Characterisation of antibacterial compounds produced by Pseudomonas spp. isolated from Hogsback wetlands, Eastern Cape, South Africa
- Authors: Obi, Akudo Odochi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/15688 , 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.
- Full Text:
- Authors: Obi, Akudo Odochi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/15688 , 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.
- Full Text:
Evaluation of some pseudomonas species isolated from Hogsback forest reserve for the production of antibacterial compounds
- Authors: Mkono, Yonela Pelokazi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape Pseudomonas
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/5961 , vital:29458
- Description: Pseudomonas species are Gram-negative bacteria most abundant in soil and water bodies, with the capacity to thrive in varied environments. They are largely associated with resistant pathogenic bacteria linked to human and plant diseases. Species such as Pseudomonas aeruginosa have been particularly targeted as case studies due to the extremity to which they pose a threat to human health. With more focus directed at using these species for biocontrol and bioremediation purposes, their role in bioactive compound production may be equally important. As the crisis on antimicrobial resistance still persists, the need for effective antimicrobial compounds is ever more urgent and solutions may possibly still be dormant in bacterial species whose potential has not been fully investigated. On a bid to source out potential antimicrobial compound producers, soil samples were collected from Hogback forest reserve in the province of the Eastern Cape, South Africa. For bacterial screening, M1 and R2A agar were used and the cultures grown at 37˚C for a period of seven days. After the presumed Pseudomonas species were identified, antimicrobial production was determined by submerged fermentation method using nutrient broth as media of choice. Active isolates were further studied to determine the optimum conditions which best facilitate for antimicrobial compound production, with parameters such as temperature (25˚C – 40˚C) and pH (4 – 9) considered. The role plasmids play in antimicrobial compound production was also investigated. Each isolate was grown in fermentation media containing Sodium dodecyl sulphate and Ethedium Bromide, at varying concentrations, to facilitate for plasmid curing. With each sample, distinct colonies were identified with varying pigmentations most dominant being a cream colour. The identity of the isolated strains was achieved through sequencing of 16S rDNA. Phylogenetic analysis showed that isolate A16 had 80 percent homology with Pseudomonas plecoglossicida strain P4 and share a close ancestor with isolates Y52 and Y81, also isolate Y89 showed a 90 percent homology with Pseudomonas sp. Co-11a. With the exception of isolate A16, the isolates which were active against Gram-negative bacteria lost activity as the screening processes continued. When looking at temperature variations, isolates Y81 and A16 were highly active with maximum activity observed at 35˚C while Y89 performed best at 25˚C and Y52 showed constant activity across all studied temperatures. The plasmids in all isolates were found to be 48.5 kb in size with the exception of isolate Y89 which was 20 kb. The plasmids were cured at concentrations of (1 mg/ml; 5 mg/ml; 7 mg/ml; 10 mg/ml; 11 mg/ml) SDS and (125 μg/ml; 6.5 μg/ml; 5μg/ml) EtBr. The curing process also showed changes in both the antimicrobial activity of the isolates as well as their physical characteristics. The isolates are the first reported Pseudomonas species from Hogsback forest reserve with the ability to produce antimicrobial compounds which are active against Gram-positive and Gram-negative bacteria. These mesophilic bacteria also show that plasmids do not pay any role in the production of antimicrobial compounds and that the biosynthesis genes are highly likely to be chromosomal borne meaning that the production cannot be linked to horizontal transfer of genes. Therefore, these isolated Pseudomonas species provide a potential reservoir of antimicrobial compounds which may play an important role in the antimicrobial resistance phenomenon.
- Full Text:
- Authors: Mkono, Yonela Pelokazi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape Pseudomonas
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/5961 , vital:29458
- Description: Pseudomonas species are Gram-negative bacteria most abundant in soil and water bodies, with the capacity to thrive in varied environments. They are largely associated with resistant pathogenic bacteria linked to human and plant diseases. Species such as Pseudomonas aeruginosa have been particularly targeted as case studies due to the extremity to which they pose a threat to human health. With more focus directed at using these species for biocontrol and bioremediation purposes, their role in bioactive compound production may be equally important. As the crisis on antimicrobial resistance still persists, the need for effective antimicrobial compounds is ever more urgent and solutions may possibly still be dormant in bacterial species whose potential has not been fully investigated. On a bid to source out potential antimicrobial compound producers, soil samples were collected from Hogback forest reserve in the province of the Eastern Cape, South Africa. For bacterial screening, M1 and R2A agar were used and the cultures grown at 37˚C for a period of seven days. After the presumed Pseudomonas species were identified, antimicrobial production was determined by submerged fermentation method using nutrient broth as media of choice. Active isolates were further studied to determine the optimum conditions which best facilitate for antimicrobial compound production, with parameters such as temperature (25˚C – 40˚C) and pH (4 – 9) considered. The role plasmids play in antimicrobial compound production was also investigated. Each isolate was grown in fermentation media containing Sodium dodecyl sulphate and Ethedium Bromide, at varying concentrations, to facilitate for plasmid curing. With each sample, distinct colonies were identified with varying pigmentations most dominant being a cream colour. The identity of the isolated strains was achieved through sequencing of 16S rDNA. Phylogenetic analysis showed that isolate A16 had 80 percent homology with Pseudomonas plecoglossicida strain P4 and share a close ancestor with isolates Y52 and Y81, also isolate Y89 showed a 90 percent homology with Pseudomonas sp. Co-11a. With the exception of isolate A16, the isolates which were active against Gram-negative bacteria lost activity as the screening processes continued. When looking at temperature variations, isolates Y81 and A16 were highly active with maximum activity observed at 35˚C while Y89 performed best at 25˚C and Y52 showed constant activity across all studied temperatures. The plasmids in all isolates were found to be 48.5 kb in size with the exception of isolate Y89 which was 20 kb. The plasmids were cured at concentrations of (1 mg/ml; 5 mg/ml; 7 mg/ml; 10 mg/ml; 11 mg/ml) SDS and (125 μg/ml; 6.5 μg/ml; 5μg/ml) EtBr. The curing process also showed changes in both the antimicrobial activity of the isolates as well as their physical characteristics. The isolates are the first reported Pseudomonas species from Hogsback forest reserve with the ability to produce antimicrobial compounds which are active against Gram-positive and Gram-negative bacteria. These mesophilic bacteria also show that plasmids do not pay any role in the production of antimicrobial compounds and that the biosynthesis genes are highly likely to be chromosomal borne meaning that the production cannot be linked to horizontal transfer of genes. Therefore, these isolated Pseudomonas species provide a potential reservoir of antimicrobial compounds which may play an important role in the antimicrobial resistance phenomenon.
- Full Text:
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