp., Thymus vulgaris or Verbena sp. (MIC 100–200 mg/ml). Alippi et al. (1996) described activity of essential oils from Cymbopogon citratus, Satureja hortensis, Lavandula intermedia, Mentha piperita, Origanum vulgare, T. vulgaris, Rosmarius officinalis (MIC 50–700 mg/ml). Essential oils from Acantholippia seriphioides, Schinus molle, Minthosta-
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Table 3 Acute oral toxicity of tested natural products to worker honey bees. Compound/extract
24 h LD50 (mg/bee)
Capsaicin Nordihydroguaiaretic acid Sanguinarine Thymoquinone trans-2-Hexenal Humulus lupulus—cone extract Myrtus communis—leaves extract Dimethoate
>100 >200 153 >100 >200 >100 >100 0.12*
* Accepted range for dimethoate according to the OECD (1998) guideline for the oral toxicity test: 0.10 to 0.35 mg/bee.
chys mollis, Tagetes minuta, Lippia turbinata showed MIC 216–800 mg/ml (Fuselli et al., 2006a), or that from Cinnamomum zeylanicum exhibited MIC 25–100 mg/ml (Gende et al., 2008). Out of pure compounds, thymol (Fuselli et al., 2006b) or allicin (Aronstein and Hayes, 2004) were tested with MICs 100–130 mg/ml and 350 mg/ml, respectively.
growth of P. larvae in vitro at applicable concentrations. The use of perspective non-toxic compounds could represent a natural alternative to synthetic antibiotics in the control of American foulbrood, lessening considerably the controversies of antibiotic residues and resistance. Because of their significant growth-inhibitory action on P. larvae and their low toxicity to bees, these plant-derived products are potentially useful alternatives in suppressing American foulbrood, while being commercially available. The subsequent work will focus on field efficacy, methods of dosage (sugar solution, candy or aerosol) and delivery. Acknowledgements This research was supported by grants GACR No. 525/ 08/H060 (Czech Science Foundation), NAZV QH72144 (Grant Agency of the Ministry of Agriculture of the Czech Republic), and MSM No. 6046070901 (Ministry of Education, Youth, and Sports of the Czech Republic). References
3.2. The acute toxicity to worker honey bees The drugs could be administered to honey bees either orally, in vapour phase or topically on the thorax, therefore determination of toxicity represents an essential step for the follow up studies. In our opinion, one of the most promising methods is administration via feed, because compounds contained in the feeds are subsequently passed to bee products including royal jelly, as young larvae are fed royal jelly, later nectar or diluted honey and pollen. Therefore the oral toxicity to adult bees is of crucial importance, as they will be affected with the most concentrated product. Toxicity of products tested expressed as LD50 could not be precisely determined, as most of them showed lethal effect to bees at very high concentrations close to the solubility limits (LD50 values of capsaicin, nordihydroguaiaretic acid, thymoquinone, trans-2-hexenal, H. lupulus, and M. communis were >100, >200, >100, >200, >100, and >100 mg per bee, respectively) and were therefore virtually non-toxic. The LD50 could have been determined by probit analysis only for sanguinarine and was 153 mg peer bee, referring to slight toxicity at much higher concentrations than the potential therapeutic dose. Results of the oral toxicity tests are summarised in Table 3. Negative control consisting of DMSO solution showed no mortality at two-fold concentration as used (10%) and LD50 of DMSO was >6000 mg/bee. The LD50 value with 95% confidence for reference compound dimethoate was 0.12 mg/bee after 24 h, which corresponds to range of dimethoate according to the OECD (1998) guideline for the oral toxicity test: 0.10–0.35 mg/bee. Tests of oral toxicity indicated slight toxicity only in case of sanguinarine (LD50 = 153 mg peer bee), other compounds were virtually non-toxic. 4. Conclusion Our study indicates that some plant products (H. lupulus and M. communis) as well as plant-derived compounds capsaicin, nordihydroguaiaretic acid, sanguinarine, thymoquinone, and trans-2-hexenal have potential to control
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