P Addo, M Quartey, M Abbas, B Adu-Addai, E Owusu, I Okang, A Dodoo, D De Souza, N Ankrah, D Ofori-Adjei
antimicrobial susceptibility testing, buruli ulcer, herbal medicines, minimum inhibitory concentration
P Addo, M Quartey, M Abbas, B Adu-Addai, E Owusu, I Okang, A Dodoo, D De Souza, N Ankrah, D Ofori-Adjei. In-Vitro Susceptibility of Mycobacterium Ulcerans to Herbal Preparations. The Internet Journal of Tropical Medicine. 2007 Volume 4 Number 2.
Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. Surgery is the main treatment option because antibiotics have mostly been ineffective. However, there are reports of successful treatment with undisclosed herbal preparations. This study screened 44 herbal preparations for (i) inhibitory activity against 7 M. ulcerans isolates, (ii) determined the minimum inhibitory concentrations (MICs) of 10 herbal preparations and (iii) screened them for extended antimicrobial activity, since BU is often associated with secondary bacterial infections. Twenty-five of the herbal preparations inhibited the growth of the M. ulcerans isolates. MICs of the 10 herbal preparations were between 0.20 and 12.50% volume/volume (1:128 - 1:2), and all 10 demonstrated extended antimicrobial activity. The findings (i) indirectly confirmed the claims that BU is being successfully treated with herbal preparations and (ii) suggest that the treatment of BU with herbal preparations might simultaneously treat secondary bacterial and fungal infections. In conclusion, there are a number of herbal preparations with anti-M. ulcerans activity, therefore there is the need to pay more attention to herbal therapy as a potential BU treatment option.
Buruli ulcer (BU) is a re-emerging disease caused by
Clinically, BU could present on any part of the body as a papule, nodule, plaque, oedema or ulcer with deeply undermined edges (WHO, 2001; Johnson et al, 2005); some of the skin conditions become cancerous with time (WHO, 2001). BU is often secondarily infected by bacteria and fungi (those often implicated include
In Ghana, there are unsubstantiated claims of the successful treatment of BU patients by traditional herbal practitioners (THPs) with undisclosed herbal preparations. This study therefore primarily sought to identify herbal preparations with inhibitory activity against
Materials and Methods
Microbial Strains other than
Nineteen microorganisms (6 Gram-positive bacteria, 10 Gram-negative bacteria and 3 fungi) were used to investigate any extended antimicrobial property the herbal preparations may possess. They microbes consisted of 11 biochemically-authenticated clinical isolates (
Medicinal Plants and Herbal Preparations
Forty-four medicinal plants and herbal preparations (Table 1) were obtained for the study, based on their purported antimicrobial and anti-inflammatory properties, which are exploited for the treatment of Buruli ulcer, various skin conditions, sores, new and old wounds, gastric and diabetic ulcers and cancers. The herbal preparations were obtained from: (i) Traditional herbal practitioners, (ii) a vendor of traditional herbal medicines, (iii) 2 herbal shops and (iv) users of traditional herbal preparations. All the plant materials were authenticated at the Botany Department of the University of Ghana, with the exception of 4 imported herbal preparations. The 4 imported herbal preparations (Swedish bitters,
Antibacterial and Antifungal Agents
Pefloxacin mesylate dehydrate and miconazole were used as reference drugs for the Gram-positive bacteria /Gram-negative bacteria and fungi respectively. Pefloxacin mesylate dehydrate is an antibiotic produced by WORKHARDT LTD of India, while miconazole is an antifungal produced by JANSSEN-CILAG LTD of Portugal.
The study was undertaken in a level 2 biosafety laboratory. Microbiological procedures such as subculturing, inoculum preparation, standardization of microbial isolates and the inoculation of media with microbial isolates were all conducted in a class IIA/B3 biosafety cabinet, while the technical team observed all institutional (NMIMR) biosafety guidelines for personnel protection and that of the laboratory.
Preparation of Inocula
Processing of Herbal Preparations
The medicinal plants were prepared into juices, infusions or decoctions and incorporated into media as such, to simulate the state in which consumers would use them. Swedish bitters, being a liquid extract was incorporated into media as such; also simulating the state in which it would be used.
Preparation of Herbal Juices
Fresh rhizomes, bulbs, thick leaves and plants containing gel were prepared into juices. Weighed amounts of each herb was washed in running water, rinsed with sterile distilled water and blended with sterile distilled water in a sterile Waring laboratory blender. The resultant 20% w/v juice was strained with a sterile tea strainer and filtered through a sterile Whatman No. 1 filter paper. The filtrate was kept at 4 C until use the following day.
Preparation of Herbal Infusions
Weighed amounts of whole plants or leaves were washed under running water, rinsed with sterile distilled water, shredded into bits, macerated overnight in sterile distilled water and subsequently boiled in a covered glass jar for 5 minutes. The infusion was strained in a sterile tea strainer, while pressing down the leaves (to get as much of the aqueous component from the plant as possible). The resultant 20% w/v infusion was left to cool and filtered through a sterile Whatman No. 1 filter paper and kept at 4 C until use the following day.
Preparation of Herbal Decoctions
Weighed amounts of stems, barks and roots were prepared as decoctions. The stems, barks and roots were scrubbed in running water, rinsed with sterile distilled water, air dried in a clean air rack and dry blended into powder. The powders were then macerated overnight in sterile distilled water and subsequently boiled in a covered glass jar for 20 minutes. Each decoction was strained in a sterile tea strainer, while pressing down the powder (to get as much of the aqueous component from the plant as possible). The resultant 20% w/v decoction was left to cool and filtered through a sterile Whatman No. 1 filter paper and kept at 4 C until use the following day.
Screening of Herbal Preparations for Anti- Activity
The herbal preparations were screened for demonstration of anti-
Determination of Minimum Inhibitory Concentrations of Herbal Preparations
The herbal preparations that exhibited anti-
Screening of Herbal Preparations for Extended Antimicrobial Activity
In view of the secondary infections that sometimes accompany BU, the herbal preparations that exhibited anti-
The Statistical Package for the Social Sciences (SPSS), version 12.0.1 was used for data analysis. The case summaries procedure was used to determine the means, standard deviations, minimum, maximum and median of the minimum inhibitory concentrations of the herbal preparations and susceptibilities of the isolates. After conducting the above-mentioned determinations the data were screened for normality and homogeneity of variance. Most of the data violated the assumptions of the normal distribution and since they could not be acceptably transformed they were analysed with nonparametric tests: (1) The Kruskal-Wallis Test was used to determine: (i) if the differences in the MIC values of the herbs were significant and (ii) if the differences in the susceptibilities of the
Demonstration of Anti- Activity
Twenty-five (56.82%) of the 44 herbal preparations inhibited the growth of all the 7
Minimum Inhibitory Concentrations of Herbal Preparations
MICs of the 10 herbal preparations are presented in Table 2. Briefly, the MICs were between 0.20 and 12.50% vol/vol (i.e. 1:640 – 1:2 dilution), with
Susceptibilities of Isolates to Herbal Preparations
Table 2 shows that all the 7
Susceptibilities of Isolates to Herbal Preparations by Community
The comparative susceptibilities of the isolates by community presented in Table 2 shows that the Amasaman group of isolates was on average (6.10 ± 4.53% vol/vol ) more susceptible to the herbal preparations than the Nsawam group of isolates (6.64 ± 5.17 %vol/vol). However, the difference in their susceptibilities was not statistically significant (P 0.05). The low eta2 coefficient (0.003 = 0.3%) further confirmed the weak effect that the source (community) of the isolates had on their susceptibilities to the herbal preparations.
Demonstration of Extended Antimicrobial Activity
Demonstration of anti- activity
Given that 56.81% (n=44) of the herbal preparations that were selected on the basis of their purported dermatological and antimicrobial properties also demonstrated anti-
Minimum inhibitory concentration (MIC) of herbal preparations
The differences in the MICs of the herbs were statistically significant, suggesting that some of the herbal preparations may be more effective than others; for which reason, there is the need to decide on the herbal preparations that merit further investigation. According to van den Berghe & Vlietinck's (1991) ‘rule of thumb', a prominent plant with antibacterial effect that is worthy of further investigation, is one that apart from the 1:2 dilution, the 1:8 and 1:32 dilutions also show inhibitory activities. Against this background, 3 of the preparations namely;
Susceptibility of isolates to herbal preparations
All the 6 Ghanaian
Herbal therapy would be worth considering as a treatment option because at some stage of BU (in both BU patients and experimentally infected animals), a large number of bacilli are present (Portaels et al., 1998; Addo et al., 2005, 2007), suggesting that a favourable condition for the selection of resistant mutants could be created. It is for this reason that combination therapy (as practised in the treatment of TB) has been adopted in the treatment of BU (WHO, 2003; WHO, 2004; Etuaful et al., 2005). Some TB drugs have also been found to be effective to some extent in BU treatment and have therefore been recommended by W.H.O as the current antibiotic treatment for BU (WHO, 2003). Unfortunately the use of the TB drugs (ex. rifampicin and streptomycin) could lead to an increase in drug resistance, especially in undiagnosed TB patients, which could eventually result in an increase in TB cases, especially in TB/BU endemic communities. In view of this, herbal therapy should be actively pursued as a BU treatment option because a single herb is never a single compound but a group of compounds, which potentiate each other or create synergy (Stermitz et al., 2000). In other words, the use of a single herb could simulate combination therapy, which may prevent, or at worse, delay the development of antimicrobial resistance. Against this background, the herbal preparations that we have identified should be used as whole plants to preserve their ‘combination therapy' property, since a lot of plant extracts are known to fail because only the active ingredient is isolated, which leads to the loss of the synergy that is conferred by the rest of the plant components (Chaudhury, 1992; Duke et al., 1998; Stermitz et al., 2000). Several cases have been documented by Beckstrom-Sternberg & Duke (1994), where synergy was lost by using the single-ingredient approach to develop drugs from plants.
Demonstration of extended antimicrobial activity
Observations in BU patients and
In conclusion, this study has shown that there are a number of herbal preparations in Ghana with anti-
The team members are grateful to the World Health Organization for funding the study; Professor Françoise Portaels for providing the reference isolates; Dr. Mensah Quianoo (Amasaman Hospital in the Greater Accra Region of Ghana) and Dr. Aninakwa (Nsawam Hospital in the Eastern Region of Ghana) for performing the surgeries and providing the biopsies and Mr. Amponsah of the Botany Department of the University of Ghana for authenticating the plants. The team members also acknowledge with gratitude the invaluable assistance of traditional herbal practitioners, vendors and users of traditional herbal medicines.
Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research
Source of Funding
World Health Organization, Geneva, Switzerland
Phyllis Addo (DVM, PHD) Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research College of Health Sciences, University of Ghana, P. O. Box LG 581, Legon. Accra, Ghana Email: firstname.lastname@example.org email@example.com Fax 233 21 502182 Telephone: 233 21 501178/9