Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Date Issued: 2020
Targeting allosteric sites of Escherichia coli heat shock protein 70 for antibiotic development
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
- Authors: Okeke, Chiamaka Jessica
- Date: 2019
- Subjects: Heat shock proteins , Escherichia coli , Allosteric proteins , Antibiotics , Molecular chaperones , Ligands (Biochemistry) , Molecular dynamics , Principal components analysis , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/115998 , vital:34287
- Description: Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation.
- Full Text:
- Date Issued: 2019
Applicability of putative probiotic bacteria to replace antibiotics as growth promoters in commercial and indigenous piglets
- Authors: Dlamini, Ziyanda Confidence
- Date: 2017
- Subjects: Antibiotics , Dietary supplements , Piglets
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/11428 , vital:39071
- Description: Antibiotic use in pig production contributes to development of antimicrobial resistance in food animals and risk of antibiotic residues in animal food products. The use of probiotics appears to be the potential alternative to antibiotics. This work is designed to validate the applicability of administered direct-fed putative probiotic strains of Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4, Lactobacillus salivarius ZJ614 and Streptococcus salivarius NBCR 13956 on growth performance, hematological parameters, and antibody stimulation in weaned commercial and indigenous pigs. Forty-five weaned piglets were divided into five treatment experimental groups as follows: Diet with: antibiotic (PC), No antibiotic and no probiotic (NC), Probiotic (P1) (P2) combination of probiotics (P3). Piglets’ performance were monitored during the trial. Faecal and ileum samples were collected for microbial count analysis. Blood samples were collected from the sacrificed piglets at the end of the trial, for the hematological and biochemical analysis and probiotics ability to stimulate immunoglobulin G (IgG). Four probiotic strains were tested for cell surface hydrophobicity against xylene, chloroform and ethyl acetate. The strains were also tested for auto-aggregation and co-aggregation abilities. Enterococcus faecalis ATCC 29212 was used as a control pathogen in the co-aggregation ability test. The probiotic strains were also screened for presence of virulence and resistance genes. DNA was extracted from all the four probiotic strains using ZR Fungal/Bacterial DNA MiniPepTM (Zymo Research, USA) following manufacturer’s instructions. The DNA samples were later amplified in PCR reactions with specific primers to detect virulence genes of adhesion collagen protein (ace), aggregation substances (agg and asa); antibiotic resistance genes of: Vancomycin vanA, Vancomycin vanC1 and Vancomycin vanC2/3. The data was analyzed by one-way ANOVA using SAS statistics software (SAS 9.3) (2003). Results from the study revealed that supplementation of probiotics had no effect on feed intake (FI) in all of the experimental groups. However, supplementation of probiotics in P3 treatment resulted in greater average daily gain (ADG) and improved feed conversion ratio (FCR) of weaned piglets (p < 0.05). Microbial count of fecal samples from all the treatment groups did not differ while ileum samples had lower enteric bacteria in P3 group as compared to other treatments. Concentration of albumin, globulin, neutrophils and basophils were high in NC treatment compared to other treatments. (p < 0.05). IgG concentration was highest in P3 compared to other treatment (p < 0.05). All probiotics strains showed high hydrophobicity to all solvents used in the test. Cell Surface Hydrophobic was highest in xylene (78percent – 84percent), followed by chloroform (68percent – 75percent) and lowest in ethyl acetate (52percent - 60percent). All of the test strains showed high auto-aggregation ability after 4 hr of incubation. L .reuteri VB4 exhibited highest auto-aggregation ability of 70percent, while the least auto-aggregation ability of 60percent was observed for L. salivarius ZJ614. In the co-aggregation assays, all probiotic bacterial strains exhibited a strong co-aggregation of 45percent to 56percent after 4 hr of incubation at 37 °C. L. reuteri VB4 exhibited highest co-aggregation ability while L. salivarius ZJ614 exhibited the lowest co-aggregation ability. No presence of virulence genes ace and asa was detected in all of the putative probiotic strains, but agg virulence gene was detected in L. reuteri VB4 strains. L. reuteri VB4 and S. salivarius NBRC13956 had resistance genes vanC 2/3 and vanC1, respectively. Overall, the results from this study, suggest that the candidate probiotics strains have enhanced the growth and measured blood parameters of the weaned piglets used in these experiments, and also, the probiotic strains have the abilities to initiate the stimulation of IgG. Probiotics could be used as a novel alternative to antibiotics in the animal nutrition and production, with the abilities to confer health benefits and promote growth. The outcome of the research advocates that these probiotics will be beneficial to pig production, and might outcompete the current antibiotics in use as growth promoters. One of the major advantages of probiotics to pig industry is the production of antibiotic free and good quality pork meat to local and international consumers, thus, enhancing South African Pork industry.
- Full Text:
- Date Issued: 2017
- Authors: Dlamini, Ziyanda Confidence
- Date: 2017
- Subjects: Antibiotics , Dietary supplements , Piglets
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/11428 , vital:39071
- Description: Antibiotic use in pig production contributes to development of antimicrobial resistance in food animals and risk of antibiotic residues in animal food products. The use of probiotics appears to be the potential alternative to antibiotics. This work is designed to validate the applicability of administered direct-fed putative probiotic strains of Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4, Lactobacillus salivarius ZJ614 and Streptococcus salivarius NBCR 13956 on growth performance, hematological parameters, and antibody stimulation in weaned commercial and indigenous pigs. Forty-five weaned piglets were divided into five treatment experimental groups as follows: Diet with: antibiotic (PC), No antibiotic and no probiotic (NC), Probiotic (P1) (P2) combination of probiotics (P3). Piglets’ performance were monitored during the trial. Faecal and ileum samples were collected for microbial count analysis. Blood samples were collected from the sacrificed piglets at the end of the trial, for the hematological and biochemical analysis and probiotics ability to stimulate immunoglobulin G (IgG). Four probiotic strains were tested for cell surface hydrophobicity against xylene, chloroform and ethyl acetate. The strains were also tested for auto-aggregation and co-aggregation abilities. Enterococcus faecalis ATCC 29212 was used as a control pathogen in the co-aggregation ability test. The probiotic strains were also screened for presence of virulence and resistance genes. DNA was extracted from all the four probiotic strains using ZR Fungal/Bacterial DNA MiniPepTM (Zymo Research, USA) following manufacturer’s instructions. The DNA samples were later amplified in PCR reactions with specific primers to detect virulence genes of adhesion collagen protein (ace), aggregation substances (agg and asa); antibiotic resistance genes of: Vancomycin vanA, Vancomycin vanC1 and Vancomycin vanC2/3. The data was analyzed by one-way ANOVA using SAS statistics software (SAS 9.3) (2003). Results from the study revealed that supplementation of probiotics had no effect on feed intake (FI) in all of the experimental groups. However, supplementation of probiotics in P3 treatment resulted in greater average daily gain (ADG) and improved feed conversion ratio (FCR) of weaned piglets (p < 0.05). Microbial count of fecal samples from all the treatment groups did not differ while ileum samples had lower enteric bacteria in P3 group as compared to other treatments. Concentration of albumin, globulin, neutrophils and basophils were high in NC treatment compared to other treatments. (p < 0.05). IgG concentration was highest in P3 compared to other treatment (p < 0.05). All probiotics strains showed high hydrophobicity to all solvents used in the test. Cell Surface Hydrophobic was highest in xylene (78percent – 84percent), followed by chloroform (68percent – 75percent) and lowest in ethyl acetate (52percent - 60percent). All of the test strains showed high auto-aggregation ability after 4 hr of incubation. L .reuteri VB4 exhibited highest auto-aggregation ability of 70percent, while the least auto-aggregation ability of 60percent was observed for L. salivarius ZJ614. In the co-aggregation assays, all probiotic bacterial strains exhibited a strong co-aggregation of 45percent to 56percent after 4 hr of incubation at 37 °C. L. reuteri VB4 exhibited highest co-aggregation ability while L. salivarius ZJ614 exhibited the lowest co-aggregation ability. No presence of virulence genes ace and asa was detected in all of the putative probiotic strains, but agg virulence gene was detected in L. reuteri VB4 strains. L. reuteri VB4 and S. salivarius NBRC13956 had resistance genes vanC 2/3 and vanC1, respectively. Overall, the results from this study, suggest that the candidate probiotics strains have enhanced the growth and measured blood parameters of the weaned piglets used in these experiments, and also, the probiotic strains have the abilities to initiate the stimulation of IgG. Probiotics could be used as a novel alternative to antibiotics in the animal nutrition and production, with the abilities to confer health benefits and promote growth. The outcome of the research advocates that these probiotics will be beneficial to pig production, and might outcompete the current antibiotics in use as growth promoters. One of the major advantages of probiotics to pig industry is the production of antibiotic free and good quality pork meat to local and international consumers, thus, enhancing South African Pork industry.
- Full Text:
- Date Issued: 2017
Sequence and structural investigation of the nonribosomal peptide synthetases of Bacillus atrophaeus UCMB 5137(63Z)
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
Comparative in-vitro activities of trimethoprimsulfamethoxazole and the new fluoroquinolones against confirmed extended spectrum beta-lactamase producing Stenotrophomonas maltophilia in Nkonkobe Municipality, Eastern Cape environment
- Adeyemi, Oluwatosin Oluwakemi
- Authors: Adeyemi, Oluwatosin Oluwakemi
- Date: 2012
- Subjects: Antibiotics , Microbial sensitivity tests , Drug resistance in microorganisms , Pathogenic microorganisms , Gram-negative bacterial infections
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11275 , http://hdl.handle.net/10353/d1007576 , Antibiotics , Microbial sensitivity tests , Drug resistance in microorganisms , Pathogenic microorganisms , Gram-negative bacterial infections
- Description: Stenotrophomonas maltophilia is increasingly emerging as an opportunistic pathogen of global concern. Due to its inherent resistance to several classes of antibiotics including carbapenems and its ability to acquire mobile resistance elements, treatment of infections caused by S. maltophilia is a constant challenge for clinicians. Trimethoprim-sulphamethoxazole (TMP-SMX) is the generally accepted antibiotic of choice for the treatment of infections caused by this organism, but resistance to the drug is increasingly being reported; hence, the need for alternative therapeutic options. In this study, the antimicrobial susceptibility profile of 110 commensal S. maltophilia isolates obtained from Nkonkobe municipality, Eastern Cape Province, Republic of South Africa was investigated. Twenty-one antibiotics including TMP-SMX and the newer fluoroquinolones; levofloxacin, gatifloxacin and moxifloxacin were included in the antibiotic panel. About 63.4 percent of the isolates were susceptible to TMP-SMX with a resistance rate of 28.2 percent. The fluoroquinolones were more effective with susceptibilities ranging from 76 percent to 94.7 percent. Resistance to the fluoroquinolones ranged from 1.3 percent to 2.7 percent. Levofloxacin was the most effective fluoroquinolone tested. Phenotypic dectection of extended spectrum β-lactamases (ESBLs) showed double disc synergy test (DDST) positivity in 59.5 percent of the isolates. Cefepime was the most sensitive indicator cephalosporin in the DDST with 77.3 percent of suspected ESBL-producing isolates showing cefepime-clavulanic acid synergy. Isolates exhibited nine different ESBL phenotypes, however, PCR amplification of the bla genes revealed four isolates that possessed genes belonging to the CTX-M group (CTX-M-1 and CTX-M-8 groups). ESBL genes are usually carried on mobile elements such as plasmids and transposons which may also bear genes that mediate resistance to aminoglycosides, tetracyclines, TMP-SMX and fluoroquinolones. ESBL positive isolates appeared more susceptible to the fluoroquinolones compared to TMP-SMX but there was no significant relationship between ESBL production and susceptibility to these drugs (p > 0.05). The newer fluoroquinolones are a possible alternative treatment option for S. maltophilia infections in this environment but further studies and clinical investigations are needed to determine the in vivo efficacy of these drugs.
- Full Text:
- Date Issued: 2012
- Authors: Adeyemi, Oluwatosin Oluwakemi
- Date: 2012
- Subjects: Antibiotics , Microbial sensitivity tests , Drug resistance in microorganisms , Pathogenic microorganisms , Gram-negative bacterial infections
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11275 , http://hdl.handle.net/10353/d1007576 , Antibiotics , Microbial sensitivity tests , Drug resistance in microorganisms , Pathogenic microorganisms , Gram-negative bacterial infections
- Description: Stenotrophomonas maltophilia is increasingly emerging as an opportunistic pathogen of global concern. Due to its inherent resistance to several classes of antibiotics including carbapenems and its ability to acquire mobile resistance elements, treatment of infections caused by S. maltophilia is a constant challenge for clinicians. Trimethoprim-sulphamethoxazole (TMP-SMX) is the generally accepted antibiotic of choice for the treatment of infections caused by this organism, but resistance to the drug is increasingly being reported; hence, the need for alternative therapeutic options. In this study, the antimicrobial susceptibility profile of 110 commensal S. maltophilia isolates obtained from Nkonkobe municipality, Eastern Cape Province, Republic of South Africa was investigated. Twenty-one antibiotics including TMP-SMX and the newer fluoroquinolones; levofloxacin, gatifloxacin and moxifloxacin were included in the antibiotic panel. About 63.4 percent of the isolates were susceptible to TMP-SMX with a resistance rate of 28.2 percent. The fluoroquinolones were more effective with susceptibilities ranging from 76 percent to 94.7 percent. Resistance to the fluoroquinolones ranged from 1.3 percent to 2.7 percent. Levofloxacin was the most effective fluoroquinolone tested. Phenotypic dectection of extended spectrum β-lactamases (ESBLs) showed double disc synergy test (DDST) positivity in 59.5 percent of the isolates. Cefepime was the most sensitive indicator cephalosporin in the DDST with 77.3 percent of suspected ESBL-producing isolates showing cefepime-clavulanic acid synergy. Isolates exhibited nine different ESBL phenotypes, however, PCR amplification of the bla genes revealed four isolates that possessed genes belonging to the CTX-M group (CTX-M-1 and CTX-M-8 groups). ESBL genes are usually carried on mobile elements such as plasmids and transposons which may also bear genes that mediate resistance to aminoglycosides, tetracyclines, TMP-SMX and fluoroquinolones. ESBL positive isolates appeared more susceptible to the fluoroquinolones compared to TMP-SMX but there was no significant relationship between ESBL production and susceptibility to these drugs (p > 0.05). The newer fluoroquinolones are a possible alternative treatment option for S. maltophilia infections in this environment but further studies and clinical investigations are needed to determine the in vivo efficacy of these drugs.
- Full Text:
- Date Issued: 2012
Phytochemical analysis and bioactivity of Garcinia Kola (Heckel) seeds on selected bacterial pathogens
- Seanego, Christinah Tshephisho
- Authors: Seanego, Christinah Tshephisho
- Date: 2012
- Subjects: Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants , Microbial sensitivity tests , Streptococcal infections , Streptococcus , Staphylococcus aureus infections , Salmonella typhimurium , Traditional medicine
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11259 , http://hdl.handle.net/10353/420 , Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants , Microbial sensitivity tests , Streptococcal infections , Streptococcus , Staphylococcus aureus infections , Salmonella typhimurium , Traditional medicine
- Description: Garcinia kola is one of the plants used in folklore remedies for the treatment of microbial infections. Bacterial resistance to commonly used antibiotics has necessitated the search for newer and alternative compounds for the treatment of drug resistant microbial infections. This study focuses on the bioactivity of G. kola seeds on Streptococcus pyogenes (ATCC 49399), Staphylococcus aureus (NCTC 6571), Plesiomonas Shigelloides (ATCC 51903) and Salmonella typhimurium (ATCC 13311), organisms which can cause illnesses from mild to severe with potentially fatal outcomes. The crude ethyl acetate, ethanol, methanol, acetone and aqueous extracts were screened by agar-well diffusion method and the activities of the extract were further determined by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. The inhibition zones ranged from 0 - 24 mm, while MIC and MBC of the extract ranged between 0.04 - 1.25 mg/mL and 0.081 - 2.5 mg/mL respectively. Chloroform/ Ethyl Acetate/ Formic acid (CEF) solvent system separated more active compounds followed by Ethyl Acetate/ Methanol/ Water (EMW) and Benzene/ Ethanol/ Ammonium Hydroxide (BEA). The extracts were fractionated by Thin Layer Chromatography (TLC). Bioautography was used to assess the activity of the possible classes of compounds present in the more active extracts. Column chromatography was used to purify the active compounds from the mixture while Gas Chromatography-Mass Spectrometry (GC-MS) was used to identify the phyto components of the fractions. The MIC of the fractions ranged between 0.0006 - 2.5 mg/mL. CEF 3 (F3), CEF 11 (F11) and CEF 12 (F12) revealed the presence of high levels fatty acids Linoleic acid, 1, 2-Benzenedicarboxylic acid and 2, 3-Dihydro-3, 5-dihydroxy-6-methyl, respectively. The results obtained from this study justify the use of this plant in traditional medicine and provide leads which could be further exploited for the development of new and potent antimicrobials.
- Full Text:
- Date Issued: 2012
- Authors: Seanego, Christinah Tshephisho
- Date: 2012
- Subjects: Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants , Microbial sensitivity tests , Streptococcal infections , Streptococcus , Staphylococcus aureus infections , Salmonella typhimurium , Traditional medicine
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11259 , http://hdl.handle.net/10353/420 , Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants , Microbial sensitivity tests , Streptococcal infections , Streptococcus , Staphylococcus aureus infections , Salmonella typhimurium , Traditional medicine
- Description: Garcinia kola is one of the plants used in folklore remedies for the treatment of microbial infections. Bacterial resistance to commonly used antibiotics has necessitated the search for newer and alternative compounds for the treatment of drug resistant microbial infections. This study focuses on the bioactivity of G. kola seeds on Streptococcus pyogenes (ATCC 49399), Staphylococcus aureus (NCTC 6571), Plesiomonas Shigelloides (ATCC 51903) and Salmonella typhimurium (ATCC 13311), organisms which can cause illnesses from mild to severe with potentially fatal outcomes. The crude ethyl acetate, ethanol, methanol, acetone and aqueous extracts were screened by agar-well diffusion method and the activities of the extract were further determined by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. The inhibition zones ranged from 0 - 24 mm, while MIC and MBC of the extract ranged between 0.04 - 1.25 mg/mL and 0.081 - 2.5 mg/mL respectively. Chloroform/ Ethyl Acetate/ Formic acid (CEF) solvent system separated more active compounds followed by Ethyl Acetate/ Methanol/ Water (EMW) and Benzene/ Ethanol/ Ammonium Hydroxide (BEA). The extracts were fractionated by Thin Layer Chromatography (TLC). Bioautography was used to assess the activity of the possible classes of compounds present in the more active extracts. Column chromatography was used to purify the active compounds from the mixture while Gas Chromatography-Mass Spectrometry (GC-MS) was used to identify the phyto components of the fractions. The MIC of the fractions ranged between 0.0006 - 2.5 mg/mL. CEF 3 (F3), CEF 11 (F11) and CEF 12 (F12) revealed the presence of high levels fatty acids Linoleic acid, 1, 2-Benzenedicarboxylic acid and 2, 3-Dihydro-3, 5-dihydroxy-6-methyl, respectively. The results obtained from this study justify the use of this plant in traditional medicine and provide leads which could be further exploited for the development of new and potent antimicrobials.
- Full Text:
- Date Issued: 2012
Assessment of the antibacterial properties of n-Hexane extract of Cocos Nucifera and its interactions with some conventional antibiotics
- Authors: Akinyele, Taiwo Adesola
- Date: 2011
- Subjects: Coconut palm , Microbial sensitivity tests , Gram-negative bacterial infections , Vibrio infections , Antibiotics , Hexane , Extracts
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11245 , http://hdl.handle.net/10353/416 , Coconut palm , Microbial sensitivity tests , Gram-negative bacterial infections , Vibrio infections , Antibiotics , Hexane , Extracts
- Description: Cocos nucifera belong to the family Aracaceae (palm Family). The English name is coconut and it is used extensively as medicinal remedies against infections such as urinary tract infections, gastro intestinal infections, skin and wound infections. The in vitro antibacterial (including anti-listerial and anti-vibrio) properties as well as the evaluation of the combination potentials of the plant extract with six front-line antibiotics were evaluated in this study using standard procedures. The in vitro anti-listerial properties of the crude aqueous and n-Hexane extract of the husk of Cocos nucifera were carried out against 37 Listeria isolates. Twenty-nine of the test organisms were susceptible to the aqueous extract while thirty were susceptible to the n-Hexane extract both at the screening concentration of 25 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.6 - 5.0 mg/ml. For the aqueous extract, average log reduction in viable cell count ranged between 0.32 Log10 and 4.8 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 2.4 Log10 and 6.2 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. The time-kill characteristics of the two extracts suggest that at higher concentration (2 × MIC) and longer duration of interaction (8 hr), more bacteria were killed. In vitro anti-vibrio and antibacterial properties experiment revealed that of all the 45 vibrio and 25 bacteria strains that was tested, 37 were susceptible to the aqueous extract and 38 to the n-Hexane extract, while 17 were susceptible to the aqueous extract and 21 to the n-Hexane extract. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.3 - 5.0 mg/ml. viii The time kill studies revealed that for the aqueous extract, average log reduction in viable cell count in time kill assay ranged between 0.12 Log10 and 4.2 Log10 CFU/ml after 8 hr interaction at 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 0.56 Log10 and 6.4 Log10 CFU/ml after 8 hr interaction in 1 × MIC and 2 × MIC. In the test for the combination interactions, the checkerboard method revealed synergy of 67% and indifferent of 33%, while the time kill assay detected synergy in 72% and indifferent in 28% of the combinations tested. The synergy detected was not specific to any of the antibiotics or the Gram reaction of the bacteria, and no antagonism was detected. We conclude that the aqueous and n-Hexane extract of the husk of C. nucifera contains potential broad spectrum antibiotics resistance modulating compounds that could be relevant in the treatment of infections caused by these pathogens. In addition, the husk which is being discarded as agro waste will opens up a vista of opportunities for utilization for therapeutic purposes
- Full Text:
- Date Issued: 2011
- Authors: Akinyele, Taiwo Adesola
- Date: 2011
- Subjects: Coconut palm , Microbial sensitivity tests , Gram-negative bacterial infections , Vibrio infections , Antibiotics , Hexane , Extracts
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11245 , http://hdl.handle.net/10353/416 , Coconut palm , Microbial sensitivity tests , Gram-negative bacterial infections , Vibrio infections , Antibiotics , Hexane , Extracts
- Description: Cocos nucifera belong to the family Aracaceae (palm Family). The English name is coconut and it is used extensively as medicinal remedies against infections such as urinary tract infections, gastro intestinal infections, skin and wound infections. The in vitro antibacterial (including anti-listerial and anti-vibrio) properties as well as the evaluation of the combination potentials of the plant extract with six front-line antibiotics were evaluated in this study using standard procedures. The in vitro anti-listerial properties of the crude aqueous and n-Hexane extract of the husk of Cocos nucifera were carried out against 37 Listeria isolates. Twenty-nine of the test organisms were susceptible to the aqueous extract while thirty were susceptible to the n-Hexane extract both at the screening concentration of 25 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.6 - 5.0 mg/ml. For the aqueous extract, average log reduction in viable cell count ranged between 0.32 Log10 and 4.8 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 2.4 Log10 and 6.2 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. The time-kill characteristics of the two extracts suggest that at higher concentration (2 × MIC) and longer duration of interaction (8 hr), more bacteria were killed. In vitro anti-vibrio and antibacterial properties experiment revealed that of all the 45 vibrio and 25 bacteria strains that was tested, 37 were susceptible to the aqueous extract and 38 to the n-Hexane extract, while 17 were susceptible to the aqueous extract and 21 to the n-Hexane extract. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.3 - 5.0 mg/ml. viii The time kill studies revealed that for the aqueous extract, average log reduction in viable cell count in time kill assay ranged between 0.12 Log10 and 4.2 Log10 CFU/ml after 8 hr interaction at 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 0.56 Log10 and 6.4 Log10 CFU/ml after 8 hr interaction in 1 × MIC and 2 × MIC. In the test for the combination interactions, the checkerboard method revealed synergy of 67% and indifferent of 33%, while the time kill assay detected synergy in 72% and indifferent in 28% of the combinations tested. The synergy detected was not specific to any of the antibiotics or the Gram reaction of the bacteria, and no antagonism was detected. We conclude that the aqueous and n-Hexane extract of the husk of C. nucifera contains potential broad spectrum antibiotics resistance modulating compounds that could be relevant in the treatment of infections caused by these pathogens. In addition, the husk which is being discarded as agro waste will opens up a vista of opportunities for utilization for therapeutic purposes
- Full Text:
- Date Issued: 2011
In-vitro anti-vibrio activities of crude extracts of Garcinia Kola seeds
- Authors: Penduka, Dambudzo
- Date: 2011
- Subjects: Microbial sensitivity tests , Drug resistance in microorganisms , Antibiotics , Garcinia , Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11256 , http://hdl.handle.net/10353/405 , Microbial sensitivity tests , Drug resistance in microorganisms , Antibiotics , Garcinia , Medicinal plants
- Description: The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.
- Full Text:
- Date Issued: 2011
- Authors: Penduka, Dambudzo
- Date: 2011
- Subjects: Microbial sensitivity tests , Drug resistance in microorganisms , Antibiotics , Garcinia , Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11256 , http://hdl.handle.net/10353/405 , Microbial sensitivity tests , Drug resistance in microorganisms , Antibiotics , Garcinia , Medicinal plants
- Description: The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.
- Full Text:
- Date Issued: 2011
Phytochemical analysis and bioactivity of the stem bark of Combretum Molle on some selected bacterial pathogens
- Authors: Nyenje, Mirriam, E
- Date: 2011
- Subjects: Drug resistance in microorganisms , Materia medica, Vegetable , Antibiotics , Microbial sensitivity tests , Gram-negative bacterial infections
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11261 , http://hdl.handle.net/10353/391 , Drug resistance in microorganisms , Materia medica, Vegetable , Antibiotics , Microbial sensitivity tests , Gram-negative bacterial infections
- Description: Antimicrobial resistance is a worldwide problem that has deleterious long-term effects as the development of drug resistance outpaces the development of new drugs. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive outcomes. This study was aimed at isolating and characterizing the major active antimicrobial compounds present in the stem bark of C. molle, in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. Various solvents (hexane, ethyl acetate, dichloromethane, acetone, ethanol and methanol) were used for extraction. The agar well diffusion technique was used to screen for antimicrobial activity of C. molle extracts against Streptococcus pyogenes ATCC 49399, Plesiomonas shigelloides ATCC 51903, Pseudomonas aeruginosa ATCC 15442, Helicobacter pylori ATCC 43526 and Helicobacter pylori 252C (clinical isolate); minimum inhibition concentration (MIC) of the most active extracts was determined by the broth dilution method. Fractionation of acetone extract was done by thin layer chromatography (TLC) and bioautography to determine the compounds present and their antimicrobial activity respectively. The acetone extract was purified by column chromatography and their MIC determined. The most potent fraction (EA4) was subjected to Gas chromatography- Mass spectrometry (GC-MS) and High performance liquid chromatography (HPLC) for identification of the active compounds. Results were analyzed by the Fisher‟s exact test. All the extracts tested demonstrated antimicrobial activity with zone diameters of inhibition ranging from 0–32 mm. Acetone was the most potent extract with its MIC ranging from 0.078–5.0 mg/mL. Seventeen fractions were collected from column chromatography and the most active fraction against all the organisms was EA 4 (eluted with 100 percent ethyl acetate), with its MIC ranging from 0.078 - 2.5mg/mL. There was no statistically significant difference (P>0.05) in the potency of the xii four extracts (acetone, methanol, ethanol and ethyl acetate) and antibiotic (ciprofloxacin) on the different bacterial strains tested, likewise the crude extract and the fractions. No compound was detected by GC-MS whereas numerous peaks were identified by HPLC implying that the active compounds in this plant are non volatile. We could not identify the compounds thereby proposing further studies using Nuclear magnetic resonance to identify the compounds. The study revealed that the acetone extract of C. molle was the most active against all the test organisms and therefore justifies the use of this plant in traditional medicine.
- Full Text:
- Date Issued: 2011
- Authors: Nyenje, Mirriam, E
- Date: 2011
- Subjects: Drug resistance in microorganisms , Materia medica, Vegetable , Antibiotics , Microbial sensitivity tests , Gram-negative bacterial infections
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11261 , http://hdl.handle.net/10353/391 , Drug resistance in microorganisms , Materia medica, Vegetable , Antibiotics , Microbial sensitivity tests , Gram-negative bacterial infections
- Description: Antimicrobial resistance is a worldwide problem that has deleterious long-term effects as the development of drug resistance outpaces the development of new drugs. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive outcomes. This study was aimed at isolating and characterizing the major active antimicrobial compounds present in the stem bark of C. molle, in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. Various solvents (hexane, ethyl acetate, dichloromethane, acetone, ethanol and methanol) were used for extraction. The agar well diffusion technique was used to screen for antimicrobial activity of C. molle extracts against Streptococcus pyogenes ATCC 49399, Plesiomonas shigelloides ATCC 51903, Pseudomonas aeruginosa ATCC 15442, Helicobacter pylori ATCC 43526 and Helicobacter pylori 252C (clinical isolate); minimum inhibition concentration (MIC) of the most active extracts was determined by the broth dilution method. Fractionation of acetone extract was done by thin layer chromatography (TLC) and bioautography to determine the compounds present and their antimicrobial activity respectively. The acetone extract was purified by column chromatography and their MIC determined. The most potent fraction (EA4) was subjected to Gas chromatography- Mass spectrometry (GC-MS) and High performance liquid chromatography (HPLC) for identification of the active compounds. Results were analyzed by the Fisher‟s exact test. All the extracts tested demonstrated antimicrobial activity with zone diameters of inhibition ranging from 0–32 mm. Acetone was the most potent extract with its MIC ranging from 0.078–5.0 mg/mL. Seventeen fractions were collected from column chromatography and the most active fraction against all the organisms was EA 4 (eluted with 100 percent ethyl acetate), with its MIC ranging from 0.078 - 2.5mg/mL. There was no statistically significant difference (P>0.05) in the potency of the xii four extracts (acetone, methanol, ethanol and ethyl acetate) and antibiotic (ciprofloxacin) on the different bacterial strains tested, likewise the crude extract and the fractions. No compound was detected by GC-MS whereas numerous peaks were identified by HPLC implying that the active compounds in this plant are non volatile. We could not identify the compounds thereby proposing further studies using Nuclear magnetic resonance to identify the compounds. The study revealed that the acetone extract of C. molle was the most active against all the test organisms and therefore justifies the use of this plant in traditional medicine.
- Full Text:
- Date Issued: 2011
Antibiotic use in two hospitals in West Wollega, Ethiopia
- Authors: Banja, Wakweya Dugassa
- Date: 2010
- Subjects: Antibiotics , Bacterial diseases -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:10141 , http://hdl.handle.net/10948/1263 , Antibiotics , Bacterial diseases -- Chemotherapy
- Description: In the last decades, there has been an escalating consumption of antibiotics with the number of antibiotic prescriptions increasing worldwide. Overuse or inappropriate use of antibiotics has resulted in a major increase in the development of multi-drug resistant pathogens. Antimicrobial resistance is one of the world’s most serious public health problems with great implication in terms of morbidity, mortality, and costs. To date, there has been no formal antibiotic use study conducted in the West Wollega zone of Ethiopia to assess antibiotic utilization. The objective of this study was to determine the pattern of antibiotic use in two hospitals in the West Wollega zone of Ethiopia, namely Gimbie Adventist Hospital and Nedjo Hospitals, using drug utilization metrics and the costs associated. In addition it was to assess the correlation between diagnosed infectious diseases and antibiotic prescriptions. This study was a cross-sectional, retrospective, descriptive review of antibiotic usage in the two hospitals in the year 2007. Prescriptions dispensed in the first month of each quarter of 2007 were reviewed. The number of prescriptions screened, antibiotic courses started, antibiotic days by specific agent and overall, the cost of individual and all antibiotics, the number and type of infectious diseases diagnosed were collected from which core drug use indicators were calculated. The correlation between infectious disease diagnosed and the antibiotic days prescribed were analyzed. A total of 18568 antibiotic and non-antibiotic prescriptions were reviewed retrospectively in the four months of the study period, 47 percent of which contained at least one antibiotic. The average number of antibiotics per prescription was 1.33 and 1.09 whilst the percentage of injectable antibiotics prescribed was 83.2 percent and 3.76 percent to outpatients and inpatients respectively. Antibiotics prescribed from the Essential Drug List (EDL) and List of Drugs for District Hospital (LDDH) were 63.0 percent, 74.8 percent, and 90.8 percent and 76.1 percent for outpatients and inpatients respectively. 98.6 percent of outpatient and 97.0 percent inpatient prescribed antibiotics were actually dispensed. Penicillins and quinolones were the most prescribed antibiotics in both inpatient and outpatient departments constituting 43.46 percent and 24.08 percent respectively. The antibiotic utilization ratio, incidence of outpatient antibiotic use, incidence of inpatient antibiotic use, the number of Defined Daily Doses (DDD)/1000inhabitants/year and DDD/100 Occupied Bed Days (OBD) for the zone was 0.16, 17.25, 23.56, 158.61, and 70 respectively. Antibiotic cost constituted 33.7 percent of all expenditure on drug, cost of antibiotic per patient care day and cost per antibiotic day was 3.84 Ethiopian Birr (ETB) ($0.40) and 6.29 ETB ($0.66) respectively. The correlation between infectious diseases diagnosed and antibiotic prescription shows significant variation. At outpatient departments alone an average number of antibiotic courses started was 2.7 at Gimbie Adventist Hospital and 7.6 for Nedjo Hospital. When overall antibiotic days prescribed and required was compared in both hospitals, there were 2.4 and 5 times more antibiotic days prescribed than were required for Gimbie and Nedjo Hospitals respectively. This suggests that the overuse of antibiotic is worse in the government hospital (Nedjo Hospital) than in the mission hospital (Gimbie Adventist Hospital). This study suggested that there was overuse of antibiotics in the West Wollega hospitals although further investigation is needed to identify its underlying causes and nature. It is recommended that the health personnel, the hospital management, the zonal and regional Health Bureau, the regulatory bodies and Non-Governmental Organizations (NGOs) work hand-in-hand to promote the rational use of antibiotics in this region so that the consequences and financial cost of antimicrobial resistance can be prevented.
- Full Text:
- Date Issued: 2010
- Authors: Banja, Wakweya Dugassa
- Date: 2010
- Subjects: Antibiotics , Bacterial diseases -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:10141 , http://hdl.handle.net/10948/1263 , Antibiotics , Bacterial diseases -- Chemotherapy
- Description: In the last decades, there has been an escalating consumption of antibiotics with the number of antibiotic prescriptions increasing worldwide. Overuse or inappropriate use of antibiotics has resulted in a major increase in the development of multi-drug resistant pathogens. Antimicrobial resistance is one of the world’s most serious public health problems with great implication in terms of morbidity, mortality, and costs. To date, there has been no formal antibiotic use study conducted in the West Wollega zone of Ethiopia to assess antibiotic utilization. The objective of this study was to determine the pattern of antibiotic use in two hospitals in the West Wollega zone of Ethiopia, namely Gimbie Adventist Hospital and Nedjo Hospitals, using drug utilization metrics and the costs associated. In addition it was to assess the correlation between diagnosed infectious diseases and antibiotic prescriptions. This study was a cross-sectional, retrospective, descriptive review of antibiotic usage in the two hospitals in the year 2007. Prescriptions dispensed in the first month of each quarter of 2007 were reviewed. The number of prescriptions screened, antibiotic courses started, antibiotic days by specific agent and overall, the cost of individual and all antibiotics, the number and type of infectious diseases diagnosed were collected from which core drug use indicators were calculated. The correlation between infectious disease diagnosed and the antibiotic days prescribed were analyzed. A total of 18568 antibiotic and non-antibiotic prescriptions were reviewed retrospectively in the four months of the study period, 47 percent of which contained at least one antibiotic. The average number of antibiotics per prescription was 1.33 and 1.09 whilst the percentage of injectable antibiotics prescribed was 83.2 percent and 3.76 percent to outpatients and inpatients respectively. Antibiotics prescribed from the Essential Drug List (EDL) and List of Drugs for District Hospital (LDDH) were 63.0 percent, 74.8 percent, and 90.8 percent and 76.1 percent for outpatients and inpatients respectively. 98.6 percent of outpatient and 97.0 percent inpatient prescribed antibiotics were actually dispensed. Penicillins and quinolones were the most prescribed antibiotics in both inpatient and outpatient departments constituting 43.46 percent and 24.08 percent respectively. The antibiotic utilization ratio, incidence of outpatient antibiotic use, incidence of inpatient antibiotic use, the number of Defined Daily Doses (DDD)/1000inhabitants/year and DDD/100 Occupied Bed Days (OBD) for the zone was 0.16, 17.25, 23.56, 158.61, and 70 respectively. Antibiotic cost constituted 33.7 percent of all expenditure on drug, cost of antibiotic per patient care day and cost per antibiotic day was 3.84 Ethiopian Birr (ETB) ($0.40) and 6.29 ETB ($0.66) respectively. The correlation between infectious diseases diagnosed and antibiotic prescription shows significant variation. At outpatient departments alone an average number of antibiotic courses started was 2.7 at Gimbie Adventist Hospital and 7.6 for Nedjo Hospital. When overall antibiotic days prescribed and required was compared in both hospitals, there were 2.4 and 5 times more antibiotic days prescribed than were required for Gimbie and Nedjo Hospitals respectively. This suggests that the overuse of antibiotic is worse in the government hospital (Nedjo Hospital) than in the mission hospital (Gimbie Adventist Hospital). This study suggested that there was overuse of antibiotics in the West Wollega hospitals although further investigation is needed to identify its underlying causes and nature. It is recommended that the health personnel, the hospital management, the zonal and regional Health Bureau, the regulatory bodies and Non-Governmental Organizations (NGOs) work hand-in-hand to promote the rational use of antibiotics in this region so that the consequences and financial cost of antimicrobial resistance can be prevented.
- Full Text:
- Date Issued: 2010
Antimicrobial activity of selected Eastern Cape medical plants
- Authors: Mohlakoana, Keneuoe
- Date: 2010
- Subjects: Materia medica, Vegetable -- South Africa , Drug resistance in microorganisms -- South Africa , Anti-infective agents -- South Africa , Antibiotics
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10120 , http://hdl.handle.net/10948/1199 , Materia medica, Vegetable -- South Africa , Drug resistance in microorganisms -- South Africa , Anti-infective agents -- South Africa , Antibiotics
- Description: Bacterial resistance to antibiotics has been a great problem for many years. The degree of resistance and the speed with which resistance develops varies with different organisms and different drugs. Enzymes called β-lactamases are produced by bacteria and are one mechanism in which bacteria develop antimicrobial resistance. Gram-negative bacteria producing enzymes called ESBLs because of their wide substrate range are of a particular concern in nosocomial infections. In many countries people still use traditional medicine derived from plants as an alternative to the Western medicine due to increased cost of Western medicine and microbial resistance of antibiotic treatments. Biologically active compounds isolated from plants species are used in herbal medicine. Because of the high prevalence of the ESBLs and their increasing resistance to the antibiotics, this research study was done to test the antimicrobial activities of selected medicinal plants of the Eastern Cape; G. incanum, D. angustifolia and E. autumnalis which were traditionally used to treat various infections. The in vitro antimicrobial activity of three different extracts (acetone, methanol & distilled water) and the traditional preparations of the three plants were tested against the selected strains of ESBL-producing bacteria, non β-lactamase producers and the different fungal species. The extracts were screened against 26 Gram-positive bacterial strains, 53 Gram-negative bacterial strains and 15 fungal strains. The Gram-positive bacteria included strains from S. aureus, B. cereus and E. faecalis. The Gram-negative bacteria included strains from E. ii coli, E. cloacae, K. pneumoniae, P. aeruginosa and Acinetobacter spp. The fungal strains included 9 strains of Candida albicans and a single strain of each of the following opportunistic fungi, Mucor sp, Geotrichium sp, Penicillium sp, Fusarium sp and Rhizopus sp. The agar dilution assay was used for the antimicrobial screening of the plants extracts and for the determination of the MICs. The Ames test was performed for the determination of probable carcinogenicity of the extracts of G. incanum and D. angustifolia. The distilled water extracts followed by acetone extracts of the plants revealed the highest antimicrobial activity against the different microbial strains. The extracts of G. incanum followed by the extracts of D. angustifolia inhibited the highest number of microbial strains. The extracts of E. autumnalis did not show any antimicrobial activity against all the pathogens in this study. More of the Gram-positive bacteria were inhibited by the plant extracts. The lowest MIC was obtained with Gram-positive bacteria. The bacterial strains of E. faecalis and P. aeruginosa were not inhibited by any of the plants extracts in the agar dilution assay yet Acinetobacter species which are MDR were inhibited by the distilled water and methanol extracts of G. incanum. A single strain of Mucor sp was the only spore forming fungi that was inhibited by the distilled water extracts of G. incanum. None of the plants extracts showed any mutagenic effects on the TA100 S. typhimurium strains incorporated on the Ames test. Apart from revealing of new antimicrobial agents that may be used against resistant organisms, the proper use of antimicrobial agents should be recommended. The study has highlighted a need for further investigations on the properties of the medicinal plants used in this study.
- Full Text:
- Date Issued: 2010
- Authors: Mohlakoana, Keneuoe
- Date: 2010
- Subjects: Materia medica, Vegetable -- South Africa , Drug resistance in microorganisms -- South Africa , Anti-infective agents -- South Africa , Antibiotics
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10120 , http://hdl.handle.net/10948/1199 , Materia medica, Vegetable -- South Africa , Drug resistance in microorganisms -- South Africa , Anti-infective agents -- South Africa , Antibiotics
- Description: Bacterial resistance to antibiotics has been a great problem for many years. The degree of resistance and the speed with which resistance develops varies with different organisms and different drugs. Enzymes called β-lactamases are produced by bacteria and are one mechanism in which bacteria develop antimicrobial resistance. Gram-negative bacteria producing enzymes called ESBLs because of their wide substrate range are of a particular concern in nosocomial infections. In many countries people still use traditional medicine derived from plants as an alternative to the Western medicine due to increased cost of Western medicine and microbial resistance of antibiotic treatments. Biologically active compounds isolated from plants species are used in herbal medicine. Because of the high prevalence of the ESBLs and their increasing resistance to the antibiotics, this research study was done to test the antimicrobial activities of selected medicinal plants of the Eastern Cape; G. incanum, D. angustifolia and E. autumnalis which were traditionally used to treat various infections. The in vitro antimicrobial activity of three different extracts (acetone, methanol & distilled water) and the traditional preparations of the three plants were tested against the selected strains of ESBL-producing bacteria, non β-lactamase producers and the different fungal species. The extracts were screened against 26 Gram-positive bacterial strains, 53 Gram-negative bacterial strains and 15 fungal strains. The Gram-positive bacteria included strains from S. aureus, B. cereus and E. faecalis. The Gram-negative bacteria included strains from E. ii coli, E. cloacae, K. pneumoniae, P. aeruginosa and Acinetobacter spp. The fungal strains included 9 strains of Candida albicans and a single strain of each of the following opportunistic fungi, Mucor sp, Geotrichium sp, Penicillium sp, Fusarium sp and Rhizopus sp. The agar dilution assay was used for the antimicrobial screening of the plants extracts and for the determination of the MICs. The Ames test was performed for the determination of probable carcinogenicity of the extracts of G. incanum and D. angustifolia. The distilled water extracts followed by acetone extracts of the plants revealed the highest antimicrobial activity against the different microbial strains. The extracts of G. incanum followed by the extracts of D. angustifolia inhibited the highest number of microbial strains. The extracts of E. autumnalis did not show any antimicrobial activity against all the pathogens in this study. More of the Gram-positive bacteria were inhibited by the plant extracts. The lowest MIC was obtained with Gram-positive bacteria. The bacterial strains of E. faecalis and P. aeruginosa were not inhibited by any of the plants extracts in the agar dilution assay yet Acinetobacter species which are MDR were inhibited by the distilled water and methanol extracts of G. incanum. A single strain of Mucor sp was the only spore forming fungi that was inhibited by the distilled water extracts of G. incanum. None of the plants extracts showed any mutagenic effects on the TA100 S. typhimurium strains incorporated on the Ames test. Apart from revealing of new antimicrobial agents that may be used against resistant organisms, the proper use of antimicrobial agents should be recommended. The study has highlighted a need for further investigations on the properties of the medicinal plants used in this study.
- Full Text:
- Date Issued: 2010
Assessment of antibiotic production by some marine Streptomyces isolated from the Nahoon Beach
- Ogunmwonyi, Isoken Nekpen Henrietta
- Authors: Ogunmwonyi, Isoken Nekpen Henrietta
- Date: 2010
- Subjects: Streptomyces , Actinobacteria , Gram-positive bacteria , Antibiotics , Antibiotics -- Testing , Drug resistance in microorganisms
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11243 , http://hdl.handle.net/10353/264 , Streptomyces , Actinobacteria , Gram-positive bacteria , Antibiotics , Antibiotics -- Testing , Drug resistance in microorganisms
- Description: Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease.
- Full Text:
- Date Issued: 2010
- Authors: Ogunmwonyi, Isoken Nekpen Henrietta
- Date: 2010
- Subjects: Streptomyces , Actinobacteria , Gram-positive bacteria , Antibiotics , Antibiotics -- Testing , Drug resistance in microorganisms
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11243 , http://hdl.handle.net/10353/264 , Streptomyces , Actinobacteria , Gram-positive bacteria , Antibiotics , Antibiotics -- Testing , Drug resistance in microorganisms
- Description: Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease.
- Full Text:
- Date Issued: 2010
In vitro bioactivity of crude extracts of Lippia javanica on clinical isolates of Helicobacter pylori: preliminary phytochemical screening
- Authors: Nkomo, Lindelwa Precious
- Date: 2010
- Subjects: Extracts , Helicobacter pylori , Antibiotics , Drug resistance in microorganisms , Materia medica, Vegetable
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11257 , http://hdl.handle.net/10353/508 , Extracts , Helicobacter pylori , Antibiotics , Drug resistance in microorganisms , Materia medica, Vegetable
- Description: Helicobacter pylori classified as a class 1 carcinogen is a common human pathogen implicated in certain gastrointestinal diseases. Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries. H. pylori infection causes peptic ulcer, duodenitis, gastritis and cancer. The growing resistance of H. pylori to antibiotics used in its treatment as well as other innate limitations of the triple therapy has necessitated a search for alternative treatment from natural sources which could be readily available, less cost effective. The antimicrobial activity of solvents (acetone, ethanol, methanol, chloroform and water) crude extracts of Lippia javanica were investigated against 31 H. pylori strains by the agar well diffusion technique. The minimum inhibitory concentration (MIC) was determined by spectrophotometric analysis at 620 nm using the broth micro dilution method and the rate of kill by broth dilution method. Phytochemical analysis was also performed. H. pylori standard strain NCTC 11638 was included as a positive control. Metronidazole and amoxicillin were used as positive control antibiotics. The ANOVA test was used to analyze the results using SPSS version 17.0. The strains were inhibited by all the extracts with inhibition zones of diameter ranging from 0-36 mm and 0-35 mm for the control antibiotic, clarithromycin. The MIC90 ranged from 0.039- 0.625 mg/mL for acetone; 0.039-1.25mg/mL for methanol, 0.00195-0.313 mg/mL for ethanol; 0.01975-2.5 mg/mL for metronidazole and 0.0048-2.5 mg/mL for amoxicillin. Acetone extract completely inhibited strain PE369C at MIC (0.1 mg/mL) and 2× MIC (0.2 mg/mL) in 18h and at ½× MIC (0.05 mg/mL) in 36h. Strain PE466C was completely inhibited at 4× MIC in 72h. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids and alkaloids. The results indicate that the extracts of the leaves of L. javanica may contain compounds with anti-H. pylori activity and merits further study to identify the compounds.
- Full Text:
- Date Issued: 2010
- Authors: Nkomo, Lindelwa Precious
- Date: 2010
- Subjects: Extracts , Helicobacter pylori , Antibiotics , Drug resistance in microorganisms , Materia medica, Vegetable
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11257 , http://hdl.handle.net/10353/508 , Extracts , Helicobacter pylori , Antibiotics , Drug resistance in microorganisms , Materia medica, Vegetable
- Description: Helicobacter pylori classified as a class 1 carcinogen is a common human pathogen implicated in certain gastrointestinal diseases. Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries. H. pylori infection causes peptic ulcer, duodenitis, gastritis and cancer. The growing resistance of H. pylori to antibiotics used in its treatment as well as other innate limitations of the triple therapy has necessitated a search for alternative treatment from natural sources which could be readily available, less cost effective. The antimicrobial activity of solvents (acetone, ethanol, methanol, chloroform and water) crude extracts of Lippia javanica were investigated against 31 H. pylori strains by the agar well diffusion technique. The minimum inhibitory concentration (MIC) was determined by spectrophotometric analysis at 620 nm using the broth micro dilution method and the rate of kill by broth dilution method. Phytochemical analysis was also performed. H. pylori standard strain NCTC 11638 was included as a positive control. Metronidazole and amoxicillin were used as positive control antibiotics. The ANOVA test was used to analyze the results using SPSS version 17.0. The strains were inhibited by all the extracts with inhibition zones of diameter ranging from 0-36 mm and 0-35 mm for the control antibiotic, clarithromycin. The MIC90 ranged from 0.039- 0.625 mg/mL for acetone; 0.039-1.25mg/mL for methanol, 0.00195-0.313 mg/mL for ethanol; 0.01975-2.5 mg/mL for metronidazole and 0.0048-2.5 mg/mL for amoxicillin. Acetone extract completely inhibited strain PE369C at MIC (0.1 mg/mL) and 2× MIC (0.2 mg/mL) in 18h and at ½× MIC (0.05 mg/mL) in 36h. Strain PE466C was completely inhibited at 4× MIC in 72h. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids and alkaloids. The results indicate that the extracts of the leaves of L. javanica may contain compounds with anti-H. pylori activity and merits further study to identify the compounds.
- Full Text:
- Date Issued: 2010
The investigation of novel marine microorganisms for the production of biologically active metabolites
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
Assessment of antibacterial potentials of Garcinia Kola seed extracts and their interactions with antibiotics
- Authors: Sibanda, Thulani
- Date: 2007
- Subjects: Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11242 , http://hdl.handle.net/10353/71 , Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants
- Description: The antibacterial potency of the extracts of the seed of Garcinia kola (bitter kola) was investigated in this study against a panel of referenced, environmental and clinical bacterial strains. The killing rates of the active extract as well as their potential for combination antibacterial therapy with standard antibiotics were also elucidated using standard procedures. The aqueous and acetone extracts of the seed were screened for activity against 27 bacterial isolates. The aqueous extract exhibited activity mainly against Gram positive organisms with Minimum inhibitory concentration (MIC) values ranging from 5 mgml-1 – 20 mgml-1, while the acetone extract showed activity against both Gram negative and Gram positive organisms with MIC values ranging from 10 mgml-1 - 0.156 mgml-1. The acetone extract also showed rapid bactericidal activity against Staphylococcus aureus ATCC 6538 with a 3.097 Log10 reduction in counts within 4 hours at 0.3125 mgml-1 and a 1.582 Log10 reduction against Proteus vulgaris CSIR 0030 at 5 mgml-1 after 1 hour. In addition, the aqueous, methanol and acetone extracts of the seeds also exhibited activity against four clinical strains of Staphylococcus isolated from wound sepsis specimens. The MIC values for the aqueous extract were 10 mgml-1 for all the isolates while the acetone and methanol extracts had lower values ranging from 0.3125 - 0.625 mgml-1. The acetone extract was strongly bactericidal against Staphylococcus aureus OKOH3 resulting in a 2.70 Log10 reduction in counts at 1.25 mgml-1 within 4 hours of exposure and a complete elimination of the organism after 8 hours. The bactericidal vi activity of the same extract against Staphylococcus aureus OKOH1 was weak, achieving only a 2.92 Log10 reduction in counts at 1.25 mgml-1 (4× MIC) in 24 hours. In the test for interactions between the acetone extract of the seeds and antibiotics, synergistic interactions were observed largely against Gram positive organisms using the FIC indices, (indices of 0.52 - 0.875) with combinations against Gram negatives yielding largely antagonistic interactions (indices of 2.0 to 5.0). Synergy (≥ 1000 times or ≥ 3 Log10 potentiation of the bactericidal activity) against both Gram negative and Gram positive organisms was detected by time kill assays mainly involving the antibiotics tetracycline, chloramphenicol, amoxycillin and penicillin G. Combinations involving erythromycin and ciprofloxacin consistently gave antagonistic or indifferent interactions. We conclude that the acetone extract of Garcinia kola seeds possess strong bactericidal activities against both Gram positive and Gram negative organisms and can be therapeutically useful in the treatment of bacterial infections including the problematic staphylococcal wound infections. In addition, the acetone extract can be a potential source of broad spectrum resistance modifying compounds that can potentially improve the performance of antibiotics in the treatment of drug resistant infections.
- Full Text:
- Date Issued: 2007
- Authors: Sibanda, Thulani
- Date: 2007
- Subjects: Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: vital:11242 , http://hdl.handle.net/10353/71 , Drug resistance in microorganisms , Garcinia , Antibiotics , Medicinal plants
- Description: The antibacterial potency of the extracts of the seed of Garcinia kola (bitter kola) was investigated in this study against a panel of referenced, environmental and clinical bacterial strains. The killing rates of the active extract as well as their potential for combination antibacterial therapy with standard antibiotics were also elucidated using standard procedures. The aqueous and acetone extracts of the seed were screened for activity against 27 bacterial isolates. The aqueous extract exhibited activity mainly against Gram positive organisms with Minimum inhibitory concentration (MIC) values ranging from 5 mgml-1 – 20 mgml-1, while the acetone extract showed activity against both Gram negative and Gram positive organisms with MIC values ranging from 10 mgml-1 - 0.156 mgml-1. The acetone extract also showed rapid bactericidal activity against Staphylococcus aureus ATCC 6538 with a 3.097 Log10 reduction in counts within 4 hours at 0.3125 mgml-1 and a 1.582 Log10 reduction against Proteus vulgaris CSIR 0030 at 5 mgml-1 after 1 hour. In addition, the aqueous, methanol and acetone extracts of the seeds also exhibited activity against four clinical strains of Staphylococcus isolated from wound sepsis specimens. The MIC values for the aqueous extract were 10 mgml-1 for all the isolates while the acetone and methanol extracts had lower values ranging from 0.3125 - 0.625 mgml-1. The acetone extract was strongly bactericidal against Staphylococcus aureus OKOH3 resulting in a 2.70 Log10 reduction in counts at 1.25 mgml-1 within 4 hours of exposure and a complete elimination of the organism after 8 hours. The bactericidal vi activity of the same extract against Staphylococcus aureus OKOH1 was weak, achieving only a 2.92 Log10 reduction in counts at 1.25 mgml-1 (4× MIC) in 24 hours. In the test for interactions between the acetone extract of the seeds and antibiotics, synergistic interactions were observed largely against Gram positive organisms using the FIC indices, (indices of 0.52 - 0.875) with combinations against Gram negatives yielding largely antagonistic interactions (indices of 2.0 to 5.0). Synergy (≥ 1000 times or ≥ 3 Log10 potentiation of the bactericidal activity) against both Gram negative and Gram positive organisms was detected by time kill assays mainly involving the antibiotics tetracycline, chloramphenicol, amoxycillin and penicillin G. Combinations involving erythromycin and ciprofloxacin consistently gave antagonistic or indifferent interactions. We conclude that the acetone extract of Garcinia kola seeds possess strong bactericidal activities against both Gram positive and Gram negative organisms and can be therapeutically useful in the treatment of bacterial infections including the problematic staphylococcal wound infections. In addition, the acetone extract can be a potential source of broad spectrum resistance modifying compounds that can potentially improve the performance of antibiotics in the treatment of drug resistant infections.
- Full Text:
- Date Issued: 2007
Antimicrobial resistance patterns in a Port Elizabeth hospital
- Authors: Meiring, Jillian A
- Date: 1993
- Subjects: Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4043 , http://hdl.handle.net/10962/d1004104 , Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Description: Antibiotic resistance in clinical bacterial isolates remains an ongoing problem requiring continuous monitoring to effect some form of control. Comparative studies have not been previously reported for the Eastern Cape Region, South Africa and this study was undertaken to monitor resistance patterns in clinical isolates from Provincial Hospital, Port Elizabeth. Over the three year period 1989 to 1991, 9888 susceptibility results from isolates examined in the SAIMR pathology laboratory were analysed and collated using a stand-alone computer program. Resistance patterns for a range of nineteen antibiotics were collated for isolates from various sampling points within the hospital. Results were reported as resistance patterns in individually isolated species. Levels of resistance in each species were compared to those reported from South Africa and abroad, and changing patterns of resistance were noted within the three year period at the Provincial Hospital, Port Elizabeth.
- Full Text:
- Date Issued: 1993
- Authors: Meiring, Jillian A
- Date: 1993
- Subjects: Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4043 , http://hdl.handle.net/10962/d1004104 , Antibiotics , Drug resistance in microorganisms , Hospitals -- Drug distribution systems -- South Africa -- Port Elizabeth
- Description: Antibiotic resistance in clinical bacterial isolates remains an ongoing problem requiring continuous monitoring to effect some form of control. Comparative studies have not been previously reported for the Eastern Cape Region, South Africa and this study was undertaken to monitor resistance patterns in clinical isolates from Provincial Hospital, Port Elizabeth. Over the three year period 1989 to 1991, 9888 susceptibility results from isolates examined in the SAIMR pathology laboratory were analysed and collated using a stand-alone computer program. Resistance patterns for a range of nineteen antibiotics were collated for isolates from various sampling points within the hospital. Results were reported as resistance patterns in individually isolated species. Levels of resistance in each species were compared to those reported from South Africa and abroad, and changing patterns of resistance were noted within the three year period at the Provincial Hospital, Port Elizabeth.
- Full Text:
- Date Issued: 1993
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