Many South Africans continue to use traditional medicine in their daily lives as an alternative form of health care, also as part of their cultural heritage. Medicinal plants are proving to be an important source of novel drugs, and the knowledge provided by traditional healers is a useful tool in the search for antimicrobials. The antimicrobial activity of <M. villosus was investigated against ten bacteria and six fungi. The antibacterial assay showed that the root extract had the highest inhibition against the Gram-positive bacteria at the minimum inhibition concentration of o.1 mg/ml, as well as against the Gram-negative, E. coli, at the MIC of 1.0 mg/ml. Antifungal activity was witnessed against Cladosporium cladosporoide, C. cucumerinum&C sphaerosperum all at the minimum inhibitory concentration of 1.0 mg/ml. An attempt was made to isolate and identify the active antimicrobial compounds. A flavonol, quercetin was isolated and identified by means of UV spectral graphs, and TLC comparison of the plant extract and standard. However, a second isolated antibacterial compound could not be identified fully but it can be said that it is a triterpenoid. / Dissertation (MSc (Botany))--University of Pretoria, 2006. / Plant Science / unrestricted

The thesis presents the development of functionalized electrospun nylon 6 nanofibers for the eradication of pathogenic microorganisms in drinking water. Imidazole derivatives were synthesized as the antimicrobial agents and were characterized by means of NMR spectroscopy, IR spectroscopy, elemental analysis and X-ray crystallography. The first set of compounds (2-substituted N-alkylimidazoles) consisted of imidazole derivatives substituted with different alkyl groups (methyl, ethyl, propyl, butyl, heptyl, octyl, decyl and benzyl) at the 1-position and various functional groups [carboxaldehyde (CHO), alcohol (CH2OH) and carboxylic acid (COOH)] at the 2-position. It was observed that the antimicrobial activity of the compounds increased with increasing alkyl chain length and decreasing pKa of the 2-substituent. It was also observed that the antimicrobial activity was predominantly against a Gram-positive bacterial strains [Staphylococcus aureus (MIC = 5-160 μg/mL) and Bacillus subtilis subsp. spizizenii (MIC = 5-20 μg/mL)], with the latter being the more susceptible. However, the compounds displayed poor antimicrobial activity against Gram-negative bacterial strain, E. coli (MIC = 150- >2500 μg/mL) and did not show any activity against the yeast, C. albicans. The second set of compounds consisted of the silver(I) complexes containing 2-hydroxymethyl-N-alkylimidazoles. The complexes displayed a broad spectrum antimicrobial activity towards the microorganisms that were tested and their activity [E. coli (MIC = 5-40 μg/mL), S. aureus (MIC = 20-80 μg/mL), Bacillus subtilis subsp. spizizenii (MIC = 5-40 μg/mL) and C. albicans (MIC = 40-80 μg/mL)] increased with the alkyl chain length of the 2-hydroxymethyl-N-alkylimidazole. The third set of compounds consisted of the vinylimidazoles containing the vinyl group either at the 1-position or at the 4- or 5- position. The imidazoles with the vinyl group at the 4- or 5-position contained the alkyl group (decyl) at the 1-position. For the fabrication of the antimicrobial nanofibers, the first two sets of imidazole derivatives (2-substituted N-alkylimidazoles and silver(I) complexes) were incorporated into electrospun nylon 6 nanofibers while the third set (2-substituted vinylimidazoles) was immobilized onto electrospun nylon 6 nanofibers employing the graft polymerization method. The antimicrobial nylon nanofibers were characterized by IR spectroscopy and SEM-EDAX (EDS). The electrospun nylon 6 nanofibers incorporated with 2-substituted N-alkylimidazoles displayed moderate to excellent levels of growth reduction against S. aureus (73.2-99.8 percent). For the electrospun nylon 6 nanofibers incorporated with silver(I) complexes, the levels of growth reduction were >99.99 percent, after the antimicrobial activity evaluation using the shake flask method. Furthermore, the grafted electrospun nylon 6 nanofibers showed excellent levels of growth reduction for E. coli (99.94-99.99 percent) and S. aureus (99.93-99.99 percent). The reusability results indicated that the grafted electrospun nylon 6 nanofibers maintained the antibacterial activity until the third cycle of useage. The cytotoxicity studies showed that grafted electrospun nylon 6 nanofibers possess lower cytotoxic effects on Chang liver cells with IC50 values in the range 23.48-26.81 μg/mL. The thesis demonstrated that the development of antimicrobial electrospun nanofibers, with potential for the eradication of pathogenic microoganisms in water, could be accomplished by incorporation as well as immobilization strategies.

Triclosan and triclocarban are antimicrobials found in numerous consumer products, while caffeine is the most commonly consumed stimulant by humans. This study was undertaken to determine the effects of triclosan, triclocarban, and caffeine on the development and physiology of amphibian larvae. LC50 values of triclosan and triclocarban were determined after 96 hours for three North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a common amphibian developmental model: Xenopus laevis. Amphibian larvae were most sensitive to triclosan and triclocarban exposure during early development based upon 96-hour LC50 values. Heart rates for X. laevis and North American larvae exposed to triclosan were variable throughout development. However, significantly lower heart rates were observed in all larvae exposed to triclocarban. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to triclosan were significantly affected in larvae exposed to ½ LC50 and the LC50 concentration. Metabolic rates of X. laevis larvae exposed to triclocarban were significantly affected by exposure to ½ LC50 concentrations in three of four stages investigated. No significant differences were observed in North American larvae exposed to triclocarban. Tissue uptake, lipid uptake, tissue bioconcentration factor (BCF) and lipid BCF of triclosan and triclocarban were investigated in three developmental stages of X. laevis, and in one developmental stage of B. woodhousii woodhousii, and R. sphenocephala. For most tissue and lipid uptake values, a significant increase was observed as exposure concentration increased. Tissue and lipid BCF values were dependent upon both stage and species. Chronic and acute effects of caffeine were determined in X. laevis larvae. Acute 96-hour LC50 values in four developmental stages were > 75,000 ug L-1 caffeine and heart rates were significantly different at the two earliest developmental stages. Larvae chronically exposed to caffeine reached metamorphosis at the same time as controls. Changes in chronic heart rate were dependent upon stage of development and exposure concentration. This research indicates that the toxicity of amphibian larvae exposed to triclosan, triclocarban, and caffeine appears to be dependent upon species and developmental stage, with early developmental stages being most sensitive to contaminant exposure.

Hipóteses do estudo: 1) A magnitude da ação antimicrobiana difere entre os princípios ativos cloreto de cetilpiridíneo (CPC), digluconato de clorexidina (CHX), e óleos essenciais (OE) quando utilizados em dose única no bochecho pré-operatório. 2) A redução bacteriana é mais evidente nos pacientes periodontalmente doentes. Objetivos: Avaliou-se a ação antimicrobiana de bochechos pré-operatórios em dose única em diferentes condições periodontais. Método: Sessenta indivíduos saudáveis, sessenta com gengivite e sessenta com periodontite foram randomizados nos grupos: CPC 0,07%, CHX 0,12%, OE (timol 0,064%, mentol 0,042%, eucaliptol 0,092% e salicilato de metila 0,06%) e solução controle negativo. Para o diagnóstico periodontal foram avaliados índice de placa (IP), índice sangramento gengival (ISG), profundidade de sondagem (PS) e nível de inserção clínica (NIC). A quantificação da contagem bacteriana total e das espécies Porphyromonas gingivalis, Tannerella forsythia,Treponema denticola e Streptococcus oralis foi realizada pela PCR em tempo real (qPCR) em amostras de saliva. Os dados foram analisados utilizando-se os testes t pareado e t Student (p<0,05). Resultados: CHX, OE e CPC acarretaram reduções das contagens totais bacterianas e das espécies bacterianas específicas superiores ao controle negativo. A magnitude do efeito variou entre os agentes testados e foi influenciada pela condição periodontal. Para as reduções das contagens totais bacterianas OE exibiram os melhores resultados já que foram eficazes na saúde, gengivite e periodontite. CHX mostrou esse mesmo padrão, exceto no grupo gengivite. CPC não foi eficaz estatisticamente no grupo periodontite para esse parâmetro. As maiores reduções das espécies Gram negativas agrupadas no complexo vermelho (Pg, Td e Tf simultaneamente) foram observadas após o uso de CHX, no grupo saúde e OE, nos grupos gengivite e periodontite. Quanto às reduções da espécie Gram positiva So, elas foram maiores no grupo saúde após o uso de OE, no grupo gengivite após o uso de CHX e no grupo periodontite após o uso em dose única de CPC. Conclusões: O bochecho pré-procedimento tem ação antimicrobiana que sofre influência da condição periodontal e difere entre os agentes testados. Para pacientes saudáveis o clínico pode optar por CHX, OE ou CPC, mas para pacientes com doença periodontal OE ou CHX devem ser a primeira escolha. / The propouse of this study: 1) The extent the dimension of the antimicrobial action differs among the active agents cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), and essential oils (EO) when used as a single dose in the preoperative rinse. 2) The bacterial reduction after the use of a single dose mouthwash is more evident in patients with periodontal diseases. Goals: The present study evaluated by microbiological parameters the antimicrobial effectiveness of the preoperative rinses in differents periodontal conditions. Method: Sixty periodontal healthy individuals, sixty individual with gingivitis, sixty individual with periodontitis were randomized into the following groups: CPC(0,07%), CHX(0,12%), EO(timol 0,064%,menthol 0,042%,eucalyptol 0,092%,methyl salicylate 0,06%) and negative control solution. Plaque index (IP), gingival bleeding index (IG), probing depth (PS) and clinical attachment level (NIC) were evaluated clinically. The bacterial identification and the quantification (Porphyromonas gingivalis, Tannerella forsythia,Treponema denticola, Streptococcus oralis) were realized for PCR real time(qPCR) on salivary samples. The data were analysed by the paired t-test and Student t-test(p<0,05). Results: CHX, EO and CPC have resulted in reduction in bacterial total counts and specifc bacterial species, wich were higher than the negative control. The magnitude of the effect varied between the tested agents and it was influenced by periodontal condition. The reductions in bacterial total counts EO showed the best results since they were effective in healthy, gingivitis and periodontitis. CHX showed the same pattern, except in the gingivitis group. CPC was not statistically effective in periodontitis group for this parameter. The largest reductions of the species Gram negative grouped in the red complex (Pg, Tf and Td simultaneously) were observed after the use of CHX in health, and EO in the gingivitis and periodontitis groups. The reductions in Gram positive species So, were higher in the group health after using EO, in the gingivitis group after the use of CHX and in the periodontitis group after using CPC. Conclusions: The pre-procedure rinses has antimicrobial action suffering influence of periodontal condition and varies from agents tested. In healthy patients the clinician may choose CHX, EO or CPC but for patients with periodontal disease EO or CHX should be the first choice.

Orientador: Márcio Garcia Ribeiro / Banca:: Liliane de Oliveira Dantas / Banca: Paulo Francisco Domingues / Resumo: Trueperella pyogenes são bactérias oportunistas caracterizadas por infecções piogênicas em animais, em geral refratárias aos tratamentos com antimicrobianos convencionais. Recentemente, os genes que codificam a exotoxina piolisina (plo), e fatores relacionados à adesão de T. pyogenes às células do animal susceptível, como fímbrias (fimA, fimC, fimE, fimG), neuraminidases (nanH, nanP), e proteína ligada ao colágeno (cbpA) têm sido associados a virulência do patógeno. O presente estudo investigou a ocorrência de multirresistência dos isolados aos antimicrobianos e a presença dos genes plo, fimA, fimC, fimE, fimG, nanH, nanP e cbpA em 41 linhagens de T. pyogenes isoladas de diferentes afecções em bovinos, caprinos, ovinos, equino e cão. T. pyogenes foi identificado predominantemente em casos de mastite (46,3%), abscessos (19,5%), pneumonia (9,8%), metritie (9,8%), linfadenite (7,3%), seguidos em menor frequência por encefalite (4,9%) e orquite (2,4%). A maior sensibilidade dos isolados foi observada para florfenicol (97,6%), azitromicina (97,6%), tetraciclina (95,2%), ceftiofur (92,7%), penicilina (92,7%), ampicilina (92,7%), gentamicina (90,3%) e eritromicina (87,8%). Em contraste, alta frequência de resistência dos isolados foi observada para bacitracina (46,3%), neomicina (31,7%), lincomicina (31,7%) e sulfametoxazole/trimetoprim (24,3%). O índice de resistência múltipla aos antimicrobianos - IRMA (>0,3) foi encontrado em 13 (31.7%) isolados, particularmente nos casos de mastite bovina. Dentre os isolados multirresistentes a três ou mais grupos de antimicrobianos, 12 (92,3%) foram identificados na espécie bovina, notadamente em infecções mamárias. Os genes mais frequentes detectados nos isolados foram: plo (40/41=97,6%), fimA (36/41=87,9%), nanP (34/41=82,9%), fimE (32/41=78,0%), nanH (28/41=62,3%), fimC (23/41=56,0%) e cbpA (5/41=12,2%). As principais associações de genes nos isolados foram observadas entre... / Abstract: Trueperella pyogenes are opportunistic bacterium characterized by suppurative infections in domestic animals, commonly refractory to conventional therapy. Recently, genes which encode exotoxin pyolysin (plo), and factors that promote adhesion of T. pyogenes to host cells, such as fimbriae (fimA, fimC, fimE, fimG), neuraminidases (nanH, nanP), and collagen-binding protein (cbpA) have been associated to virulence of pathogen. The aim of present study was investigate occurrence of multi-drug resistance of isolates, as well the presence of genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA in 41 T. pyogenes strains obtaned from bovine, goats, sheep, horses, and dog isolated among different clinical manifestations. T. pyogenes was identified predominantly in mastitis (46.3%), abscesses (19.5%), pneumonia (9.8%), metritis (9.8%), lymphadenitis (7.3%), followed by encephalitis (4.9%) and orchitis (2.4%). The strains showed major in vitro sensitivity to florfenicol (97.6%), azithromicin (97.6%), tetracycline (95.2%), ceftiofur (92.7%), penicillin (92.7%), ampicillin (92.7%), gentamicin (90.3%), and erythromycin (87.8%). In contrast, highest frequency of resistance among isolates was observed to bacitracin (46.3%), neomycin (31.7%), lincomycin (31.7%), and trimethropim/sulfamethoxazole (24.3%). Antimicrobial multiple resistance index - AMRI (>0.3) was found in 13 (31.7%) isolates, particularly in bovine mastitis cases. Among multi-drug resistant isolates to 3 or more group of antimicrobials, 12 (92.3%) were identified in bovine, predominantly in mammary infections. The most common genes detected among isolates were: plo (40/41=97.6%), fimA (36/41=87.9%), nanP (34/41=82.9%), fimE (32/41=78.0%), nanH (28/41=62.3%), fimC (23/41=56.0%), and cbpA (5/41=12.2%). The major associations between genes of isolates were plo, fimA, fimE, nanH, and nanP (10/41=24.4%), and plo, fimA, fimE, fimC, nanH, and nanP (8/41=19.5%), mainly in bovine mastitis. To ... / Mestre

Background: Antimicrobial resistance (AMR) is a global public health challenge since infection with resistant organisms may cause death, can spread across the community, and increase health care costs at individual, community and government level as more expensive antimicrobials will have to be made available for the treatment of infections caused by resistant bacteria. This calls for urgent and consolidated efforts in order to effectively curb this growing crisis, to prevent the world from slipping back to the pre-antibiotic era. The World Health Organization made a call in 2011 advocating for strengthening of surveillance and laboratory capacity as one-way of detecting and monitoring trends and patterns of emerging AMR. Knowledge of AMR guides clinical decisions regarding choice of antimicrobial therapy, during an episode of bacteraemia and forms the basis of key strategies in containing the spread of resistant bacteria. The current study focused on Staphylococcus aureus (SA), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA), as they are common hospital acquired infections which are prone to developing resistance to multiple antibiotics. Aim: The aim of this project was to assess and utilize the laboratory information system (LIS) at the National Health Laboratory Services (NHLS), as a tool for reporting AMR and monitoring resistance patterns and trends over time of clinical isolates of SA, KP and PA, cultured from the blood of patients admitted to seven tertiary public hospitals in three provinces in South Africa. Methods: A retrospective and prospective analysis was done on isolates of SA, KP, PA from blood specimens collected from patients with bacteraemia and submitted to diagnostic microbiology laboratories of the NHLS at seven tertiary public hospitals in three provinces in South Africa. These hospitals comprised the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), Chris Hani Baragwanath Hospital (CBH), Helen Joseph Hospital (HJH), Steve Biko Pretoria Academic Hospital (SBPAH), Groote Schuur Hospital (GSH), Tygerberg Hospital (TH) and the Universitas Hospital of the Free State (UH). For retrospective analysis, data submitted during the period July 2005 to December 2009 were used and for prospective analysis, data relating to AMR in SA, KP, PA, collected by the Group for Enteric, Respiratory and Meningeal disease Surveillance in South Africa, (GERMS-SA) from July 2010 to June 2011 were used. AMR in these three pathogens to commonly used antimicrobial drugs was systematically investigated. Multivariate logistic regressions models were used to assess factors associated with AMR. In addition, a systematic review of research done to date on AMR in bacterial pathogens commonly associated with hospital-acquired infections was conducted in order to understand the existing antimicrobial surveillance systems and baseline resistance patterns in South Africa. Results: A total of 9969 isolates were reported from the retrospective dataset. These were 3942 (39.5%) SA, 4466 (44.8%) KP and 1561 (15.7%) PA. From the prospective dataset, a total of 3026 isolates were reported, 1494 (49.4%) SA and 1532 (50.6%) KP isolates respectively. The proportion of invasive bacteraemia was higher in the <5 year old children. Nearly all strains of SA in South Africa were resistant to penicillin, and >30% up to as high as 80% were resistant to methicillin-related drugs among~560 invasive SA isolates over the two year period. Methicillin resistant Staphylococcus aureus (MRSA) rates significantly differed between hospitals (p=<0.001). The proportion of MRSA isolates in relation to methicillin-susceptible strains showed a declining trend from 22.2% in 2005 to 10.5% in 2009 (p=0.042). Emerging resistance was observed for vancomycin: 1 isolate was identified in 2006 and 9 isolates between July 2010-June 2011, and all except 1 were from Gauteng hospitals. The study found increasing rates of carbapenem-resisant KP of 0.4% in 2005 to 4.0% in 2011 for imipenem. The mean rate of extended spectrum beta lactamase (ESBL-KP) producing KP was 74.2%, with the lowest rate of 62.4% in SBPAH and the highest rate of 81.3% in UH, showing a significant geographical variation in rates of resistance (p=0.021). PA showed a tendency for multi-drug resistance with resistance rates of >20% to extended spectrum cephalosporins, fluoroquinolones and aminoglycosides respectively. Emerging resistance in PA isolates was observed to colistin, showing a resistance rate of 1.9% over the 5 years period. In the multivariate model, age <5 years, male gender, and hospital location were factors significantly associated with MRSA, while ESBL-KP was significantly associated with age <5 years and hospital location. Concluding remarks: The study has clearly demonstrated that AMR is relatively common in South Africa among children <5 years. Enhancement of continued surveillance of nosocomial infections through use of routine laboratory data should be reinforced as this will facilitate effective interpretation and mapping of trends and patterns of AMR. Therefore, the LIS as a tool for gathering such data should be strengthened to provide reliable AMR data for improved understanding of the extent of the AMR, and present evidence on which future policies and practices aimed at containing AMR could be based. Key words: Laboratory information system, Trends, Patterns, Antimicrobial resistance, Bacterial pathogens, Nosocomial infections, Surveillance, Bacteraemia, Blood culture.

Indiana University-Purdue University Indianapolis (IUPUI) / Root canal therapy is a recommended treatment for apical periodontitis. Root canal failure can occur as a result of microbial leakage. Resilon, a resin based root canal obturating cone material introduced in 2004 attempts to minimize leakage by a unique bonding method of the resin sealer to both the core material and to the dentin of the canal walls. Resilon has no bactericidal or antimicrobial effect15. Furthermore, it has been shown that Resilon is susceptible to alkaline and enzymatic hydrolysis as well as bacterial degradation.73, 184-186 It has been suggested that Resilon may be susceptible to degradation by microorganisms found in the infected root canal space. This work focuses on the susceptibility of root canal obturating materials to be degraded by endodontic pathogens seen in root canal treated teeth with apical periodontitis. The aim of this study was to determine if Resilon could be degraded by selected pathogenic bacteria found in the infected root canal system, and if this degradation is more severe than with gutta-percha, a conventional obturating material. P. intermedia, E. faecalis and P. aeruginosa, known endodontic pathogens were inoculated on discs of obturating material (Resilon or gutta-percha) mounted on a platform and placed in wells containing TSB incubated at 37°C under aerobic conditions. The discs were polished, examined by SEM, profilometry, and elemental analysis prior to inoculation to establish a baseline, and were then re-examined by these methods one month after inoculation. The overall results were inconclusive; and using these methods it cannot be determined that the selected bacteria can degrade Resilon. An ideal future study would utilize SEM with gold coated samples as well as atomic force microscopy to evaluate for changes in topographical features of these obturating materials. A notable finding was that Resilon turns black when exposed to bacteria, and the significance of this finding should be addressed in future studies.

Mangrove ecosystems are found in tropical coastal areas and play vital roles in shoreline protection, habitat support, and medicinal value. These ecosystems are under threat from human activities and climate change. This thesis explored the biodiversity and evolutionary ecology of mangroves in the Indonesian Archipelago, home to the world’s richest composition of mangrove species. Indonesia's geography, including over 17,500 islands and the intersection of Laurasian and Gondwanan flora, makes it an ideal location for studying mangrove evolution. Sea level changes and the presence of land and oceanic barriers have shaped the distribution and evolution of mangroves, with the Indonesian Throughflow (ITF) playing an essential role in genetic exchange. The study analyzed 14 populations of Lumnitzera littorea and 21 populations of L. racemosa, utilizing DNA sequencing to assess genetic diversity, structure, and dispersal in relation to geographical distance and sea surface currents. Both species showed low genetic variation, and significant effects of distance and sea current connectivity were observed. Sulawesi acted as a barrier in genetic differentiation, while other barriers were discovered affecting population structure. Divergent evolutionary trajectories were noted between L. littorea and L. racemosa. The study also investigated the relationship between phylogeny and the chemophenetic patterns of their natural products, given their anti-microbial, antioxidant, and other properties. Techniques such as chromatographic separation and high-resolution ESI-MS were used to link molecular phylogeny with phytochemical characterizations. Root samples were collected for chemical analysis, revealing an unusual diversity in sulfated constituents, with some compounds found for the first time in mangroves. Specific geographical areas showed antibacterial potential, and a previously unknown compound, Lumnitzeralactone, was identified. The conclusion highlights this study as the first to reveal molecular evidence of intraspecific phylogroups in Lumnitzera mangroves, with distinctions defined by geographical lines such as Wallace's line. Additionally, the study found promising sources for anti-bacterial agents and potential therapeutic benefits to human health. The observed genetic differentiation emphasizes the need for conservation strategies at the population level, and the potential for new medicines underscores the importance of mangrove conservation in their natural habitats across Indonesia.:Preface 4 Summary 5 Zusammenfassung 10 1. Introduction 15 1.1. Characteristics, significance, and threat of mangroves 15 1.1.1. Characteristics of mangroves 15 1.1.2. Significance of Indonesian mangroves 16 1.1.3. Threats to mangroves 18 1.2. Evolutionary processes and diversity of mangroves 19 1.2.1. Evolution of mangroves 19 1.2.2. Sea surface currents in Indonesia shape genetic structure 20 1.2.3. The relevance of Wallace’s line to mangrove evolution 22 1.2.4. Isolation by geographical distance 24 1.2.5. Genetic diversity and population structure 25 1.3. Diversity of bioactive compounds of mangrove genus Lumnitzera 27 1.3.1. Sulfur-containing metabolites 27 1.3.2. Phylogenetics 28 1.3.3. Anti-infective potential 29 1.4. Study species 30 1.5. Aim of the thesis 33 2. Material and Methods 37 2.1. Sampling and sample design 37 2.2. Laboratory procedures and genetic analysis 38 2.2.1. DNA isolation 38 2.2.2. ddRADseq sequencing, and bioinformatics 38 2.2.3. Genetic diversity, population structure and differentiation 40 2.2.4. Identifying barriers and areas of connectivity 41 2.2.5. Isolation by distance and sea surface current connectivity 42 2.2.6. Polymerase Chain Reaction (PCR), and phylogenetic analyses 43 2.3. Laboratory procedures and phytochemical analysis 44 2.3.1. Root sample extraction 44 2.3.2. TLC, Low-resolution ESI-MS spectra, HPLC, and NMR 45 2.3.3. UHPLC-ESI-QqTOF-MS and MS/MS 46 2.3.4. RP-UHPLC-ESI-LIT-Orbitrap-MS 47 2.3.5. Extraction and isolation of compounds 47 2.3.6. Anti-infective bioassays 51 3. Results 52 3.1. Genetic diversity and population structure 52 3.1.1. Genetic diversity 52 3.1.2. Population structure and genetic differentiation 54 3.1.3. Effective migration 58 3.1.4. Isolation by distance and sea surface current connectivity 59 3.2. Unusual-sulfated constituent and anti-infective properties 61 3.2.1. Phytochemical screening 61 3.2.2. Phylogenetic tree of Lumnitzera 69 3.2.3. Evaluation of anti-infective properties 70 4. Discussion 74 4.1. Population genomics of Lumnitzera mangroves in Indonesia 74 4.1.1. The genetic diversity paradox in mangroves 74 4.1.2. Phylogroups and the Sunda-Wallacea biogeographical pattern 76 4.1.3. Limited mixture among phylogroups and populations by sea surface currents 78 4.1.4. Restricted gene flow by geographical distance 80 4.1.5. Evolutionary ecology of Lumnitzera inferred by genetics and chemodiversity 81 4.2. Bioactive compounds and anti-infective potential of Lumnitzera 82 4.2.1. Diversity of bioactive compounds 82 4.2.2. Sulfated and nonsulfated ellagic acid supported by phylogenetic pattern 83 4.2.3. Anti-infective properties and their restriction to particular locations 86 5. Conclusion and future perspective 89 6. References 94 7. Appendix 111 Curriculum vitae 119 Declaration of independent work 122 Acknowledgments 123 Author contributions statement 125 / Viele Mangrovenarten haben neben Holz- und Nichtholzprodukten auch einen medizinischen Wert und werden traditionell genutzt. Doch durch menschliche Aktivitäten und den Anstieg des Meeresspiegels durch den Klimawandel verschwinden sie rasch. Diese Arbeit erforscht die biologische Vielfalt der Mangroven im indonesischen Archipel, insbesondere die Arten Lumnitzera littorea und Lumnitzera racemosa. Der indonesische Archipel hat die weltweit reichste Mangrovenzusammensetzung und dient als Übergangsregion für verschiedene Pflanzenlinien. Der Meeresspiegelanstieg und dessen mögliche Auswirkungen auf Mangroven wurden genau untersucht. Mangroven besitzen schwimmfähige Fortpflanzungsorgane, und die Meeresströmungen des Indonesian Throughflow (ITF) könnten den genetischen Austausch zwischen verschiedenen Populationen ermöglichen. In der Studie wurden 14 Populationen von L. littorea und 21 von L. racemosa genotypisiert. Die Ergebnisse zeigten eine geringe genetische Variation auf Populationsebene und eine signifikante genetische Differenzierung, beeinflusst von Entfernung und Meeresströmung. Es wurden auch divergierende Entwicklungsverläufe und zwei Phylogruppen bei beiden Arten beobachtet. Die Studie untersuchte auch die phylogenetischen Beziehungen und die Chemophenetik der beiden Arten. Mangrovenböden weisen einen hohen Sulfatgehalt auf, und medizinisch wirksame Verbindungen werden oft von Mikroorganismen wie endophytischen Pilzen produziert. Schwefelverbindungen in Mangroven wurden bisher vernachlässigt. Durch Kombination von phylogenetischen Daten mit chemischen Analysen wurde die Entwicklung der Mangrovenarten und die Vielfalt der Wurzelmetaboliten untersucht. Es wurden chromatographische und tandemmassenspektrometrische Techniken eingesetzt, um eine molekulare Phylogenie mit phytochemischen Charakterisierungen zu verbinden. Die Untersuchung ergab eine ungewöhnliche Vielfalt an sulfatierten Bestandteilen und antibakterielles Potenzial. Zusammenfassend liefert die Studie molekulare Beweise für intraspezifische Phylogruppen in Lumnitzera-Mangroven, definiert durch die biogeografische Trennung von Sunda und Wallacea. Beide Arten stellen eine vielversprechende Quelle für antibakterielle Wirkstoffe dar, einschließlich sulfatierter Ellagsäurederivate. Die populationsgenomischen Ergebnisse liefern Informationen über die Erhaltungsstrategie von Lumnitzera-Arten, und die metabolomischen Ergebnisse berichten über potenzielle neue Arzneimittel, was die Bedeutung der Erhaltung von Mangroven in ihren natürlichen Lebensräumen im gesamten indonesischen Archipel unterstreicht.:Preface 4 Summary 5 Zusammenfassung 10 1. Introduction 15 1.1. Characteristics, significance, and threat of mangroves 15 1.1.1. Characteristics of mangroves 15 1.1.2. Significance of Indonesian mangroves 16 1.1.3. Threats to mangroves 18 1.2. Evolutionary processes and diversity of mangroves 19 1.2.1. Evolution of mangroves 19 1.2.2. Sea surface currents in Indonesia shape genetic structure 20 1.2.3. The relevance of Wallace’s line to mangrove evolution 22 1.2.4. Isolation by geographical distance 24 1.2.5. Genetic diversity and population structure 25 1.3. Diversity of bioactive compounds of mangrove genus Lumnitzera 27 1.3.1. Sulfur-containing metabolites 27 1.3.2. Phylogenetics 28 1.3.3. Anti-infective potential 29 1.4. Study species 30 1.5. Aim of the thesis 33 2. Material and Methods 37 2.1. Sampling and sample design 37 2.2. Laboratory procedures and genetic analysis 38 2.2.1. DNA isolation 38 2.2.2. ddRADseq sequencing, and bioinformatics 38 2.2.3. Genetic diversity, population structure and differentiation 40 2.2.4. Identifying barriers and areas of connectivity 41 2.2.5. Isolation by distance and sea surface current connectivity 42 2.2.6. Polymerase Chain Reaction (PCR), and phylogenetic analyses 43 2.3. Laboratory procedures and phytochemical analysis 44 2.3.1. Root sample extraction 44 2.3.2. TLC, Low-resolution ESI-MS spectra, HPLC, and NMR 45 2.3.3. UHPLC-ESI-QqTOF-MS and MS/MS 46 2.3.4. RP-UHPLC-ESI-LIT-Orbitrap-MS 47 2.3.5. Extraction and isolation of compounds 47 2.3.6. Anti-infective bioassays 51 3. Results 52 3.1. Genetic diversity and population structure 52 3.1.1. Genetic diversity 52 3.1.2. Population structure and genetic differentiation 54 3.1.3. Effective migration 58 3.1.4. Isolation by distance and sea surface current connectivity 59 3.2. Unusual-sulfated constituent and anti-infective properties 61 3.2.1. Phytochemical screening 61 3.2.2. Phylogenetic tree of Lumnitzera 69 3.2.3. Evaluation of anti-infective properties 70 4. Discussion 74 4.1. Population genomics of Lumnitzera mangroves in Indonesia 74 4.1.1. The genetic diversity paradox in mangroves 74 4.1.2. Phylogroups and the Sunda-Wallacea biogeographical pattern 76 4.1.3. Limited mixture among phylogroups and populations by sea surface currents 78 4.1.4. Restricted gene flow by geographical distance 80 4.1.5. Evolutionary ecology of Lumnitzera inferred by genetics and chemodiversity 81 4.2. Bioactive compounds and anti-infective potential of Lumnitzera 82 4.2.1. Diversity of bioactive compounds 82 4.2.2. Sulfated and nonsulfated ellagic acid supported by phylogenetic pattern 83 4.2.3. Anti-infective properties and their restriction to particular locations 86 5. Conclusion and future perspective 89 6. References 94 7. Appendix 111 Curriculum vitae 119 Declaration of independent work 122 Acknowledgments 123 Author contributions statement 125

The use of traditional medicine is a popular practice in South Africa especially among rural dwellers due to several reasons such as availability of natural products, cultural beliefs, preference of natural products to synthetically derived drugs and the high cost of modern drugs. Traditional healers in South Africa play key roles in administering treatment for all sorts of ailments using plants. The aim of this study was to evaluate the efficacy of seven selected medicinal plants that are used in traditional medicine to treat stomach-related ailments for their pharmacological and phytochemical properties. Plant material was extracted sequentially with ethyl acetate (EtOAc), ethanol (EtOH) and water. The extracts were evaluated for their antimicrobial activities using the microdilution technique against two Gram-positive (Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 12600) bacteria and a Gram-negative (Escherichia coli ATCC 11775) bacterium. A modified microdilution technique was used to screen for antifungal activity against a yeast-like fungus (Candida albicans ATCC 10231). Only the EtOAc extract of Tetradenia riparia demonstrated good antibacterial activity against the Gram-negative E. coli, all the other extracts that were active only showed good antibacterial activity against the two Gram-positive (E. faecalis and S. aureus) bacteria with MIC values <1 mg/ml. None of the extracts that exhibited good inhibitory activity showed corresponding bactericidal activity against the bacterial test strains, suggesting that the observed activity were all inhibitory. Good antifungal activity with an MIC value <1 mg/ml was observed in only 5 extracts, and none of the extracts exhibited corresponding fungicidal activity. The in vitro colorimetric assay for anthelmintic activity against Caenorhabditis elegans revealed that almost all the extracts possessed moderate to high anthelmintic properties. The EtOAc extract of T. riparia had the best activity at MLC value of 0.004 mg/ml. The anti-inflammatory activity of the plant extracts was tested using the cyclooxygenase assays to determine their inhibitory potential against COX-1 and COX-2 enzymes. All the EtOAc extracts demonstrated both COX-1 and COX-2 inhibitory activity in the range of 50.7 ± 2.4 to 99.5 ± 0.5%. Apart from the EtOH extracts of C. multicava that showed high inhibitory activity against both COX-1 and COX-2, all the other EtOH extracts were COX-2 selective. Aqueous extracts exhibited poor inhibitory activity against both COX-1 and COX-2 enzymes with the exception of T. riparia and Coddia rudis that showed good inhibitory activity (69.1 ± 0.9 and 92.65 ± 0.7%) against COX-1 and COX-2 respectively. The standard plate incorporation assay for the Ames test was carried out to determine the potential genotoxic effects of the plant extracts and this revealed that all the extracts were non-mutagenic towards Salmonella typhimurium tester strains TA98, TA100 and TA1537 without metabolic activation. However, further studies incorporating metabolizing enzymes are needed to confirm the safe use of the studied plants. Phytochemical analysis revealed relatively high amounts of total phenolics, gallotannins and flavonoids in all the evaluated plants. Total and steroidal saponins were detected in only two plant samples, Canthium spinosum and Cassinopsis ilicifolia (bark). These findings present useful information on the types of bioactive compounds that could be responsible for the pharmacological activities observed among some of the plant extracts. The results obtained in this study showed different levels of pharmacological activities among all the evaluated medicinal plants which provide scientific validation for their use in traditional medicine as antimicrobial agents. Phytochemical analysis provides valuable information for further study that will be aimed at isolation and identification of the bioactive principles in the evaluated plant species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

M. Tech. (Chemistry, Department of Chemistry), Vaal University of Technology / Infectious diseases, a group of illnesses caused by specific pathogens or its toxins is a leading cause of death globally. Treatment with antibiotics is a key intervention in the control and management of many infectious disease. However, the increasing incidence of antibiotics failure, due to the emergence of drug resistant pathogens, is rendering the use of antibiotics chemotherapy ineffective. A possible solution is to synthesize new compounds with broad spectrum characteristics and superior drug performances as alternative to conventional antibiotics. Schiff Bases are biologically active ligands. They form metal complexes with superior biological activities. This research aims to synthesize some Schiff Base metal complexes and investigate their biological effects on Staphylococcus aureus and Enterococcus faecalis. Metal acetylacetonates of Vanadium, Copper, Cobalt, Zinc, Magnesium, Manganese, Cadmium, Nickel and Iron were synthesized and characterized by Fourier transform infrared spectroscopy. Four Schiff bases, LI, L2, L3 and L4 were also synthesized by the condensation of 4- (diethylamino)-2-hydroxybenzaldehyde with 4-nitrobenzohydrazide and 4-methoxybenzohydrazide to form L1 and L2. 4-(dimethylamino) benzaldehyde was reacted with 4-nitrobenzohydrazide and 4-methoxybenzohydrazide to form L3 and L4 respectively. The Schiff base ligands were then reacted with synthesized Vanadium, manganese, cobalt and magnesium acetylacetonates to form Schiff base complexes (SBC 1A to 4D). Schiff bases ligands and complexes were characterized by FT-IR, 1H-NMR, 13C-NMR, TGA and DTA. Fourier Transform infrared spectroscopy (FTIR) of the acetylacetonates showed the formation of metal acetylacetonates as characterized by the absence of the carbonyl stretching n(C=O) vibration in metal acetylacetonate spectra as compared to pure acetylacetone. Metal acetylacetonates also showed the presence of metal oxygen vibration frequency, n(M-O-C), in the spectra obtained. Thermogravimetric analysis (TGA) and Derivative or Differential Thermogravimetric analysis (DTA) of the Schiff base ligands showed the presence of a single decomposition product in L1, L2, L3 and L4 indicating the formation of a single reaction product while those of Schiff base complexes showed the formation of several decomposition products. Proton and carbon thirteen Nuclear Magnetic Resonance (1H- and 13C-NMR) spectroscopy of the Schiff base ligands indicated the presence of hydrogen and carbon-13 in different environments. The chemical shifts of the hydrogens and carbon-13 provided evidence that Schiff base ligands were formed. The strongest evidence is the presence of the azomethine hydrogen and carbon in the spectra of the Schiff base ligands. The presence of aromatic hydrogens and carbon at chemical shift environments found in literature also confirmed the formation of Schiff base ligand. The NMR spectra of Schiff base complexes showed the presence of azomethine (HC=N) and aromatic hydrogens at expected chemical shifts. The synthesized Schiff bases and their corresponding metal complexes were screened for their invitro antibacterial activities against two Gram-positive (Staphylococcus aureues and Enterococcus feacalis) bacterial strains by the Agar-well diffusion methods.The ligands and complexes were tested against confirmed S. aureus and E. faecalis strains and only 4 exhibited antimicrobial activities. The ligands and complexes were effective against the S. aureusand E. faecalis isolates. / VUT