Screening of antibiotic-producing Streptomyces from marine sediments of Bangladesh

Abstract

Antimicrobial resistance is a rising concern in the treatment of infectious diseases and the discovery of potential antimicrobial compounds is needed to combat against it. The focus of this study was the in vitro antimicrobial activities of Streptomyces obtained from the soil samples collected from different places of Cox’s Bazar, Bangladesh. A total of 156 isolates was obtained from thirty soil samples using two selective media namely yeast malt agar and starch casein agar. The isolates were morphologically distinct on the basis of spore mass color, reverse slide color, aerial and substrate mycelia formation and production of diffusible pigment. Among the isolates, 12 exhibited good antimicrobial activity against the tested microorganisms. Isolates were subjected to biochemical characterization and identified as Streptomyces spp. The results suggest that the Streptomyces species could be a promising source for potential antibacterial agents.

Introduction

The discovery and application of antibiotics in the treatment of bacterial diseases had been a noteworthy medical success of the 20th century.¹ The demand for new antibiotics continues to grow due to the rapidly emerging of multiple antibiotic resistant pathogens causing life-threatening infection and nature still remains the richest and the most versatile source of new antibiotics.^2-4 Natural products having novel structures have been observed to perform inherent biological activities. The soil is a natural reservoir for microorganisms and their antimicrobial products. Soil Actinomycetes are of special interest because of their known property to produce chemically diverse compounds with a wide range of biological activities.⁵ A huge number of currently used antibiotics including erythromycin, streptomycin, rifamycin, and gentamycin, are all products isolated from soil Actinomycetes.⁶ The two major groups of soil Actinomycetes that serve as important sources of antibiotics are Streptomyces and Micromonospora. It has been stated that Streptomyces account for about 80% of the total antibiotic products; while Micromonospora closely follows with less than one-tenth as much as Streptomyces.⁷

The Actinomycetes are classified as a group of Gram-positive bacteria with high G+C content (>55%) that are unique for their spore-forming abilities and formation of mycelia structures.^{7, 8} The most important characteristic of the genus Streptomyces that they are potential sources for secondary metabolites possessing a variety of biological activities, including antibacterial, antifungals, antivirals, antitumoral, antihypertensives, and mainly antibiotics and immunosuppressives, which is used for human and animal treatment.⁷

Marine environments are a largely untapped source for the isolation of new micro organisms with the potentiality to produce active secondary metabolites.⁹ Researchers have already isolated more than 11,000 marine-derived natural products and several compounds have been shown to possess significant bactericidal activity. The extreme environment of salinity and pressure exist in the marine environment cause these Streptomyces to adapt and produce natural compounds.¹⁰

Bangladesh is a tropical country and contains great ecological diversity. Relatively few scientific studies have been carried out for new antibiotics from microorganisms from the marine soil, where Actinomycetes, in particular, Streptomyces spp. are found abundantly. Streptomyces bangladeshensis, is a new species of Streptomyces, from the soil samples of Bangladesh producing bis-(2-ethylhexyl)-phthalate.¹¹ Actinomycin D was isolated from a new type strain of Streptomyces parvulus strain MARS-17, from the soils collected from Rajshahi, Bangladesh.¹²

Looking for the new and safe antibiotics to tackle the antibiotic resistance problem, the aim of this study was to explore few new regions of soil samples in Cox’s Bazar marine ecosystems of the Bay of Bengal which contains a vast diversity of microbial community and also to isolate Streptomyces which may produce exceptional bioactive compounds as antibiotics against the selective human pathogenic microorganisms.

Materials and Methods

Sample collection and processing

Thirty soil samples were collected from the different places of Cox’s Bazar including Moheshkhali Island, Moheshkhali bridge area, Moheshkhali Shooting area, Moheshkhali ghat, Sugondha beach and Laboni beach of the Bay of Bengal (Table I).

Results

Isolation of Streptomyces

A total of 156 isolates were recovered from the marine soil samples collected from the different location of the Cox’s Bazar in Bangladesh.

The colors of the aerial and substrate mycelium are described in Table II. Aerial hyphae arrangements, spore chain ornamentation and spore surface of the isolates were subsequently observed by light microscopy, indicating variation among the sporophore sizes and ornamentation and even in the spore surface. The morphological examination of these isolates indicates that these belong to the Streptomyces genus.^{14, 16-18} Classical biochemical test evidenced that all the isolates were positive in starch hydrolysis, casein hydrolysis, and catalase and oxidase test.

Antimicrobial activity of Streptomyces isolates

Results indicated that out of 156 isolates, only 12 were shown to have potent antimicrobial activity against the test pathogens and designated as MS6, MS27, LB63, MB92, MI104, LS105, LS106, SB121, SB126, MB131, MS136 and MB141 (Table III). No growth of the tested pathogenic organisms after 24 hours adjacent to the streaking of Actinomycetes was detected indicating good antimicrobial activity of the isolates. The antibacterial activity of the test isolates was varied among the 12 isolates, only 3 isolates showed no zone of inhibition against S. aureus, but showed good inhibition zone against E. coli, S. typhi and P. aeruginosa.

Discussion

Streptomyces are widely represented in nature by the largest number of species and varieties. They differ greatly in their morphology, physiology and biochemical activities in producing the majority of known antibiotics.¹⁹ This study was aimed to isolate antibiotic producing Streptomyces from the soil collected from different places of Cox’s Bazar. The soil contains a diversity community of organisms differentiated by morphology, biochemical and antibacterial activity.

The collected samples were varied in their physico-chemical, biochemical and biological nature, which consisted of sediment, seawater and intestinal tract of shrimp. For that reason, pretreatment methods such as heat treatment, air drying and serial dilution were done which was an important step for reducing contamination and enhancing the isolation of Streptomyces spp. The present results revealed that simply air drying and heat treatment coupled with shaking successfully enhanced the isolation of Streptomyces. In this study, we used two selective media namely yeast malt agar and starch casein agar supplemented with antifungal and antibacterial agents (nystatin and cyclohexamide) which allowed preferential growth of Streptomyces spp. over other species. Commonly, media with minimal nutrients containing high molecular weight compounds are used for the isolation of actinomycetes. In this respect, the obtained results confirmed the effectiveness of yeast malt agar and starch casein agar as previously demonstrated by Okazaki and Okami.²⁰ A total of 156 isolates were obtained with abundant colonies with white, yellow, grey and orange/brown color. Results indicated that most of the isolates were slow-growing, aerobic, chalky, and contain both aerial and substrate mycelia with a variety of colors relevant to that of Streptomyces. In particular, Streptomyces genus can be easily distin-guished from all other bacterial groups based on their distinctive phenotypic features that derived from chemotaxonomic markers and a wide range of other stable expressed features such as micro- and macro-morphology, physiology and biochemical properties.^14,21 The gross of the obtained morphological, chemotaxonomical and physiological as well as biochemical properties clearly confirmed that the marine isolates under study are belonging to the genus Streptomyces.

Most of the isolates were obtained from Moheshkhali shooting area (37 isolates) and Moheshkhali Island (30 isolates) and, others from Moheshkhali bridge area, Laboni beach and Sugondha beach. Since the Streptomyces are highly tolerant to salinity, in this study they were found abundantly in the salty soil of Cox’s Bazar marine ecosystem having salinity of 32% to 34.5%. The single streak method or perpendicular streak method was used to screen the antibiotic producing Streptomyces using Muller Hinton agar medium. Out of 156 isolates, the antibacterial activity was exhibited by 12 of the isolates belonging to the sample source of Moheshkhali shooting area (MS6 -surface; MS27 & MS136- 1.5 inch depth), Moheshkhali bridge area (MB92 & MB141 -surface; MB131 -1.5 inch depth), Moheshkhali island (MI104 -1.5 inch depth), Laboni beach (LB63 & LB106 -surface; LB105 -1.5 inch depth) and Sugongha beach (SB121 & SB126 -1.5 inch depth).

In this study, out of 12 isolates 9 were found to have potent inhibition zone against all the four tested pathogenic bacteria, although 3 isolates (MB131, MS136 and MB141) were not able to inhibit gram positive S. aureus, but showed good inhibition zone against three remaining gram negative pathogenic bacteria. E. coli, S. typhi and P. aeruginosa were inhibited by all the 12 Streptomyces isolates (100%) and S. aureus were inhibited by 9 isolates (75%). In this study among 12 isolates with antimicrobial activity, some exhibited highly potential results against tested organisms. For example, five soil isolates MS6, LB63, MI104, LB105, and LB106 were found to have combined potent antimicrobial activity against S. aureus, E. coli and S. typhi; LB63, LB106, SB121, MS136, and MB141 showed good inhibition zone against P. aeruginosa. Most of the highly potent isolates belong to the source of Laboni beach, Moheshkhali Island, Sugondha beach and Moheshkhali bridge respectively. This is may be due to the physical, biochemical and biological parameters of the sample sources.

The extent of inhibition zone was relatively larger and clearer leading to the conclusion that the tested pathogens are susceptible to the active compounds produced by these isolates. It is therefore suggested that a combination of several molecular analysis methods such as DNA re-association and PCR-based fingerprinting techniques may extremely help to provide broader information about the total genetic diversity of soil Streptomyces community. Perhaps, such methods may lead to the improvement in isolating antibiotic producing strains of soil Streptomyces obtained in this study.

Conclusion

Our results indicated the tremendous potential of marine Streptomyces species as a useful and sustainable source of powerful antibiotic and bioactive natural products. The present study was successful in selecting effective sampling site for discovery of novel antimicrobial agent.

References

1. Enright M. The evolution of a resistant pathogen: The case of MRSA. Curr Opin Pharmacol. 2003; 3: 474–79.

2. Coates ARM, Hu Y. Novel approaches to developing new antibiotics for bacterial infections. Br J Pharmacol. 2007; 152: 1147–54.

3. Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nat Rev Drug Discov. 2005; 4: 206-20.

4. Lam KS. New aspects of natural products in drug discovery. Trends Microbiol. 2007; 15: 279–89.

5. Bredholt H, Fjærvik E, Johnsen G, Zotchev SB. Actinomycetes from sediments in the Trondheim Fjord, Norway: Diversity and biological activity. Mar Drugs. 2008; 6: 12-24.

6. Jeffrey LSH. Isolation, characterization and identification of actinomycetes from agriculture soils at Semongok, Sarawak. Afr J Biotechnol [Internet]. 2008 Jan 1 [cited 2017 Oct 17];7(20). Available from: https://www.ajol.info/index.php/ajb/article/view/59415

7. Arifuzzaman M, Khatun MR, Rahman H. Isolation and screening of actinomycetes from Sundarbans soil for antibacterial activity. Afr J Biotechnol. 2010; 9: 4615-19.

8. Kutzner H. Über die bildung von huminstoffen durch streptomyceten. Landwirtsch Forsch. 1968; 21.

9. Baskaran R, Vijayakumar R, Mohan PM. Enrichment method for the isolation of bioactive Actinomycetes from mangrove sediments of Andaman Islands, India. Malaysian J Microbiol. 2011; 7: 22-28.

10. Poosarla A, L VR, Krishna RM. Isolation of potent antibiotic producing Actinomycetes from marine sediments of Andaman and Nicobar Marine Islands. J Microbiol Antimicrob. 2013; 5: 6–12.

11. Al-Bari MAA, Bhuiyan MSA, Flores ME, Petrosyan P, García-Varela M, Islam MAU. Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis-(2-ethylhexyl)phthalate. Int J Syst Evol Microbiol. 2005; 55: 1973–77.

12. Rahman M, Zahidul Islam M, Khondkar P, Islam MAU. Characterization and antimicrobial activities of a polypeptide antibiotic isolated from a new strain of Streptomyces parvulus. Bangladesh Pharm J. 2010; 13: 14-16.

13. Rahman MA, Islam MZ, Islam MAU. Antibacterial activities of Actinomycete isolates collected from soils of Rajshahi, Bangladesh. Biotechnol Res Int. 2011; 2011.

14. Shirling EB, Gottlieb D. Method for characterization of Streptomyces species. Int J Syst Bacteriol. 1966; 16: 313-40.

15. Madigan MT, Martinko JM, Parker J. Brock biology of microorganisms. 8th ed. USA, Prentice Hall, 1997.

16. Waksman SA. Classification, identification and descriptions of genera and species. The Actinomycetes. 1961 [cited 2017 Oct 17];2. Available from: http://ci.nii.ac.jp/naid/10005882694/

17. Nonomura H. Key for classification and identification of 458 species of the Streptomycetes included in ISP. J Ferment Technol. 1974; 52: 78-92.

18. LOCCI R. Streptomycetes and related genera. Bergeys Man Syst Bacteriol. 1989; 4: 2451-508.

19. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997; 25: 3389-402.

20. Okazaki T, Okami Y. Studies on marine microorganisms. II. J Antibiot (Tokyo). 1972; 25: 461-66.

21. Bergey DH, Holt JG. Bergey’s manual of determinative bacteriology. Baltimore, Williams & Wilkins, 1994.