- Title
- Molecular characterization, antibiograms and activity of medicinal plants against streptococcus pneumoniae and haemophilus influenzae isolates from clinical samples of patients in the Eastern Cape province, South Africa
- Creator
- Morobe, Isaac Christopher
- Subject
- Medicinal plants
- Date Issued
- 2015-00
- Date
- 2015-00
- Type
- Master's/Doctoral theses
- Type
- text
- Identifier
- http://hdl.handle.net/11260/6469
- Identifier
- vital:46340
- Description
- H. influenzae and S. pneumoniae are important causes of community acquired respiratory tract infections including, pneumonia, acute sinusitis, otitis media, meningitis, bacteremia, sepsis, osteomyelitis, pericarditis, septic arthritis, endocarditis, peritonitis, cellulitis and brain abscesses. The ability to effectively treat bacterial infections has been compromised in recent years due to the acquisition of antibiotic resistance, particularly to β-lactam drugs. The increasing trends in antibiotic resistance have prompted incessant searches aimed at unraveling new effective sources for the management of microbial infections. Plant derived antimicrobial compounds that have no or minimal toxicity to host cells are considered candidates for developing new antimicrobial drugs. Safety is therefore critical in the development and formulation of such antimicrobials. In addition to studies on the structural elucidation of active compounds of selected medicinal plants and determination of their toxicity levels, concerted investigations on the molecular landscape of the designated bacteria, including genes coding for resistance and virulence, the phylogenetic profiles of isolates from different sources and the abilities of isolates to withstand the normal bactericidal activities of human serum samples from different blood groups are critical for a thorough understanding of the management, pathogenetic and clinico- epidemiological trajectories of the pathogens. Therefore, the aims of the various studies were to characterize local H. influenzae and S. pneumoniae isolates by serological and molecular methods; ascertain the antibiotic susceptibility profiles of isolates in order to provide updated data and guide clinicians and other health care workers on the empiric management of patients; determine genes coding for virulence and phylogenetic relatedness of isolates of H. influenzae and S. pneumoniae from diverse sources; ascertain the bactericidal activity of human serum samples from different blood groups against H. influenzae and S. pneumoniae and also to determine the activity of active compounds and toxicity levels of selected medicinal plants. In order to achieve these goals, relevant samples were collected and screened using an array of microbiological, serological, molecular and phytochemical methods, which would be espoused in the relevant chapters, presented hereunder. Key findings of the various chapters including their contributions to knowledge are highlighted. The studies are presented in eight chapters and each chapter, with the exception of chapter one (General Introduction and Literature Review) consists of an introduction, materials and methods, results, discussions, conclusions and references. Each chapter is therefore designed as a publishable unit. Chapter 1 gives an account of the background to the study and the literature review. The morphology, cultural characteristics, laboratory diagnosis, pathogenesis, antibiogram and clinical manifestations of H. influenzae and S. pneumoniae were reviewed. Furthermore, the activities of medicinal plants and their various applications in the management of infections in different countries, including their possible active compounds and toxicity levels were also explored in order to provide a suitable background for the study. Similar reviews were undertaken for molecular aspects of both pathogens as well as the activities of human serum samples against microbial infections. In Chapter 2 the prevalence and antibiotic resistance profiles of H. influenzae and, S. pneumoniae isolates from clinical samples of patients in Mthatha, Eastern Cape Province were investigated. Clinical samples were obtained randomly from individuals attending different hospitals in Mthatha district. Samples were analysed using the Kirby Bauer disk diffusion test (antibiotic susceptibility testing) and MIC breakpoints were determined using E-test strips. From a total of 475 clinical samples tested, 323 (68.0%) were positive for both H. influenzae and S. pneumoniae. Most of the positive isolates were obtained from children under 9 years. Out of 323 isolates, 187 (57.89%) were positive for H. influenzae and 136 (42.1%) were positive for S. pneumoniae. From 10 hospitals selected for sampling in this study, Mthatha General Hospital recorded the highest number of isolates, 42 (25.15%) and 31 (22.79%) of H. influenzae and S. pneumoniae positive isolates respectively, followed by Nelson Mandela Academic Hospital 33 (19.76%) and 26 (19.12%) respectively while ST. Patricks 8 (4.79%) recorded the least number of isolates for H. influenzae and Khotsong 4 (2.94%) recorded the least number of isolates for S. pneumoniae. Antibiotic susceptibility tests revealed Amoxicillin (MIC50, 0.125μg/ml) and Vancomycin (MIC50,0.12μg/ml) as the most effective antibiotics against S. pneumoniae isolates and Co-amoxiclav (MIC50,0.3µg/ml) and Cefuroxime (MIC50,0.15µg/ml) against H. influenzae isolates. These data highlight the need for education and to consider predominant resistance when choosing empiric therapies to treat bacterial infections. Chapter 3 was designed to investigate the virulence factors of H. influenzae and S. pneumoniae isolates from clinical specimens of patients with respiratory tract infections in Mthatha district, the Eastern Cape Province. PCR and sequencing methods were used to verify the genetic determinants responsible for virulence in H. influenzae and S. pneumoniae strains. Results indicated that, of the 187 H. influenzae isolates studied, 26 (13.9%) were typeable, positive by genotypic determination, while 161 (86.1%) were non typeable (NTHi) strains. On the other, out of the 136 S. pneumoniae isolates 24 (17.6%) were typeable while 112 (82.4%) were non typeable strains. All isolates tested contained the metS2 gene for both H. influenzae and S. pneumoniae. The phylogenetic clusters identified by maximum-parsimony analysis were also compared to the results of 16S rRNA sequences. Twenty five percent of none typeable strains were typed by 16S rRNA sequencing. The phylogenetic tree yielded 7.7% H. influenzae similarities while S. pneumoniae yielded 25% similarities with other typeable strains. The presence of genes coding for virulence in this study suggest a significant contribution of genes encoding for virulence to antimicrobial resistance among respiratory tract organisms studied. This study underlines the importance of understanding the virulence composition and diversity of pathogens for enhanced clinico-epidemiological monitoring and health care delivery. The findings will also provide a genetic foundation for future research into mechanisms of pathogenesis of H. influenzae and S. pneumoniae and may accelerate the development of safe and effective vaccines to prevent and control diseases caused by H. influenzae and S. pneumoniae. In Chapter 4, cytotoxic effects and safety profiles of extracts of active medicinal plants from the OR Tambo District Municipality in the Eastern Cape of South Africa were carried out. The most prominent families of medicinal plants (Solanacea and Euphorbiaceae) were used. Extracts of nine South African medicinal plants were screened for cytotoxic activities against MAGI CC5+ cells using MTT assay. Results indicated that nine plant extracts (methanolic and aqueous) used in the MTT assay revealed Herb 2 (Cyanthula inculata) as the most potent extract identified with activity of 1.4 Cc50 values of 25.6 mg/mL and induced over 50% of cell deaths, followed by herb 3 (Croton grattismus) and Herb 4 (Cassine trasvaalensis) with activity of 0.2 Cc50 values of 3.7 mg/mL each. The herbs that induced the least cell death, were herbs 5 (Capris tomentosa) and 7 (Hypoxis hemerocallidea), with the activity of 0.05 Cc50 values of 0.9 mg/mL each. Of the nine plant extracts 2(22%) exhibited minimal toxicity on MAGI cells and 7(77.8%) exhibited 50% toxicity. Two (22%) of the methanolic extracts exhibited anti-HIV1 IIIB activities and against Mycobacterium tuberculosis (TB) only one medicinal plant extract (Lysium inerme) exhibited 29% activity. Cytotoxicity tests will provide comprehensive reference data bases for the profiling and eventual considerations of medicinal plants as potential templates for drug designs and medical applications. In chapter 5 Chemical Components of the volatile and non-volatile extractives of Croton species and their microbial activities were screened. Isolation of the essential oils from the leaves of Croton pseudopulchellus and C. gratissimus from the Eastern Cape and KwaZulu-Natal Provinces in South Africa were performed using an all glass Clevenger apparatus according to the British Pharmacopeia method. The minimum inhibitory concentrations of the oils were assessed against the seven different standard strains of bacteria: H. influenzae, Bacillus pimitus, Staphylococcus aureus, S. pneumoniae, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Entarobacter cloacae using micro dilution technique on a 96 well microtitre plate. Results showed golden to very light yellow oils obtained with percentage yield of 1.03 -1.25 respectively (w/w). Analysis of the oils was performed using Gas Chromatography and Gas Chromatography/Mass Spectrophotometry. The leaf oil of Croton pseudopulchellus had germacrene (24.2%),β-phellandrene (17.4%), myrcene (13.4%) and β-caryophyllene (11.4%) as the prominent compounds identified in the oil. The chemical composition of the leaf oil of C. gratissimus was characterized by sabinene (14.6%), β-phellandrene (12.3%), α-phellandrene (10.7%), α-pinene (6.0%) and germacrene D (5.9%). Chemical profiles of the essential oils of Croton species reported in literatures are specific to their geographical location. The oils from C. pseudopulchellus and C. grattisimus were found to have significant antibacterial activities and therefore could be used as natural antimicrobial agents for the treatment of several infectious diseases caused by pathogenic and antibiotic resistant organisms. Chapters 6 and 7 were designed to isolate two bioactive compounds from the stem bark of Lycium inerme and the leaves of Croton grattisimus and were screened for their biological activities against H. influenzae and S. pneumoniae. A qualitative phytochemical screening and bioassay of the plants extracts was carried out. Antimicrobial screening was by broth microdilution and bioautography. Bioassay results showed that compounds with Rf –values between 0.67 to 0.80 were very active against H. influenzae and S. pneumoniae. However, the most active of these compounds was observed at 0.70 for H. influenzae and 0.69 for S. pneumoniae from the dichloromethane extract. Column chromatography, Preparative Thin Layer Chromatography (PTLC) and Sephadex LH20 were used for isolation, sample clean-up and purification of this extract. Two active compounds: a coumarin (7-hydroxy-6-methoxy-coumarin) and two triterpenoids, Ursolic acid (3-oxo-19-hydroxyl-6-methoxylpomolic acid) and Moronic acid (3-oxoolean-12-en-28-oic acid) were isolated from the dichloromethane fraction. The presence of Coumarins and Terpenoids in this herb was observed from the TLC fingerprints. NMR spectroscopic methods were used for the structural elucidation of the active compounds while the GC-MS was used to determine the presence of essential oils from volatile samples obtained from the leaves of C. grattisimus and L. inerme. C. grattisimus extracts possess strong free radicals with scavenging, antimicrobial and antifungal activities; therefore, further studies are needed to determine their efficacies in vivo or clinical usefulness. L. inerme stem bark can therefore be used as a source of alternative medicine or new pharmaceutical and health care product or as a starting material for synthesis of drugs. In chapter 8, general conclusions and perspectives of various parts of the findings were captured. The multi-drug resistance was observed among the emerging respiratory tract bacterial pathogens. It was recommended that measures should be put in place to control the spread of drug resistance in pathogens through improved and standardized laboratory practices, proper and regular surveillance to help guide against the indiscriminate use of antibiotics in empirical treatment. The recognition but cautious use of medicinal plants as alternative sources of therapies and a probable means to solve the emerging resistance problem was recommended. Improved standard of hygiene in hospital settings and the communities is important to prevent the spread of infections. The thesis provides a novel reference document on the genes coding for resistance, antibiograms and phylogenetic profiles of local isolates of H. influenzae and S. pneumoniae as well as the activities, active compounds and toxicity levels of medicinal plants investigated in an endeavour to effectively understand the possible therapeutic, molecular and epidemiological trends in respect of the designated pathogens.
- Description
- Thesis (Phd) -- Faculty of Health Sciences, 2015
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (177 pages)
- Format
- Publisher
- Walter Sisulu University
- Publisher
- Faculty of Health Sciences
- Language
- English
- Rights
- Faculty of Health Sciences
- Rights
- All Rights Reserved
- Rights
- Open Access
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