- Title
- Characterisation of antibacterial compounds produced by Pseudomonas spp. isolated from Hogsback wetlands, Eastern Cape, South Africa
- Creator
- Obi, Akudo Odochi
- Subject
- Bacteriology -- South Africa -- Eastern Cape
- Date Issued
- 2017
- Date
- 2017
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- http://hdl.handle.net/10353/15688
- Identifier
- vital:40507
- Description
- With microbial pathogens developing resistance against currently existing antimicrobials, there is an earnest need to discover and develop new therapeutic compounds with unique and/or precise modes of action. Natural products have provided the inspiration for most of the active ingredients in medicines and thus are regarded as a potential screening resource lead for discovery of new drugs. Pseudomonads are ubiquitous, Gram-negative, motile bacteria that flourish and inhabit a diversity of environments. Literature has vastly revealed that some members of the genus Pseudomonas are capable of producing a plethora of biologically active metabolites and thus are regarded as “attractive” bacteria for testing as potential sources for new therapeutic compounds. In this regard, this study was undertaken with an aim to isolate and characterize antibacterial compounds produced by Pseudomonas species isolated from aquatic milieu of the Hogsback wetlands, Eastern Cape, South Africa. A total of 12 water samples were aseptically collected from Hogsback wetlands. To ascertain antimicrobial potential of the isolates, 9 test bacterial (sensitive) strains, namely Escherichia coli ATCC 3695, Vibrio fluvialis, Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa ATCC 27853, Escherichia coli (2), Listeria ivanovii, Listeria monocytogenes and Enterococcus faecalis ATCC 19433 were used for antimicrobial screening. A total of 500 presumptive Pseudomonas spp. were isolated and screened for the production of antibacterial compound through the Agar-well diffusion method. About 8percent of the presumptive Pseudomonas spp. produced compound with activity against the test bacteria and based on the levels of activity shown, three isolates coded as A53, Y55 and Y138 were selected and identified by 16S rDNA sequence analysis and The Basic Local Alignment Search Tool analysis revealed that the three isolates (A53, Y55 and Y138) belonged to the Pseudomonas genus. Using MEGA6 software, a molecular phylogenetic tree was constructed by Maximum Likelihood Method. The phylogenetic analysis by this method showed that all three isolate codes (A53, Y55 and Y138) formed the same clade with Pseudomonas viridiflava strain P3 (Accession number: AY972186) and Pseudomonas plecoglossicida strain P9 (Accession number: AY972231) at a high bootstrap value of 100percent. Isolate code A53 and Y55 also grouped together at a bootstrap value of 96percent. Factors affecting antibacterial compound production by the three isolates were elucidated. The effect of pH on the three isolates revealed that isolate code A53 and Y55 could produce antibacterial compounds over all pH ranges (pH 4-9), however, antibacterial production was produced optimally at acidic conditions (pH 4) and a less acidic-neutral condition (pH 6) respectively. Isolate Y138 showed optimal production at pH 5. The effect of temperature on all three isolates was studied at different temperatures ranging between 25°C-40°C. Study on effect of temperature variations on antibacterial production revealed that all three isolates are capable of growing and producing antibacterial compounds over all the different temperature ranges, however, maximum/optimum production was observed at temperature of 30°C. In order to establish the role of plasmids in antibacterial production, two curing agents; Sodium dodecyl sulfate (SDS) and Ethidium bromide under different sub-lethal concentrations (1 mg/ml -11mg/ml and 2.5 μg/ml- 125 μg/ml respectively) were used to cure the selected isolates. Plasmid DNA samples of A53, Y55 and Y138, were separated by electrophoresis on a 0.7percent (w/v) agarose. Agarose gel electrophoresis revealed that not all the concentrations used were effective in curing the plasmid. A Quick Load 1 kb Extend DNA ladder marker was used for determining the size of the plasmids. Isolate code A53 and Y138 had a plasmid size of 48.5 kb while isolate Y55 had a plasmid size of approximately 20 kb. Antibacterial activity by agar-well diffusion method after the curing process revealed that antibacterial production was not lost or prevented, but rather increased. Thus, it is suggested that the genes responsible for antibiotic production in this study were chromosomal-encoded and not plasmid-encoded. This study revealed that Hogsback wetlands may represent a previously unexplored environment/source of discovering novel and potent antibacterial compounds.
- Format
- 109 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | SOURCE1 | MSc (Microbiology) OBI, AO.pdf | 2 MB | Adobe Acrobat PDF | View Details Download |