• Hassan Mohamed Awad Chemistry of Natural and Microbial Products Dept. Pharmaceutical Industries Div. National Research Centre, 33 EL Bohouth St. (Former EL Tahrir St.) Dokki, Giza, Egypt. P.O.Box:12622
  • Mousa O Germoush Department of Biology, College of Science, Aljouf University, Sakaka, Al-Jouf, Kingdom of Saudia Arabia



 Objective: Clavulanic acid (CA) is a vital agent in the treatment of bacterial infections since it is a potent inhibitor of an extensive variety of β-lactamase enzymes. Its production from Streptomyces strains is fact of expanding clinical significance. This study aimed to isolate and characterize a promising Streptomyces (S) species strain which produced an effective β-lactamase inhibitor.

Methods: Streptomyces sp. NRC-88 was isolated from an Egyptian soil sample. The phenotypic and phylogenetic examinations of 16S rRNA gene were investigated. The active metabolite of this strain (CA) was determined by particular synergistic bioassay, spectrophotometric assay, recognized by thin layer chromatography, and structure of the CA affirmed by high-performance liquid chromatography (HPLC) method.

Results: A phylogenetic examination of the 16S rRNA gene sequence of the NRC-88 strain consistent with conventional taxonomy was carried out, and confirmed that the strain NRC-88 was most similar to S. aburaviensis S-66 (99%). The active metabolite of this strain (CA) was determined by different methods and confirmed the structure of the CA by the HPLC method. It produced up to 87 mg/l in a specific CA production medium.

Conclusion: A new species of Streptomyces sp. NRC-88 isolated and recognized by phenotypic and genotypic proof. This strain suggested the name, Streptomyces sp. NRC-88 (accession number KM014489). It was able to produce CA as the β-lactamase inhibitor.

Keywords: Isolation, Streptomyces sp. NRC-88, Phenotypic and phylogenic identification, Clavulanic acid production.


1. Alanis AJ. Resistance to antibiotics: Are we in the post-antibiotic era? Arch Med Res 2005;36(6):697-705.
2. Page MG. Beta-lactamantibiotics. In Antibiotic Discovery and Development. In: Dougherty TJ, Pucci MJ, editors. NewYork, NY: Springer; 2012. p. 79-117.
3. Shahid M, Sobia F, Singh A, Malik A, Khan HM, Jonas D, et al. Beta-lactams and beta-lactamase-inhibitors in current-or potential-clinical practice: A comprehensive update. Crit Rev Microbiol 2009;35(2):81-8.
4. Neu HC. The crisis in antibiotic resistance. Science 1992;257(5073):1064-73.
5. Toussaint KA, Gallagher JC. ß-lactam/ß-lactamase inhibitor combinations: From then to now. Ann Pharmacother 2015;49(1):86-98.
6. Saudagar PS, Survase SA, Singhal RS. Clavulanic acid: A review. Biotechnol Adv 2008;26(4):335-51.
7. Gor P, Ajbani A, Dalal K. Use of fixed dose combinations of antibiotics in a surgical department of a tertiary care teaching hospital alpa. Int J Pharm Pharm Sci 2015;7(11):259-62.
8. Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. J Antibiot (Tokyo) 2009;62(1):5-16.
9. Lazzarini A, Cavaletti L, Toppo G, Marinelli F. Rare genera of actinomycetes as potential producers of new antibiotics. Antonie Van Leeuwenhoek 2000;78(3-4):399-405.
10. Tan LT, Ser HL, Yin WF, Chan KG, Lee LH, Goh BH. Investigation of antioxidative and anticancer potentials of Streptomyces sp. MUM256 isolated from Malaysia mangrove soil. Front Microbiol 2015;6:1316.
11. Sathya R, Ushadevi T. Industrially important enzymes producing Streptomyces species from mangrove sediments. Int J Pharm Pharm Sci 2014;6(10):233-7.
12. Brown AG, Butterworth D, Cole M, Hanscomb G, Hood JD, Reading C, et al. Naturally-occurring beta-lactamase inhibitors with antibacterial activity. J Antibiot (Tokyo) 1976;29(6):668-9.
13. Jensen SE, Paradkar AS. Biosynthesis and molecular genetics of clavulanic acid. Antonie Van Leeuwenhoek 1999;75(1-2):125-33.
14. Cook MA, Wilkins RB. Process for the Preparation of Potassium Clavulanate; EP0672669B1; 1997.
15. Kitano K, Kintaka K, Katamoto K. Clavulanic acid production by Streptomyces katsurahamanus. Chem Abstr 1979;90:119758b.
16. Ocean Co. Ltd. (1981). Clavulanic Acid. Chem Abstr 94:137-803z.
17. Awad HM, El-Shahed KY, El-Nakkadi AE. Isolation, screening and identification of newly isolated soil Streptomyces (Streptomyces sp. NRC-35) for β-lactamase inhibitor production. World Appl Sci J 2009;7(5):637-46.
18. Awad HM, El-Shahed KY. A novel Actinomycete sp. isolated from Egyptian soil has β-lactamase inhibitor activity and belongs to the Streptomyces rochei phylogenetic cluster. World Appl Sci J 2013;3:360-70.
19. Guda IS, Abdelwahed NA, Awad HM, Shallan MA, El-Shahed KY, Abdel-Rahim AE. Enhancement of clavulanic acid production by Streptomyces sp. Mu-NRC77 via mutation and medium optimization. Trop J Pharm Res 2017;16(1):31-42.
20. Romero J, Liras P, Martin JF. Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 1984;20:318-25.
21. Reading C, Cole M. Clavulanic acid: A beta-lactamase-inhiting beta-lactam from Streptomyces clavuligerus. Antimicrob Agents Chemother 1977;11(5):852-7.
22. Bird AE, Bellis JA, Gasson BC. Spectrophotometric assay of clavulanic acid by reaction with imidazole. Analyst 1982;107:1241-5.
23. Foulstone M, Reading C. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob Agents Chemother 1982;22(5):753-62.
24. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacteriol l966;16(3):313-40.
25. Tresner HD, Backus EJ. System of color wheels for Streptomyces taxonomy. Appl Microbiol 1963;11:335-8.
26. Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST. Bergey’s Manual of Determinative Bacteriology. 9th ed. Baltimore: Williams & Wilkins; 2000.
27. Locci R. Streptomycetes and related genera. In: Williams ST, Sharpe ME, Holt JG, editors. Bergey’s Manual of Systematic Bacteriology. Vol. 4. Baltimore: The Williams, Wilkins Co.; 1989. p. 2451-508.
28. Lechevalier MP, Lechevalier HA. The chemotaxonomy of actinomycetes. Actinomycete Taxonomy. In: Dietz A, Thayer DW, ediors. Vol. 6. Arlington SIM, USA: Special Publication; 1980. p. 227-91.
29. Lee YK, Kim HW, Liu CL, Lee HK. A simple method for DNA extraction from marine bacteria that produce extracellular materials. J Microbiol Methods 2003;52(2):245-50.
30. Rintala H, Nevalainen A, Rönkä E, Suutari M. PCR primers targeting the 16S rRNA gene for the specific detection of Streptomycetes. Mol Cell Probes 2001;15(6):337-47.
31. Kim J, Lee J. Cloning, DNA sequence determination and analysis of growth-associated expiration of the SodF gene coding for Fe- and Zn containing superoxide dismutase of Streptomyces griseous. J Microbiol Biotechnol 2000;10:700-6.
32. Brodskii LI, Ivanov VV, Kalaidzidis IaL, Leontovich AM, Nikolaev VK, Feranchuk SI, et al. GeneBee-NET: An internet based server for biopolymer structure analysis. Biokhimiia 1995;60(8):1221-30.
33. Vincze T, Posfai J, Roberts RJ. NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic Acids Res 2003;31(13):3688-91.
34. Kathiresan K, Balagurunathan R, Masilamaiselvam M. Fungicidal activity of marine actinomycetes against phyotopathogenic fungi. Ind J Bioethanol 2005;4:271-6.
35. Chen KC, Lin HY, Wu JY, Hwang SC. Enhancement of clavulanic acid production in Streptomyces clavuligerus with ornithine feeding. Enzyme Microb Technol 2003;32:152-6.
36. Neto AB, Hirata DB, Cassiano Filho LC, Bellao C, Badino Junior AC, Hokka CO. A study on clavulanic acid production by Streptomyces clavuligerus in batch, fed-batch and continuous processes. Braz J Chem Eng 2005;22(4):557-63.
37. Rosselló-Mora R, Amann R. The species concept for prokaryotes. FEMS Microbiol Rev 2001;25(1):39-67.
38. Chen K, Lin Y, Tsai C, Hsieh C, Houng J. Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding. Biotechnol Lett 2002;24:455-8.
39. Williams ST, Goodfellow M, Wellington EM, Vickers JC, Alderson G, Sneath PH, et al. A probability matrix for identification of some Streptomycetes. J Gen Microbiol 1983;129(6):1815-30.
40. Rosa JC, Baptista Neto A, Hokka CO, Badino AC, Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus. Bioprocess Biosyst. Eng. 2005; 27:99-104.
41. Anderson AS, Wellington EM. The taxonomy of Streptomyces and related genera. Int J Syst Evol Microbiol 2001;51:797-814.
42. Kim J, Lee J. Cloning, DNA sequence determination, and analysis of growth-associated expiration of the SodF gene coding for Fe-and Zn containing superoxide dismutase of Streptomyces griseous. J Microbiol Biotechnol 2000;10:700-6.
43. Bull AT, Ward AC, Goodfellow M. Search and discovery strategies for biotechnology: The paradigm shift. Microbiol Mol Biol Rev 2000;64(3):573-606.
44. Kim HJ, Lee SC, Hwang BK. Streptomyces cheonanensis sp. nov. a novel streptomycete with antifungal activity. Int J Syst Evol Microbiol 2006;56:471-5.
45. Raytapadar S, Paul AK. Production of an antifungal antibiotic by Streptomyces aburaviensis 1DA-28. Microbiol Res 2001;155(4):315-23.
46. Jensen PR, Williams PG, Oh DC, Zeigler L, Fenical W. Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 2007;73(4):1146-52.
47. Mizui Y, Sakai T, Iwata M, Uenaka T, Okamoto K, Shimizu H, et al. Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities. J Antibiot (Tokyo) 2004;57(3):188-96.
245 Views | 425 Downloads
How to Cite
Mohamed Awad, H., and M. O. Germoush. “MOLECULAR AND MORPHOLOGICAL IDENTIFICATION OF STREPTOMYCES SP. NRC-88 NOVA SPECIES AS β-LACTAMASE INHIBITOR FOR PHARMACEUTICAL APPLICATION”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 10, no. 10, Sept. 2017, pp. 376-83, doi:10.22159/ajpcr.2017.v10i10.20201.
Original Article(s)