Department of Biochemistry, Cell and Molecular Biology
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Item Pathogenomics And Antimicrobial Resistance Analysis In Neisseria Gonorrhoeae(University Of Ghana, 2021-01) Agbodzi, B.Gonorrhoea is a poorly controlled public health problem. With the global emergence of resistance to first line antibiotic treatment options, the infection has been predicted to be untreatable in the near future. This emerging trend highlights the need for constant genetic surveillance to unravel the mechanisms of resistance and inform therapy. This study therefore, sought to perform whole genome characterization of N. gonorrhoeae collected in Ghana to identify lineages of circulating strains, their antimicrobial resistance (AMR) and some virulence determinants. Gonococci isolates were cultured on gonococcal (GC) medium and identified using the API NH kit (Biomerieux, France). Genomic DNA was extracted from N. gonorrhoeae isolates using the QIAamp® DNeasy Ultraclean Microbial kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) and PubMLST Neisseria database were used to catalogue chromosomal and plasmid genes implicated in AMR and assign sequence types (STs). The core genome MLST (cgMLST) approach was used for comparative genomics. The Virulence Factors of Pathogenic Bacteria Database (VFDB) was used to annotate virulence factors. In vitro resistance measured by disc diffusion revealed that (56)100%, (51)91% and (50)89.3% of the isolates were resistant to tetracycline, penicillin and ciprofloxacin respectively, while for the E-test method, (54)96.4%, (51)91% and (49)87.5% respectively were recorded. Four isolates exhibited reduced susceptibility to both cefixime and ceftriaxone as measured by disc diffusion. For these isolates, MIC ranges of 0.004 – 0.016 μg/ml and 0.016 - 0.75 μg/ml for ceftriaxone and cefixime respectively were recorded. No spectinomycin and azithromycin resistance was recorded using the E-test method. A total of 22 STs were identified by Multi-Locus Sequence Typing (MLST), with ST-14422 (n=10), ST-1927 (n=8) and ST-11210 (n=7) being the most prevalent. Six novel STs were also identified and submitted for the assignment of new sequence types (ST-15634-115641). Seven clusters of isolates with distinct AMR genotypes were identified after the cgMLST analysis, highlighting the presence of genome wide genetic variation. All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A total of (49)87.5% and (13)23% isolates contained fluoroquinolone and macrolide resistance markers respectively. Plasmids were highly prevalent: pTetM and pBlaTEM were found in 96%, and 92% of isolates, respectively. All isolates possessed the PI (B) variant of the porB gene which is associated with localized infection while high antigenic variations in the pillin genes was also detected. The study highlighted the need for constant genomic surveillance with the looming possible emergence of cephalosporin resistant isolates and isolates with highly variable antigens which could severely impact disease treatment.Item In Vitro Studies Of The Effect Of Anopheles Gambiae Midgut Bacteria On The Development Of Plasmodium Falciparum(University Of Ghana, 2021-12) Ametsi, Williams GodwinDuring blood feeding, female Anopheles mosquitoes may ingest Plasmodium gametocytes which undergo transformation in the gut and develop into sporozoites that are infectious to humans. Bacteria inhabit the mosquito gut, and the number and diversity of these bacteria change following blood feeding. The presence of some bacteria species results in the reduced intensity of developing Plasmodium parasites. Little attention has been given to understanding this direct mechanism of bacteria on Plasmodium parasites, and the effects of bacteria on malaria parasite developmental genes are not completely understood. This limits the scope of how gut bacteria, for example Enterobacter and Serratia, which have been found with anti-Plasmodium effects can be further explored for alternative disease control strategies. Therefore, this study investigated the lethal effect of cell-free secreted bio-products of E. cloacae and S. marcescens on a key Plasmodium parasite developmental gene (Gamete release gene, GAMER) for its potential as a target for malaria transmission-blocking. Plasmodium falciparum 3D7 and Dd2 cultures at 1% parasitaemia were independently exposed to spent Luria-Bertani (LB) medium from varying concentrations of Enterobacter cloacae and Serratia marcescens. The parasite killing effect of the bacteria were assessed with SYBR green fluorescent assay after 48 hours of co-culture. Spent media with final bacteria concentration between 10e+10-10e+20 reduced parasitaemia (P<0.001) compared to parasite culture without bacteria treatment. Using real-time (quantitative) PCR, it was found that the expression of GAMER was down regulated by 2 folds after 1 hour of screening P. falciparum 3D7 with cell-free spent medium of E. cloacae cultured for 8 hours in LB broth (Ec-8). However, the expression of GAMER was unaffected after 6 and 12 hours of screening P. falciparum 3D7 with Ec-8. These data provide information for further studies on gene and protein targets for transmission blocking interventions.Item Selection Dynamics Of Circumsporozoite Protein (Csp) Vaccine Target In Ghana: The Contribution Of Human Leukocyte Antigen (Hla) Variation(University Of Ghana, 2021-07) Kpaka, J.N.Implementation of RTS,S/AS01 vaccine for malaria is underway in three (3) African countries, Ghana, Kenya, and Malawi. This vaccine, which targets the Plasmodium falciparum circumsporozoite protein (CSP) provides partial protection for infants and children against clinical and severe malaria infections. Reasons for this reduced efficacy or immunogenicity are poorly understood, but CSP variation has been implicated. Human leukocyte antigen (HLA) has also been observed to influence RTS,S-mediated protection. This study aims to define the variants of CSP and determine its distribution between Begoro and Cape Coast in Ghana over three years. Further, the influence of HLA genotype in terms of parasite frequency and RTS,S/AS01 response was assessed. About 50μl of peripheral blood was collected from participants in Begoro and Cape Coast in 2014, 2015, and 2016, dried blood spot (DBS) prepared and DNA was extracted. The C-terminal of Plasmodium falciparum CSP and the human leukocyte antigen (HLA) class II gene in humans were deep sequenced. The translated amino acid haplotypes of the CSP were aligned and compared to the reference 3D7 vaccine strain. The HLA class II haplotypes were grouped into superfamily and their association with the CSP variants was ascertained. The CSP haplotypes are evenly distributed between Begoro and Cape Coast. There were 31 Th2R haplotypes in Begoro and 30 Th2R haplotypes in Cape Coast; 15 Th3R haplotypes in Begoro and 13 in Cape Coast. About 83.9% of Th2R and 96.5% of Th3R haplotypes in Begoro are shared with Cape Coast. The amino acid changes with reference to the 3D7 vaccine strain at the Th2R epitope range from 1 to 6 and 1 to 4 at theTh3R epitope. There is a 53% and 60% reduction in the 3D7 Th2R and Th3R haplotypes, respectively, from 2014 to 2016, but 3D7 is still common in Ghana, Kenya, and Malawi. The 3D7 haplotype does not correlate with HLA-DRB1, but there is with HLA-DQA1 and HLA-DPB1. Begoro and Cape Coast are two different ecological zones in Ghana but the parasite population is homogenous. The Th2R epitope of CSP is polymorphic than the Th3R epitope and this higher polymorphism is driving a higher non-synonymous amino acid substitution at the Th2R epitope than the Th3R epitope which may have vaccine implication. A decline in frequency of 3D7 parasite population may also affect the performance in the vaccine in Begoro and Cape Coast. Initial correlations indicate that HLA-DPB1 (01:01/17:01) correlates with the 3D7 vaccine strain, but HLA-DPB1 (01:01/17:01) and other variants of HLA-DQA1 also correlates with other Th2R haplotypes and may compete with the vaccine haplotype for antigen presentation to CD+4 T cells. This may have implications for the efficacy of the RTS,S/AS01 vaccine in Ghana.Item Immunological Characterization Of A Novel Merozoite Surface Protein (PFMSP11)(University Of Ghana, 2020-12) Asiedu, K.Background: Plasmodium falciparum merozoite surface protein 11 (PfMSP11) is a novel surface protein that plays a critical role during the parasite’s blood stage development. This project aimed at immunologically characterizing the Plasmodium falciparum Merozoite Surface protein (PfMSP11). Method: Antibody (IgG) responses from 283 child clinical malaria samples Accra, Kintampo and Navrongo which represents different transmission intensities, were measured against the 20 synthetic peptides of PfMSP1 using ELISA. Overall 27 synthetic peptides were tested against PBMCs from 10 apparently healthy subjects to measure IFN-γ responses using ELISpot. Results: Peptides at the C-terminus of PfMSP11 gave higher antibody responses than peptides at the N-terminus. Peptide 2 to Peptide 12, and Peptide 17 recorded low antibody responses across all sites. Peptide 16 recorded the highest antibody responses across all sites. One subject (M14) showed a highest response across all pools in measuring IFN-γ responses . Subject M9 tested positive for pools 1 and 3 while subject M10 and M12 tested positive for pool 2Peptide 7 in pool 2 against subject M10 was the only peptide that tested positive. In conclusion, this study has shown that there are more antibody epitopes at the C-terminus than the N-terminus and more immunogenic T cell epitopes at the N-terminus.Item Emergence Of HIV-1 Drug Resistance Mutations in Patients After 12 Months of Antiretroviral Therapy(University Of Ghana, 2021-07) Asamoah, B.The United Nations Program on HIV and AIDS (UNAIDS) in 2013 set an ambitious target of 90-90-90, thus 90% of all people living with HIV must have been diagnosed, 90% of those diagnosed put on treatment and 90% of the patients on treatment must achieve viral suppression. The commonest cause of therapeutic failure in HIV patients is the presence of drug resistance mutations. This leads to viral rebound, reduction in CD4 count, and predisposes the patients to opportunistic infections. In addition, chances of transmission increase as a result of increased viral load. Occasionally, viruses carrying drug resistant mutations are transmitted from one person to the other. This study sought to investigate the emergence of HIV drug resistance mutations in protease and reverse transcriptase genes, twelve months after starting antiretroviral treatment.Item Membrane Vesicles Of Mycobacterium Ulcerans And Their Role In Buruli Ulcer Pathogenesis(University Of Ghana, 2021-01) Osei, E.N.The release of bacterial extracellular membrane vesicles (EMVs) is essential for pathogen’s adaptation and virulence. Mycobacterium ulcerans, the causative agent of Buruli ulcer, remains with queries in its pathogenic mechanism. The current study interrogated biological functions and protein content of EMVs from viable M. ulcerans’ cells as potential medium of virulence in BU pathogenesis. Here, we demonstrate release of intact EMVs from the thick cell wall of log-phase M. ulcerans (Nm 209) as well as M. marinum (Sa 200695) in respective liquid cultures. Size distributions of isolated EMVs were similar between the two strains and did not differ from EMVs released by M. smegmatis used as a positive controlled strain. Mycolactone could not be detected in isolated EMVs from M. ulcerans (Nm 209). However, presence of M. ulcerans EMVs was associated with higher total intracellular reactive oxygen species which eventually compromised viability of RAW264.7 cells through oxidative stress. After 48 hours of co-incubation, native and UV-A irradiated EMVs induced 45% and 40% loss in viability of RAW264.7 cells, respectively. Moribund phagocytes exhibited apoptotic changes. Proteomic analysis on the isolated M.ulcerans EMVs revealed an enrichment of 32 unique proteins mostly localized in the pathogen’s cell wall/membrane. A conserved hypothetical protein (MUL_2313), had the highest log2 fold change (11.92) followed by Amidase amiC, a cell-wall remodeling hydrolase (4.19). Others included integral membrane indolylacetylinositol arabinosyltransferase EmbA/B, and many conserved hypothetical proteins. Direct contributions of these proteins to EMVs cytotoxicity could not be established. Yet, protein moon-lighting or possible cross-linking could have potentially contributed to EMV-associated toxicity on RAW264.7 cells. Our results suggest that M. ulcerans EMVs can elicit toxic response from host’s macrophage cells through yet to be established mediators. This potentially reveals new dimension on macrophage-M. ulcerans interactions with possible contribution to local immunosuppression in BU and paradoxical reactions observed in its treatment.Item Investigation Of The Electrochemical Properties of Hydroxyapatite Immobilization Material for Potential Cytosensor Fabrication(University of Ghana, 2020-10) Adusei, D.Biomedical diagnostics is moving towards an effective and rapid diagnosis of which biosensor fabrication is the paradigm shift. Despite the successes obtained using sensing platforms, detachment of biorecognition elements from the transducing surface remains a hurdle to overcome. Good attachment of biorecognition elements to the transducing surface determines the sensitivity and specificity of the biosensor. In the area of cancer biosensing, gold, graphene, chitosan, and conducting polymers are among the few materials that have been exploited for effective immobilization, but they faced detachment problems. To curb these detachment problems, blends of cancer immobilizing materials and other molecules have been proposed but fabrication methods make the immobilizing material expensive. Thus, this thesis aimed at investigating the use of cost-effective hydroxyapatite (HAp) material synthesized from Achatina achatina snail shells (SHAp) for the direct immobilization of cells. SHAp was mixed with 3,4-ethylene dioxythiophene: poly 4- styrene sulfonate (PEDOT: PSS), a conductive polymer to increase the electrochemical responses of the SHAp forming a SHAp/PEDOT: PSS blend. The SHAp/PEDOT: PSS blend was used to modify a screen-printed electrode (SPCE) by a dropped coating approach after which cell-lines including pheochromocytoma (PC-12), human embryonic and kidney cells (HEK-293T) immobilized on the modified SPCE. Red blood cells (RBC) were used as a control. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed to record the cell proliferation signals. The CV results showed low peak currents for cell-lines (50 µA for HEK-293T and 120 µA for PC-12) and high peak current for the control RBC (230 µA). The EIS showed impedance values of 0.70 and 0.62 mΏ for HEK-293T and PC-12, respectively, and 0.52 mΏ for RBC. The findings demonstrate that SHAp is able to differentiate the proliferation signals of cells through potentiometric and impedimetric measurements. The unique current difference among these cells could be used as potential markers for the rapid detection of cancer cells at a low cost in future studies.Item Anti-Diabetic And Probiotic Effect Of Kombucha On Alloxan-Induced Diabetic Rats(2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. Kombucha was found to perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Development and Quality Evaluation of Pre-Gelatinized Instant Weaning Foods Based on Cereals and Legumes(University of Ghana, 1991-07) Abotsi, S.K.Item Development of Precooked Foods Process and Product Evaluation(University of Ghana, 1983-06) Masopeh, E.A pre-cooked food has developed from cornmeal and cowpead flour . The cowpea seeds were germinated and also dehulled. Control seeds of these treatments were made. Method used in the preparation of the product was steaming and traditional roasting in the earthen- ware mashing boul. Chemical analysis on the products Showed that the product developed had a higher protein content than cornmeal . Evaluation of the functional characteristics revealed that the water absorbed by the products increased with an increase in the number of germination days . The undehulled products also absorbed water and swelled more than the dehulled products. This ws attributed to the presence of the seed coat in the undehulled sead products. There was not much difference in the water absorption and swelling properties with respect to the levels of corn and cowpea. Viscoamylograph runs showed no change in the gelatinisation of the germinated seed products , however there was a little increase in the viscosity of the ungerminated seed product during the holding and cooling sections. Sensory evaluation of the corn-cowpea product showed that the product was acceptable. Anaalysis of variance calculations showed a significant (p~ 0.01) effect of dehulling and cersination on the colour, odor, flavor and tho beneral acceptability of the nroducts The panelists could detect differences in the different products subjected to the different treatments. A second product similar to the corn-cowpea product was developed from corndough. Fermented and unfermented corndough were used. Functional characteristics analysis showed a high water absorption and swelling of unfermented corndough products. Analysis on the product Showed no significant difference in the colour, odor etc . of the fermented and unfermented corndough products . However, the unfermented corndough product was more acceptable.