American Journal of Medicine and Medical Sciences

p-ISSN: 2165-901X    e-ISSN: 2165-9036

2012;  2(6): 111-113

doi: 10.5923/j.ajmms.20120206.01

In Vitro Sensitivity of Some Mycolic Acid-containing Actinomycetes to Nigella Sativa Extracts

Arif F. Fadailallah 1, Adil Mahgoub 2, Mohamed E. Hamid 2

1Department of Animal Health and Epizootic Disease Control, Federal Ministry of Animal Resources and Fisheries, Khartoum State, Sudan

2Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, KSA

Correspondence to: Mohamed E. Hamid , Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, KSA.

Email:

Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved.

Abstract

Nigella sativa L. (black seed or cumin) is an herbaceous plant, used for centuries for the treatment of various ailments, including infectious diseases. This study investigates the in vitro effect of extract of N. sativa against representative of mycolic acid containing actinomycetes notably members of the genera Gordonia, Mycobacterium, Nocardia and Rhodococcus. Methanolic and petroleum ether extracts of N. sativa at different concentrations (0.3 mg/ mL, 0.6 mg/ mL, 1.25 mg/ mL, 2.5 mg/ mL and 5 mg/ mL) were used to impregnate filter paper disks. The disks were incorporated onto Diagnostic Sensitivity Test agar, plates inoculated with test strains and incubated aerobically for up to seven days at 37 ℃. Inhibition zones around discs were measured in millimetres. At 0.6 mg/ mL all test strains showed inhibition zones of 1.5 cm except Mycobacterium phlei and R. rhodochrous. Petroleum ether extract was found to be more effective than the methanolic extracts since relatively higher concentrations from the latter were needed to achieve similar inhibitions zones produced by petroleum ether extracts. It be concluded that extracts of N. sativa is a promising candidate for the in vivo application since it is a natural food additive with no reported harmful effect.

Keywords: Nigella Sativa, Actinomycetes, Antibacterial Agents, Mycolic Acids

Cite this paper: Arif F. Fadailallah , Adil Mahgoub , Mohamed E. Hamid , "In Vitro Sensitivity of Some Mycolic Acid-containing Actinomycetes to Nigella Sativa Extracts", American Journal of Medicine and Medical Sciences, Vol. 2 No. 6, 2012, pp. 111-113. doi: 10.5923/j.ajmms.20120206.01.

Article Outline

1. Introduction
2. Methodology
3. Results
4. Discussion
ACKNOWLEDGEMENTS

1. Introduction

Different crude extracts of N. sativa have been tested for their antimicrobial effectiveness and found to have promising effects against some organisms[3, 9, 12].
The aim of this study was to investigate the in vitro effect of petroleum ether and methanolic extract of N. sativa on representative mycolic acid containing actinomycetes notably Nocardia africana which has been recently described in Sudan[8]. Also, strains from the genera: Gordonia, Mycobacterium, Nocardia and Rhodococcus were tested.

2. Methodology

Fifty grams of air dried and coarsely powdered of clean seeds of N. sativa were extracted in Soxhlet apparatus to obtain methanolic and petroleum ether extracts[5]. The extracts were filtered, and the filtrates were vaporized to dryness, and weighed in order to determine the % yield of the extracts, following the formula: % yield = (weight of extract/ weight of ground plant material) 100. The stock solutions of the crude methanolic and petroleum ether extracts were prepared by diluting the dried extracts with 50% methanol to obtain the desired final concentrations of 0.3 mg/ mL, 0.6 mg/ mL, 1.25 mg/ mL, 2.5 mg/ mL and 5 mg/ mL. These concentrations were used to impregnate filter paper disks (10 mm diameters). Disk impregnated into 50% ethanol were used as control.
Antibiotic susceptibility testing was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines as described previously[13]. The dried disks containing different concentrations and the controls were incorporated onto Diagnostic Sensitivity Test agar (Difco) onto which bacteria was inoculated, then incubated aerobically for up to seven days at 37 oC. Inhibition zones around discs were measured in millimetres.

3. Results

Figure 1. In vitro sensitivity of selected mycolic acid containing actinomycetes to petroleum ether extract of Nigella sativa using disc method on DST agar. Arrows indicate the direction of increasing concentration: 312.5μg/ml, 650μg/ml, 1,250μg/ml, 2,500μg/ml, 5,000μg/ml, and 10,000μg/m
Table 1. In vitro sensitivity of selected mycolic acid containing actinomycetes to disk impregnated at different concentration of methanolic and petroleum ether extracts of Nigella sativa
     
The effect of N. sativa in terms of inhibition zones is shown in Figure 1 and Table 1. At 312.5 µg /ml, 0.5 cm inhibition zone noticed in all test strains except Rhodococcus rhodochrous. At 625 µg/ml all test strains showed inhibition zones of 1.5 cm (+++) except Mycobacterium phlei and R. rhodochrous. Petroleum ether extract was found to be more effective than the methanolic extracts since relatively higher concentrations from the latter were needed to achieve similar inhibitions zoned produced by petroleum ether extracts.

4. Discussion

Mycolic acid containing actinomycetes (MACA) are a wide group of bacteria belonging to the genera Mycobacterium, Nocardia, Gordonia and Rhodococcus[7]. It includes some species that cause diseases such as tuberculosis, mastitis, and pulmonary infections[10]. MACA are known to be extremely tough and resistant to many therapeutic agents, both in man and animals[2, 4]. Mycobacterium is the main strong acid fast organisms, Gordonia and Nocardia are week acid fast whereas Rhodococcus is a non acid fast[11].
Throughout this study, the effects of the petroleum ether extract of N. sativa on the selected organisms were found good (Table 1). Organisms were inhibited at different concentrations. The most susceptible organisms were G. bronchialis and N. otidiscaviarum, and the inhibitory zones of 0.5 cm have been noticed on disk impregnated with the 312.5 µg/ml concentration.
On the other hand M. phlei showed 0.5 cm inhibition zone at 625µg/ml, while in case of R. rhodochrous is was 1250 µg/ml. These organisms can therefore be considered the most resistant to the anti microbial activity of N. sativa petroleum ether extract among tested strains.
From these comparisons, it is clear that the petroleum ether extract of N. sativa, as an antimicrobial agent, is more effective than the methanolic extract. Furthermore, it should be noted that the effect of the petroleum ether extract on N. otidiscaviarum and G. bronchialis was stronger than on M. phlei and R. rhodochrous (Table 1).
The study concluded that extract of N. sativa is a promising candidate for the in vivo application since it is a natural food additive with no reported harmful effect. The study needed to be supported by a wider in vitro application, not against MACA, but perhaps against a range of gram positive and gram negative organisms.

ACKNOWLEDGEMENTS

The authors are grateful to Dr H. El Subky[The Medicinal and Aromatic Plant Research Institute, Khartoum] for help with extraction and to Prof. T. Hassan[Univ. of Khartoum] for encouragement.

References

[1]  Ali, B.H., Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytother. Res., 17: 299-305.
[2]  Uhde, K.B., Pathak, S., McCullum, I.Jr, Jannat-Khah, D.P., Shadomy, S.V., Dykewicz, C.A., Clark, T.A., Smith, T.L., Brown, J.M. (2010). Antimicrobial-resistant nocardia isolates, United States, 1995-2004. Clin. Infect. Dis., 51: 1445-1448.
[3]  Salman, M.T., Khan, R.A., Shukla, I. (2008). Antimicrobial activity of Nigella sativa Linn. seed oil against multi-drug resistant bacteria from clinical isolates. Natural Product Radiance, 7: 10-14.
[4]  Brown A, Wallace RJ, Onyi G. Activities of the glycyclines N,N-dimethylglycylamido-minocycline andN,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline againstNorcardia spp. and tetracycline-resistant isolates of rapidly growing mycobacteria. Antimicrob Agents Chemoth 1996; 40: 874–878.
[5]  Kavanagh, F. (1972). Analytical Microbiology, vol. 2, Academic Press, New York.
[6]  Cowan MM. Plant products as antimicrobial agents Clin Microbiol Rev 1999; 564-582.
[7]  Goodfellow M. The genus Nocardia Trevisan 1889, 9AL, p. 464-489. In: A. Balows and B.I. Duerden (ed.), Topley and Wilson’s Microbiology and Microbial Infections, 9th edition, volume 2, Systematic Bacteriology. Edward Arnold, London, 1998.
[8]  Hamid ME, Maldonado LA, Sharaf Eldin G, Fathelrahman M, Saeed NS, Goodfellow M. Nocardia africana sp. nov. from patients with pulmonary lesions. J Clin Microbiol 2001; 39: 625-630.
[9]  Hanafy MS, Hatem ME. Studies on the antimicrobial activity of Nigella sativa seed (black cumin). J Ethnopharmacol. 1991; 34: 275-8.
[10]  McNeil MM, Brown JM. The medically important aerobic actinomycetes: Epidemiology and microbiology. Clin Microbiol Rev 1994; 7: 357-417.
[11]  Minnikin DE. Lipids, complex, their chemistry biosynthesis and roles. In: C. Ratledge and J. L. Standford (eds.). The Biology of Mycobacteria. 1, Pp. 95-184. Academic Press, London, 1982
[12]  Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant bacteria. Acta Microbiol Pol. 2000; 49: 63-74.
[13]  National Committee for Clinical Laboratory Standards (NCCLS) (2002). Susceptibility testing of mycobacteria, nocardia, and other aerobic actinomycetes. Tentative standards, 2nd ed. 2002. p. M24-T2.