Theses (PhD in Medical and Health Sciences)

Background: Bacterial biofilms within the root canal system are a major contributor in the failure of endodontic treatment, primarily attributed to the presence of significant pathogens such as Enterococcus faecalis (E. faecalis), Fusobacterium nucleatum (F. nucleatum), and Porphyromonas gingivalis (P. gingivalis), which demonstrate considerable virulence, antimicrobial resistance, and the capacity to penetrate dentinal tubules. Conventional irrigants such sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) have limitations including cytotoxicity, inadequate biofilm eradication, and adverse responses including tissue irritation and precipitate formation. Quaternary ammonium silane (codename- K21), a functionalised organosilicon compound with antibacterial and anti-inflammatory properties, not only disrupts microbial biofilms but also preserves compatibility with host tissue components, including macrophages. Comprehensive research is warranted to confirm K21's therapeutic benefits since it has potential to block Sortase Av(SrtA) and matrix metalloproteinases (MMPs), enzymes engaged in bacterial adherence and tissue breakdown. Objectives: The aim of this research was to assess K21's antibacterial and anti-inflammatory performance as an endodontic irrigant. Among specific objectives were comparing 1.00% K21 with 0.5% K21, 2.00% CHX, 6.00% NaOCl, and saline on single-species (E. faecalis, F. nucleatum and P. gingivalis respectively) and dual-species (F. nucleatum and P. gingivalis) biofilms at 200 μm and 400 μm dentinal tubule depths. Additionally evaluated in the study were K21's effects on hPDLSCs survival as well as RAW 264.7 macrophage viability and anti-inflammatory (M2) polarization. Moreover, molecular simulations were performed to assess the interaction of K21 with SrtA and MMP-9 molecular structures. While the null hypothesis predicted no significant differences, the hypotheses postulated that 1.00% K21 would show better biofilm reduction, higher macrophage and hPDLSCs viability, and greater anti-inflammatory benefits than conventional irrigants. Methods: This multimodal investigation utilized a combination of in vitro assays and imaging techniques. Biofilms of E. faecalis, F. nucleatum, and P. gingivalis were cultured on dentin specimens and exposed to different irrigants: saline, 6.00% NaOCl, 2.00% CHX, 0.5%K21, and 1.00% K21. Colony-forming unit (CFU) counts were used to assess bacterial viability, while structural changes were analyzed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Raman spectroscopy was employed to identify chemical alterations in post-treatment biofilm components. Cell viability was evaluated using the MTT assay and mitochondrial staining in RAW264.7 macrophages, while the alamar blue assay was utilised for hPDLSCs. Additionally, the expression of cytokines (TNF-α, IL-1β, TGF-β, VEGF-A) associated with macrophage polarization was evaluated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and gene expression analysis to determine the immunomodulatory impact of all the irrigants. Molecular simulations were carried out inside the Schrödinger Drug Discovery Suite using the Desmond package. Molecular simulations analyzed K21’s binding to MMP-9 and SrtA molecular structures with stability assessed via root mean square deviation (RMSD) and binding free energy (MM-GBSA). Statistical analysis used ANOVA with Tukey’s post hoc test (p < 0.05) and Pearson’s correlation for depth-dependent efficacy. Results: The CFU analysis revealed that K21, especially at a 1.00% concentration, consistently showed enhanced antibacterial efficacy relative to 6.00% NaOCl and was frequently comparable to or more effective than 2.00% CHX against E. faecalis, F. nucleatum, P. gingivalis, and dual-species biofilms. For CFU findings of E. faecalis, at a depth of 200 μm and a duration of 1-minute, 1.00% K21 exhibited the lowest CFU at 1.358 ± 0.193, in contrast to 0.5% K21 (3.453 ± 0.205), 2.00% CHX (4.65 ± 0.093), 6.00% NaOCl (5.64 ± 0.108), and saline (9.63 ± 0.031), with statistically significant differences (p < 0.001). At 400 μm and 1-minute, similar trends were observed. In 5-minute exposures, 1.00% K21 consistently exhibited the lowest CFUs (0.9687–0.9924), significantly surpassing 2.00% CHX and 6.00% NaOCl at both 200 μm and 400 μm (p < 0.001). For F. nucleatum biofilm, 1.00% K21 exhibited the lowest CFUs (2.1199 ± 0.20274 at 200 μm/5 min and 2.1782 ± 0.55736 at 400 μm/5 min), demonstrating significant reductions compared to 6.00% NaOCl (p < 0.001) and comparable efficacy to 2.00% CHX. At 1-minute application, CFU counts were markedly diminished by K21 group compared to 6.00% NaOCl. For P. gingivalis, at 200 μm/1 min, 1.00% K21 (3.9831 ± 0.10972) and 0.5%K21 were significantly better than 6.00% NaOCl (5.1798 ± 0.39327) and were comparable to 2.00% CHX (4.0792 ± 0.10074). At 400 μm/1 min and 5 min, all test irrigants demonstrated effectiveness relative to saline; nevertheless, 1.00% K21 consistently exhibited the lowest CFU counts with no statistically significant difference from 2.00% CHX. In dual-species biofilm, 1.00% K21 demonstrated significant reductions compared to saline at both 200 μm and 400 μm for 1 and 5-minute treatments. At 200 μm/5 min, 1.00% K21 (4.6173 ± 0.37708) and 0.5%K21 (4.7104 ± 0.34656) demonstrated superior efficacy compared to 6.00% NaOCl (5.1432 ± 0.48289). 1.00% K21 was comparable to 2.00% CHX (p > 0.05) and significantly better than saline (p < 0.001) and 6.00% NaOCl (p = 0.046). Morphological analyses utilising SEM and TEM corroborated the CFU findings, demonstrating that 1.00% K21 induced significant membrane rupture, cytoplasmic leakage, and biofilm matrix disintegration across all the three species. The 1.00% K21 treated groups exhibited the most pronounced intensity deterioration, and Raman spectroscopy examination indicated notable spectrum shifts in the 480–490 cm⁻¹ range, so corroborating the hypothesis of heightened structural disruption of bacterial components. For macrophage viability, all groups showed cell viability above 80%. While 0.5% and 1.00% K21 showed higher viability (87.17% and 87.4%), viability reduced for 6.00% NaOCl (82.42%) and 2.00% CHX (86.08%). K21 groups were noticeably better than 6.00% NaOCl, although all test groups had lowered viability relative to saline (p<0.05). Fluorescence imaging depicted strong, homogeneous mitochondrial signals maintained by saline and 0.5% K21 that indicated healthy, viable cells. The 1.00% K21 group maintained general function while only modestly altered mitochondrial structure. While 6.00% NaOCl produced severe cytotoxicity, low cell density, and weak fluorescence, 2.00% CHX exhibited variable effects with some mitochondrial fragmentation. At a concentration of 0.5%, K21 demonstrated superior mitochondrial preservation compared to 2.00% CHX and 6.00% NaOCl. SEM and TEM analysis indicated that macrophages treated with saline exhibited normal integrity and morphology. K21 exhibited electron-dense inclusions at elevated concentrations, signifying stress, but preserving cell morphology with slight surface alterations. The 6.00% NaOCl treatment caused severe structural damage, membrane fragmentation, and cytoplasmic debris; 2.00% CHX treated cells showed surface shrinkage and internal aggregates. K21 group displayed generally better cellular preservation than CHX and NaOCl groups. Gene expression analysis demonstrated that K21 induced modest upregulation of pro-inflammatory cytokines (TNF-α: ~4.24–4.26; IL-1β: ~1.43–1.89), whereas 2.00% CHX and 6.00% NaOCl strongly upregulated both TNF-α (~19.38 and 19.30) and IL-1β (~2.30 and 1.89). Conversely, K21-treated groups exhibited significantly higher anti-inflammatory markers, with VEGF-A reaching 58.93 ± 0.16 for 1.00% K21 and TGF-β at 6.51 ± 0.11, substantially exceeding levels observed with CHX (TGF-β: 6.31 ± 0.66) and NaOCl. Overall, K21 demonstrated a balanced immunomodulatory profile characterized by reduced inflammatory activation and enhanced pro-healing capacity compared to conventional irrigants. At 30 minutes of exposure, the viability of human periodontal ligament stem cells (hPDLSCs) remained approximately close to 80% for all tested irrigating agents. Specifically, 1.00% K21 demonstrated a viability of 81.13%, 0.5% K21 at 80.13%, CHX at 80.16%, and NaOCl at 79.34%. Saline showed the highest cell viability at 98.48%. High viability was indicated by SEM and TEM studies of hPDLSCs showing intact morphology and membrane integrity from saline-treated cells. Severe structural damage, membrane lysis, and cytoplasmic disintegration were produced by 6.00% NaOCl. Moderate membrane distortion and vacuolation caused by 2.00% CHX indicated cytotoxic effects. Although 1.00% K21 showed some structural changes while 0.5%K21 maintained better integrity. TEM verified that although K21 caused subcellular changes, it maintained membrane continuity, hence indicating better biocompatibility than 2.00% CHX and 6.00% NaOCl. The K21 group shows a specific pocket binding on several MMPs and specific SrtA structures, according to molecular simulation, which generates a classic clouting effect. This can prevent SrtA enzymes and MMPs from acting catalytically, avoiding structural changes in dentin. Conclusions The 1.00% K21 group demonstrated superior antibacterial efficacy against E. faecalis, F. nucleatum, and P. gingivalis in dentinal tubules at 200 μm and 400 μm depths compared to 6.00% NaOCl and saline, with performance comparable to 0.5% K21 and 2.00% CHX. In dual-species biofilms, 1.00% K21 outperformed NaOCl and saline while showing equivalent results to CHX and 0.5% K21. It exhibited significantly enhanced biocompatibility and anti-inflammatory properties in RAW 264.7 macrophages and hPDLSCs compared to NaOCl, with results comparable to 0.5% K21 and CHX. K21 promoted M2 macrophage polarization and a balanced immune response. Molecular docking revealed specific binding affinity to MMPs and Sortase A, indicating a unique "clouting effect" that enhances antibacterial and regenerative capabilities. Overall, 1.00% K21 represents a promising biocompatible, multifunctional endodontic irrigant with superior antibacterial and anti-inflammatory potential. Keywords: antibacterial, human periodontal ligament stem cells, K21, macrophages, molecular simulation, quaternary ammonium silane.

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.