Antibacterial and phytochemical studies of selected South African honeys on clinical isolates of Helicobacter pylori
- Authors: Manyi-Loh, Christy E
- Date: 2012
- Subjects: Helicobacter pylori , Honey--South Africa , Drug resistance in microorganisms , Bacterial diseases , Honey -- Therapeutic use , Helicobacter pylori infections , Traditional medicine -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD (Microbiology)
- Identifier: vital:11240 , http://hdl.handle.net/10353/d1001056 , Helicobacter pylori , Honey--South Africa , Drug resistance in microorganisms , Bacterial diseases , Honey -- Therapeutic use , Helicobacter pylori infections , Traditional medicine -- South Africa -- Eastern Cape
- Description: Infection with Helicobacter pylori has been associated with the pathogenesis of numerous stomach and gastroduodenal diseases that pose threats to public health. Eradicaftion of this pathogen is a global challenge due to its alarming rate of multidrug resistance. Consequently, to find an alternative treatment, the search is increasingly focused on new antimicrobial product from natural sources including honey. Honey has been used as medicine in several cultures since ancient time due to its enormous biomedical activities. Its beneficial qualities have been endorsed to its antimicrobial, antioxidant, anti-inflammatory properties added to its phytocomponents. In this study, the anti-H. pylori activity of South African honeys and their solvent extracts as well as the phytochemicals present in the two most active honeys were evaluated. Agar well diffusion test was used to investigate the antimicrobial activity of six honey varieties obtained from different locations in the country. Subsequently, the honeys were extracted with four organic solvents viz n-hexane, diethyl ether, chloroform and ethyl acetate employed in order of increasing polarity. The antibacterial activity of the different solvent extracts of each honey was evaluated by agar well diffusion; broth micro dilution and time kill assays. Different chromatographic techniques (Thin layer & column chromatography) were employed to enumerate the phytochemical constituents in the most active solvent extracts of Pure Honey (PH) and Champagne Royal Train (CRT); and were identified by gas-chromatography linked mass-spectrometry. Linalool pure compound was equally evaluated for anti-H. pylori activity in a bid to trace the antibacterial agent among the variety of compounds identified. Data were analyzed by One-way ANOVA test at 95% confidence interval. Crude honeys and their solvent extracts demonstrated potent anti-H. pylori activity with zone diameter that ranged from [16.0mm (crude) to 22.2mm (extract)] and percentage susceptibilities of test isolates between 73.3% (crude) and 93.3% (extract). The chloroform extracts of PH and CRT were most active with MIC50 in the ranges 0.01- viii 10%v/v and 0.625-10%v/v respectively, not significantly different from amoxicillin (P> 0.05); and efficient bactericidal activity (100% bacterial cells killed) at 1/2MIC and 4xMIC over different time intervals, 36-72hrs and 18-72hrs respectively. The appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts of PH and CRT; and developed with hexane: ethyl acetate: acetic acid (HEA) and methanol: acetic acid: water (MAAW) solvent systems indicated the presence of compounds. Purification of the compounds contained in these extracts over silica gel column yielded numerous fractions which were evaluated for antibacterial activity and purity. PHF5 was the most active fraction with a mean MIC50 value of 1.25mg/mL. Volatile compounds belonging to different known chemical families in honey were identified in all the active fractions obtained from PH. Conversely, only four compounds were identified in the active fractions obtained from CRT hence the non volatile constituents could be of prime relevance with respect to antibacterial activity of this honey. Of novelty was the presence of thiophene and N-methyl-D3-azirdine compounds, essential precursors used for the synthesis of natural products and pharmaceuticals with vital biomedical properties. Linalool demonstrated potent inhibitory (MIC95, 0.002- 0.0313mg/mL) and bactericidal activity (0.0039-0.313mg/mL) against the test isolates. On the other hand, a significant difference was recorded (P < 0.05) in comparing the activity of linalool compound to the fractions. PH could serve as a good economic source of bioactive compounds which could be employed as template for the synthesis of novel anti-H. pylori drugs. However, further studies are needed to determine the non volatile active ingredients in PH and CRT as well as toxicological testing
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- Date Issued: 2012
Commensal bacteria belonging to the Staphylococcus Acinetobacter and Stenotrophomonas genera as reservoirs of antibiotic resistance determinants in the environment of Nkonkobe Municipality, Eastern Cape Province , South Africa
- Authors: Adegoke, Anthony Ayodeji
- Date: 2012
- Subjects: Acinetobacter infections , Drug resistance in microorganisms , Staphylococcal infections , Bacterial diseases
- Language: English
- Type: Thesis , Doctoral , Degree
- Identifier: http://hdl.handle.net/10353/6539 , vital:30551
- Description: A study to assess the potentials of some commensal bacteria that belong to Staphylococcus, Acinetobacter and Stenotrophomonas genera as reservoirs of antibiotic resistance determinants in the environment of Nkonkobe Municipality of the Eastern Cape Province, South Africa, was carried out using standard microbiological and molecular techniques. A total of 120 Staphylococcus isolates which consisted of Staphylococcus haemolyticus (30%), Staphylococcus aureus (23.3%) from pig; Staphylococcus capitis (15%) from goat; Staphylococcus heamolyticus (5%) and Staphylococcus xylosus (15%) from cattle and other Staphylococci (11%) from dead chicken and pigs were isolated. About 23.3% of these isolates were coagulase positive and 76.7% were coagulase negative. This difference in prevalence along coagulase production divide was statistically significant (p < 0.05). Eighty-six Acinetobacter species (Acinetobacter baumannii/calcoaceticus and Acinetobacter haemolyticus) were also isolated from Alice and Fort Beaufort towns samples, while 125 Stenotrophomonas maltophilia isolates were from grass root rhizosphere (96%) and soil butternut root rhizosphere (4%). Between 75-100% of the Staphylococccus species were resistant to Penicillin G, tetracycline, sulphamethaxole and nalidixic acid; about 38 % were methicillin resistant, consisting of 12.6% methicillin resistant Staphylococcus aureus (MRSA) from pig and a total of 12% vancomycin resistant were observed. Also, 12% of the isolates were erythromycin resistant while 40.2 % were resistant to the third generation cephalosporin, ceftazidime. The antibiotic resistance genes vanA, VanB, eryA, eryB, eryC were not detected in all the phenotypically resistant Staphylococccus species, but mec A gene and mph genes were detected. In the Acinetobacter species, a wide range of 30-100% resistance to penicillin G, ceftriazone, nitrofurantoin, erythromycin, and augmentin was observed. Polymerase chain reaction (PCR) revealed the presence of Tet(B) and Tet(39) genes in these species, while Tet (A), Tet(M) and Tet(H) were absent. Also, 9.3% of the Acinetobacter species showed phenotypic production of extended spectrum beta lactamases (ESBLs) while 3.5% were positive for the presence of blaCTX-M-1 genes. The Stenotrophomonas maltophilia isolates showed varying resistance to meropenem (8.9%), cefuroxime (95.6 %), ampicillin-sulbactam (53.9%), ceftazidime (10.7%), cefepime (29.3 %), minocycline (2.2%), kanamycin (56.9%), ofloxacin (2.9%), levofloxacin (1.3%), moxifloxacin (2.8%), ciprofloxacin (24.3%), gatifloxacin (1.3%), polymyxin B (2.9 %), cotrimoxazole (26.1%), trimethoprim (98.6%), aztreonam(58%) and Polymyxin B (2.9 %). The isolates exhibited significant susceptibility to the fluoroquinolones (74.3-94.7 %), polymycin (97.1%) and meropenem (88.1%). Only sul3 genes were the only sulphonamide resistance gene detected among the trimethoprim-sulphamethoxazole resistant isolates. The observed multiple antibiotic resistance indeces (MARI) of >2 for Staphylococcus species, Acinetobacter species and Stenotrophomonas maltophilia suggest that they have arisen from high-risk sources where antibiotics are in constant arbitrary use resulting in high selective pressure. The presence of tetracycline resistance genes in Acinetobacter species justifies the observed phenotypic resistance to oxytetracycline and intermediate resistance to minocycline. High phenotypic resistance and the presence of some resistance genes in Staphylococcus species is a possible threat to public health and suggests animals to be important reservoirs of antibiotic resistance determinants in the environment. Indiscriminate use of antibiotics induces this kind of antibiotic resistance and should be discouraged. Personal hygiene is encouraged as it reduces the load of Acinetobacter species contacted from the environment that may be difficult to control. Commensal Stenotrophomonas maltophilia are as important as their clinical counterparts due to their roles in opportunistic infection, antibiotic resistance and their associated genes, especially sul gene. Personal hygiene is hereby advocated especially when in contact with soil, plants and plants’ rhizospheric soil.
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- Date Issued: 2012
Phytochemical analysis and bioactivity of selected South African medicinal plants on clinical isolates of Helicobacter pylori
- Authors: Njume, Collise
- Date: 2011
- Subjects: Helicobacter pylori , Medicinal plants -- Biotechnology , Traditional medicine -- South Africa -- Eastern Cape , Antibiotics , Drug resistance in microorganisms , Extracts , Helicobacter pylori infections
- Language: English
- Type: Thesis , Doctoral , PhD (Microbiology)
- Identifier: vital:11260 , http://hdl.handle.net/10353/449 , Helicobacter pylori , Medicinal plants -- Biotechnology , Traditional medicine -- South Africa -- Eastern Cape , Antibiotics , Drug resistance in microorganisms , Extracts , Helicobacter pylori infections
- Description: Medicinal plants have been used as traditional medicine in the treatment of numerous human diseases for thousands of years in many parts of the world. In the developing world, especially in rural areas, herbal remedies continue to be a primary source of medicine. Scientifically, medicinal plants have proven to be an abundant source of biologically active compounds, many of which have already been formulated into useful therapeutic substances or have provided a basis for the development of new lead molecules for pharmaceuticals. Antibiotic resistance, undesireable side effects and expences associated with the use of combination therapy in the treatment of Helicobacter pylori infections have generated a considerable interest in the study of medicinal plants as potential sources of new drugs against this organism. The high complexicity of bioactive compounds accumulated in plants coupled with their broad antimicrobial activity may make it difficult for pathogenic organisms, including H. pylori to acquire resistance during treatment. This study therefore evaluates the antimicrobial potential of selected South African medicinal plants employed in the treatment of H. pylori-related infections, and the subsequent isolation of the plant active principles. An ethnobotanical survey of plants used in the treatment of H. pylori-related infections was conducted in the study area. Crude extracts of Combretum molle, Sclerocarya birrea, Garcinia kola, Alepidea amatymbica and 2 Strychnos species were screened against 30 clinical strains of H. pylori and 2 standard control strains (NCTC 11638 and ATCC 43526). In the preliminary stages of this study, ethyl acetate, acetone, ethanol, methanol and water extracts of the plants were tested against H. pylori by agar well diffusion and micro broth dilution methods. The plant crude extracts that exhibited anti-H. pylori activity with a iv percentage susceptibility of 50 percent and above were considered for the rate of kill assays and the most active crude extracts selected for bio-assay guided isolation of the active ingredient. Preliminary fractionation of the crude extract was achieved by thin layer chromatography (TLC) using different solvent combinations; hexane/diethylether (HDE), ethyl acetate/methanol/water (EMW) and chloroform/ethyl acetate/formic acid (CEF) in order to determine the most suitable combination for column chromatography (CC) and subsequent testing by indirect bioautography. The extract was then fractionated in a silica gel column using previously determined solvent combinations as eluent. Active fractions obtained from column chromatography separations were further fractionated and the compounds identified by gas chromatography/mass spectrometry (GC/MS) analysis. All the plants exhibited antimicrobial activity against H. pylori with zone of inhibition diameters ranging from 0 - 38 mm and minimum inhibitory concentration (MIC) values ranging from 0.06 - 5.0 mg/mL. The most active plant extracts were the acetone extract of C. molle with a percentage susceptibility of 87.1 percent, acetone and aqueous extracts of S. birrea (71 percent each) and the ethanolic extracts of G. kola (53.3 percent). Except for the aqueous extract, these extracts also exhibited a strong bactericidal activity against H. pylori at different concentrations. TLC analysis revealed the presence of 9 components in the acetone extract of S. birrea with the EMW solvent system as opposed to 5 and 8 with HDE and CEF respectively. Bioassay-guided isolation led to the identification of 52 compounds from the acetone extract of S. birrea with n-octacosane being the most abundant (41.68 percent). This was followed by pyrrolidine (38.91 percent), terpinen-4-ol (38.3 percent), n-eicosane (24.98 percent), cyclopentane (16.76 percent), n-triacontane (16.28 percent), aromadendrene (13.63 percent) and α-gujunene (8.77 percent). Terpinen-4-ol and pyrrolidine demonstrated strong antimicrobial activity against H. pylori at all concentrations tested. These results may serve as preliminary scientific validation of the ethnomedicinal uses of the above mentioned plants in the treatment of H. pylori-related infections in South Africa. Terpinen-4-ol and pyrrolidine could be considered for further evaluation as therapeutic or prophylactic agents in the treatment of H. pylori-related infections. However, further investigations would be necessary to determine their toxicological properties, in-vivo potencies and mechanism of action against H.pylori
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- Date Issued: 2011