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| ORIGINAL ARTICLE |
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| Year : 2017 | Volume
: 2
| Issue : 4 | Page : 99-102 |
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Marine anaerobic bacterial diversity for the production of antimicrobial agents
K Muddukrishnaiah, VP Shilpa
Department of Pharmaceutical Biotechnology, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India
| Date of Submission | 29-Aug-2017 |
| Date of Acceptance | 04-Dec-2017 |
| Date of Web Publication | 22-Jan-2018 |
Correspondence Address:
K Muddukrishnaiah
Department of Pharmaceutical Biotechnology, Sanjo College of Pharmaceutical Studies Vellapara, Chitali, Palakkad - 678 702, Kerala
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ed.ed_14_17
Background: Bacterial resistance to antibiotics has led to a search for new antimicrobial agents. Infectious diseases caused by bacteria, fungi and viruses are a major threat to human health.
Materials and Methods: Sixty marine soil samples were collected from different coastal zones and different sampling sites in Kerala and kept for anaerobic enrichment. Enrichment culture was screened for antibacterial activity by crowded plate method. From the sixty marine samples, twenty bacterial strains were isolated. Among the twenty bacterial strains, KS302 showed antimicrobial activity. Further analysis was established in aqueous and organic solvents of the crude extract. These secondary metabolites were studied for antimicrobial activity against clinical isolates of S.aureus (NCIM-5345), B. Subtilis (NCIM-2920), and E. coli (NCIM-5346).
Results: Antimicrobial compound producing microorganisms were identified by anaerobic crowded plate method and purified by streak plate method. Methanolic extract as well as lipid layer did not show any antimicrobial activity but solvent layer as well as aqueous layer had shown good antimicrobial activity against all the test organisms like S.aureus (NCIM-5345), B. Subtilis (NCIM-2920), and E. coli (NCIM-5346).
Conclusion: Marine anaerobic microorganisms can be explored by isolating a novel compound, which can be used as the drug of choice against pathogenic bacteria.
Keywords: Anaerobic, antimicrobial activity, crude extract, enrichment
How to cite this article:
Muddukrishnaiah K, Shilpa V P. Marine anaerobic bacterial diversity for the production of antimicrobial agents. Environ Dis 2017;2:99-102 |
| Introduction | |  |
Anaerobic bacteria are one of the oldest terrestrial creatures. They occur ubiquitously in soil and in the intestine of higher organisms and play a major role in human health, ecology, and industry.[1] More than 70% of our planet's surface is covered by oceans. In some marine ecosystems, such as the deep-sea floor and coral reefs, experts estimate that the biological diversity is higher than that in the tropical rainforests, as marine environment conditions are extremely different from terrestrial ones. The marine environment is an exceptional reservoir of a wide variety of organisms which are capable of producing novel, bioactive, and secondary metabolites with diverse chemical structures. This diversity of chemical compounds is believed to be a consequence of competition between organisms for space and resources in most marine habitats.
| Materials and Methods | | |
Source of microorganisms: all samples were collected from marine and mangrove sources from Kerala, India.
Enrichment, crowded plating, and purification
The samples were pasteurized for 1 h at 80°C and enriched into Reinforced Clostridia medium, incubated under anaerobic conditions by flushing with ultrapure argon and incubated at room temperature for 1 week. After 1 week, the enrichment was plated on Reinforced Clostridia medium agar slants through crowded plating technique and incubated under anaerobic conditions by flushing with ultrapure argon at room temperature for 3–5 days. After incubation, colonies forming zone of inhibition (X) were selected and carefully isolated and purified by repeated sub-culturing on the same medium [Figure 1].
Colony characters
Size: medium to big, form: round, margin: entire, elevation: convex, texture: smooth, glistening, pigmentation: cream, opacity: opaque.
Cultural characters
Cells settle down into the bottom of the tube within 18–24 h. Cells do not disperse into medium even after mixing. Growth found to be very sticky when picked with a loop.
Morphological characters
The shape of cells is big rods, endospore-forming, and chain of 2–3 cells (X). Old cells form big filaments [Figure 2].
Extraction, concentration of antimicrobial compounds
Methanolic extract
A volume of 500 ml culture was grown under strictly anaerobic conditions for 1 week as mentioned in the enrichment steps above. The supernatant was collected after centrifugation of the culture at 10,000 rpm for 10 min. The supernatant thus collected was concentrated to approximately 10 ml using rotary evaporator under vacuum at 35°C–40°C. This extract was used as crude extract. The extract was then mixed with methanol (5–10 ml) to dissolve the extract completely and centrifuged at 10,000 rpm for 5 min. The powder settled at the bottom was discarded and the liquid was again concentrated to approximately 2–3 ml. This methanolic extract was checked for antimicrobial activity against various test organisms.[2],[3],[4]
Ethyl acetate extract
A volume of 500 ml was grown under strictly anaerobic conditions for 1 week as mentioned in the enrichment. The supernatant was collected after centrifugation of culture at 10,000 rpm for 10 min. The pH of the supernatant was acidified to 2–3. The collected supernatant was mixed with equal amount of ethyl acetate in a separation funnel and mixed thoroughly. The mixture was then allowed to separate overnight without disturbing it. The three layers of the aqueous layer, solvent layer and lipid layer thus formed were collected carefully, separately, and concentrated using rotary evaporator under vacuum at 35°C–40°C. All the three layers were separately tested for antimicrobial activity against various test organisms.[2],[3],[4]
| Disc Diffusion Technique | | |
Media used
Mueller Hinton Agar (MHA).
Test organisms used
All the cultures used were standardized using 0.5 McFarland standard and test microorganisms were procured from NCIM Pune.[3],[5] Staphylococcus aureus (NCIM-5345), Bacillus subtilis (NCIM-2920), Escherichia More Details coli (NCIM-5346), and Candida albicans (NCIM-3100).
Preparation of antibiotic discs
The discs were saturated with test solutions (methanolic extract and ethyl acetate extract; solvent layer, aqueous layer, and lipid layer) and allowed to dry overnight under laminar air flow. Test for each layer was performed in triplicate. Twenty-four hour-old fresh pure cultures of test organisms as mentioned above were used to make lawn over MHA plates. Discs were placed onto the lawn and kept in the refrigerator for 30 min for dispersion of test solution. After 30 min, plates were incubated in an inverted position for 24–48 h at 35°C–37°C. Along with test solution, discs saturated with solvents under test were used as negative control and standard antibiotic discs were used as positive control.
| Result | | |
Phenotypic and biochemical identification
Morphological characterization was studied based on classical macroscopic techniques of shape, size, and elevation of pure colonies. Colonies were able to grow 1–2 days of incubation at room temperature. Cells do not disperse into medium even after mixing. Growth found to be very sticky when picked with a loop. The colony morphology of the isolates is medium to big and endospore-forming. They were smooth or rough and the color ranged from white to cream and opaque. The ability of the isolates to excrete extracellular enzymes was tested through hydrolysis of gelatine and starch. The ability of the isolates to excrete intracellular enzymes was determined through tests on catalase reaction; hydrogen sulfide production, nitrate reduction, urease, Voges–Proskauer, methyl red, citrate utilization, oxidase, motility, and triple sugar-iron test. The isolates deferred greatly on their ability to excrete various enzymes [Table 1].
Antimicrobial activity
Whatman filter paper Grade 3 was used to make discs. The discs were saturated with test solutions (methanolic extract, ethyl acetate extract; solvent layer, aqueous layer, and lipid layer) and allowed to dry overnight under laminar air flow. Test for each layer was performed in duplicate [Figure 3], [Figure 4], [Figure 5].[6] | Figure 3: Zone of inhibition formed by aqueous layer (water layer) and solvent layer (ethyl acetate layer) against test organism Bacillus subtilis (NCIM-2920), (n = 3)
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 | Figure 4: Zone of inhibition formed by aqueous layer (water layer) and solvent layer (ethyl acetate layer) against test organism Staphylococcus aureus (NCIM-5345), (n = 3)
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 | Figure 5: Zone of inhibition formed by aqueous layer (water layer) and solvent layer (ethyl acetate layer) against test organism Escherichia coli (NCIM-5346), (n = 3)
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| Discussion | | |
This study concludes that marine anaerobic bacteria are capable of producing antimicrobial agents under anaerobic conditions. Samples were collected randomly from the shore of Kerala region. Microorganisms may have wide distribution in the natural environment. Different marine environment condition makes bacterial diversity. This diversity of chemical compounds is believed to be a consequence of competition between organisms. Antimicrobial compound producing microorganisms were identified by anaerobic crowded plate method and purified by streak plate method. Methanolic extract as well as lipid layer did not show any antimicrobial activity, but solvent layer as well as aqueous layer had shown good antimicrobial activity against all the test organisms such as S. aureus (NCIM-5345), B. subtilis (NCIM-2920), and E. coli (NCIM-5346).
| Conclusion | | |
These marine anaerobic microorganisms can be explored for isolating a novel compound, which can be used as the drug of choice against pathogenic bacteria.
Acknowledgment
The authors would like to thank the management of Sanjo College of pharmaceutical studies for providing the research facilities.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Behnken S, Hertweck C. Anaerobic bacteria as producers of antibiotics. Appl Microbiol Biotechnol 2012;96:61-7.  |
| 2. | Zhang Y, Mu J, Gu X, Zhao C, Wang X, Xie Z, et al. A marine sulfate-reducing bacterium producing multiple antibiotics: Biological and chemical investigation. Mar Drugs 2009;7:341-54. |
| 3. | Gokulkrishnan K, Kusuma S, Kamala B. Antimicrobial activity of marine bacteria isolated from the Mangalore coast, west coast of India. Recent Res Sci Technol 2011;3:15-7. |
| 4. | Muddukrishnaiah K, Singh S. Antimicrobial, synergistic activity and antioxidant studies on multidrug resistance human pathogen using crude extract of Azadirachta indica leaf and Withania somnifera rhizome. J Plant Pathol Microbiol 2015;S3:009. |
| 5. | Muddukrishnaiah K, Krishnan S. Study of bacterial resistance in clinical isolates of Staphylococcus aureus by comparison of conventional in vitro methods with PCR & RFLP based genotypic methods. Br J Pharm Res 2016;10:1-8. |
| 6. | Wafula EN, Kinyua J, Karuiki D, Muigai A, Mwirichia R. Isolation and characterization of Bacillus species from soil in ngere tea catchment area of Murang'a county, Kenya. Int J Life Sci Res 2014;2:27-35. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1]
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