Theses (PHD)

The emergence of Methicillin Resistant Staphylococcus aureus (MRSA) which is resistant to many available antibacterial agents, especially the β-lactam antibiotics, has become a major threat to the health sector worldwide, giving impetus to the search for novel antibacterial agents efficacious against this bacterium. Four (4) Cinnamomum species i.e. C.iners, C.altissimum, C.impressicostatum and C. porrectum were investigated against a range of multidrug resistant bacteria, including Methicillin Resistant Staphylococcus aureus, our main subject of interest, for potential antibacterial activity and elucidation of their modes and mechanisms of action. Characterisations of their bioactive fractions were performed using bioassay guided isolation techniques. C.impressiscostatum stem-bark extract recorded the highest zone of inhibition and lowest minimum inhibitory concentration against MRSA. The presence of salt enhanced the killing efficiency of the plant extract against MRSA. Cellular materials leaked from MRSA after treatment with the plant extract and this was concomitant with marked cell wall damage, as observed using scanning electron microscopy (SEM). A total of 136 genes were found to be differentially regulated by C. impressicostatum stem-bark extract. Amongst these, 73 genes were upregulated and 63 genes were downregulated. The MRSA genes that fluctuated in expression when treated with the plant extract involved various pathways, such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, membrane transport, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, biosynthesis of secondary metabolites, energy metabolism, folding, sorting and degradation, glycan metabolism, pathways in pathogenesis, replication and repair, signal transduction, transcription, translation, translational ribosomal structure and biogenesis and xenobiotic degradation and metabolism. The possible mechanisms underlying the killing action of C.impressicostatum active fraction of water extract against MRSA are via inhibition of biofilm formation, inhibition of nucleotide metabolism as well as DNA replication and repair, downregulation of the mismatch repair mechanism, thereby enabling maintenance of deleterious and lethal mutations, ultimately causing cell death, induction of increased cellular vulnerability to the effects of reactive oxygen and nitrogen species, inhibition of essential lipid biosynthesis required for cellular structure and metabolism as well as the induction of apoptosis.

Human rotaviruses (hRV) are one of the most common causes of severe diarrhoea in young children worldwide with high mortality rate especially in developing countries. Rotavirus VP6 protein has been suggested as a vaccine target due to its immunogenic and antigenic potential. Present study utilised the Agrobacterium-mediated transformation on Centella asiatica callus as the host to produce rotavirus VP6 subunit. The antibiotics - cefotaxime (250 mg/L), kanamycin (750 mg/L) and hygromycin (15 mg/L) were used in transformation process for transformant selection. Besides antibiotic susceptibility, bacterium concentrations, infection period and transformation method were also determined. The present study showed that Centella asiatica calli that were infected for45 minutes with Agrobacterium tumefaciens harbouring human rotavirus VP6genes (optical density6000.8) through needle puncture method exhibited a significant increment in its growth [5.4 ± 0.09 g fresh weight (FW) callus after four weeks of co-cultivation in antibiotic selection medium containing kanamycin and hygromycin. The transformation efficiency recorded under this optimal transformation protocols was 57.6%. The successful transformants were further confirmed using molecular techniques such as PCR(VP6gene has 1206bp)and western blot analysis(VP6 protein at 45 kDa). Quantification on the hRV-VP6 protein expressed in Centella asiatica callus recorded an amount of 0.16 to 0.46 mg/g FW callus. To the best of the knowledge, this study was the first report on the successful production of plant-based rotavirus VP6 subunit in Centella asiatica through Agrobacterium-mediated transformation. Thus, apart from adding value to the current applications of Centalla asistica callus, this study also serves as thenovel edible Centalla asiatica plants producing rotavirus vaccine that could potentially help in resolving the public health issues such as on the side effect of current available vaccines, anti-vaccine concerns and appropriate vaccination plan.

Acinetobacter baumannii is resistant to many commercially available antibacterial agents and has become a major threat to the health sector worldwide, resulting in the need to expedite the search for novel and potent antibacterial agents against this bacterium. Eugenol is a natural vanilloid present in the essential oils of Ocimum gratissimum (basil),Cinnamomum verum (cinnamon) and Myristica fragrans (nutmeg) and is also a major component of Syzygium aromaticum (clove) oil. It is used as a dental anaesthetic to relieve pain arising from pulpitis and dentinal hypersensitivity. In addition, it possesses a wide array of biological properties such as antimicrobial, anti-inflammatory, analgesic, anticancer and antidiabetic activities. Due to its broad range of antimicrobial activities, it was targeted as a valuable starting material for the synthesis of derivatives which were hoped to have enhanced antibacterial activity against multidrug resistant (MDR) A.baumannii. Thus, this thesis describes chemical synthesis of new eugenol derivatives and the evaluation of their antibacterial activity against MDR A. baumannii. A total of forty-nine eugenol derivatives were synthesised and characterised using 1H and 13C NMR spectroscopy. Representative clinical isolates of MDR A. baumannii were subjected to challenge by eugenol and its synthesised derivatives via standard antibacterial assays. All the eugenol derivatives exhibited moderate to high antibacterial potencies, with highest potency demonstrated by derivative, E43, 4-allyl-2-methoxyphenyl-2, 4, 6- trichlorobenzoate. In addition, the combined use of E43 with standard antibiotics against MDR A. baumannii yielded additive and synergistic antibacterial effects. In comparing the antibacterial effects of E43 with unmodified eugenol, E43 exhibited 2.3 fold higher potency, followed by E06 with 1.5 fold higher potency and E47 with 1.3 fold higher potency. All the synthesised eugenol derivatives also had greater predicted water solubility than unmodified eugenol and predicted cardio and neuro toxicities were within the acceptable range. Thus, these novel synthesised eugenol derivatives have immense potential for development into efficacious anti MDR A. baumannii drugs. The introductionof such drugs into the commercial market today would be a timely solution towards combating recalcitrant MDR A. baumannii infections, thereby significantly reducing associated morbidity, disability-adjusted life years (DALY) and mortality. The focus of research then pivoted towards investigating the underlying mechanism of antibacterial activity exhibited by E43. Could E43 be down regulating the efflux pumps of MDR A. baumannii? The efflux pump genes adeB, adeR and adeS of the RND efflux system, AdeABC, of MDR A. baumannii, were subsequently detected by PCR. Using RTqPCR, all three of these efflux genes were found to be significantly down – regulated in E43 treated MDR A. baumannii in comparison to untreated controls, suggesting that the additive and synergistic effects between E43 and the antibiotics was due to the down regulation in A. baumannii by E43 of this major efflux system. However, the latter findings did not explain how E43 could kill MDR A. baumannii when acting on its own. In silico studies performed using the Schrodinger small-molecule drug discovery suite 2019-2 were strongly suggestive that E43 directly interacts and binds the A. baumannii class D carbapenemase, A. baumannii outer membrane carboxylate channel porin protein, OccAB3 and the A. baumannii AdeB protein, possibly causing dysfunctionalisation of these proteins. Thus, the E43 mode of action in killing MDR A. baumannii seems to bemulti-pronged, inhibiting expression of AdeB well as, by direct binding to the protein, possibly detrimentally affecting its functionality. In addition, E43 possibly binds andrenders the class D carbapenemase of MDR. A. baumannii dysfunctional, thereby facilitating lethal effects on the bacterium in the presence of carbapenem antibiotics. Possible binding of the porin protein, OccAB3, by E43 may have modified the selectivity of this porin leading to reduced uptake of its regular amino – acid substrates, such as glycine and glutamic acid. Impediment of amino – acid uptake by E43 binding to this porin may have contributed to amino – acid starvation and its lethal effects on MDR A. baumannii when used alone. In conclusion, this study has confirmed that eugenol derivative E43 is highly biologically active in killing MDR A. baumannii. Mechanistic studies have demonstrated that this compound kills via inhibition of the AdeABC efflux pump with likely pleiotropic effects on bacterial viability.