Department of Biochemistry, Cell and Molecular Biology

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    Genetic Diversity of Mycolactone Producing Mycobacteria Causing Buruli Ulcer in Ghana and Côte D’ivoire
    (University of Ghana, 2019-07) Dogbe, M.
    Buruli ulcer remains a neglected tropical disease endemic in Africa especially Ghana and Côte d’Ivoire. It is a necrotizing skin and soft tissue infection caused by Mycobacterium ulcerans which produces mycolactone which causes adverse effects associated with disease in humans. Other mycolactone producing bacteria (MPMs) have been identified to cause granulomas in fish and frog. There are limited advanced genetic tools for studying transmission of the diseases and for carrying out molecular epidemiological studies. The mycolactone producing mycobacteria (MPM), M. shinshuense has been reported to cause Buruli ulcer and other studies have revealed several MPM infections other than M. ulcerans in clinical samples. It is therefore imperative to know the exact MPMs that are in circulation by genotypically distinguishing them using molecular tools. Patients with Buruli ulcer-like disease presentation were recruited from endemic communities in Ghana and Côte d’Ivoire. Swabs and FNA samples were screened using primers detecting the insertion sequence 2404 gene and the Enoyl reductase gene. Genotyping was achieved at 16 MU VNTR loci and length polymorphisms arising from differences in tandem repeats for samples were validated using six (6) controls and sequencing. A total one hundred and fifty-nine (159) of the 189 samples were confirmed as Buruli ulcer positive. Genotyping was successful for all controls (100%) and most samples (69%) at all sixteen (16) VNTR loci. Seven (7) MU genotypes designated, A, B, C, D, E, F and G and five MPM genotypes MLF (Mycobacterium liflandii), MHB (Mycobacterium marinum hybrid), MDL (Mycobacterium marinum DL), MCL (Mycobacterium marinum CL) and MSA (Mycobacterium marinum SA) were generated samples were assigned genotypes based on their VNTR profiles. Genotypes C, D and F were present in both Ghana and Côte d’Ivoire. However, genotypes E and MLF were only found in Ghana whiles Genotype A and G were found in only Côte d’Ivoire Genotype D has persisted over the years from 2008 to 2019 comparing this study to published data. These findings support the hypothesis that Mycolactone producing mycobacteria causing Buruli ulcer in Ghana and Côte d’Ivoire are diverse and affirms VNTR typing as a comparably useful tool for differentiating MU strains as well as other MPMs in Buruli ulcer endemic communities.
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    Discovery of Buruli Ulcer Infection Associated Metabolic Markers
    (University of Ghana, 2017-07) Laryea-Akrong, E.K.
    Buruli ulcer (BU), is a severe slow progressing necrotizing skin disease, caused by the environmental pathogen Mycobacterium ulcerans (MU). The diagnosis of the disease can be carried out in four main ways. These include microscopy which is preceded by Ziehl-Neelsen stain, PCR amplification of the IS2404 insertion sequence, histopathology and culture. The limitations of BU diagnosis include microscopy which is not sensitive and specific, culture which takes 8-12 weeks for a growth to be observed and PCR which requires skilled labour and is very expensive. Early diagnostic techniques are urgently needed for this disease at areas and communities where they are most endemic since the disease can be treated if it is diagnosed early. The aim of this study was to discover Buruli ulcer infection associated metabolic markers using a metabolomics approach. Wound swabs from 47 participants were taken and BU cases were confirmed with microscopy using Ziel-Neelson staining and PCR. Metabolites were extracted from the wound swabs with chloroform, methanol and water and derivatized for Gas Chromatography-Mass Spectrometry to determine the different metabolites of patients with or without the infection. After DNA extraction and PCR 65% of the cases were found to be positive for mycobacterial DNA, 34% of the cases were also found to be positive for M. ulcerans and 8.5% were found to be positive for H. ducreyi. Seventeen metabolites were identified, and palmitate was found to be significantly higher when the cases were compared to the controls. Palmitate and stearate were found to show significance between the cases that were on treatment and those that were not on treatment. For all cases that were positive for mycobacteria they had significantly higher levels of pentadecanoate and heptadecanoate. Finally, cases positive for H. ducreyi and had higher levels of cervonate (docosahexaenoic acid) and palmitate. Unique metabolites specific to the BU cases were found and these can be confirmed as biomarkers and utilized in the development of a possible diagnostic for this Neglected Tropical Disease.
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    Genotyping and Treatment of Secondary Bacterial Infections among Buruli Ulcer Patients in the Amansie Central District of Ghana
    (2016-09-20) Gyamfi, E.; Mosi, L; Dzudzor, B; University Of Ghana, Department of Biochemistry, Cell and Molecular Biology, School of Biological Sciences College of Basic and Applied Sciences
    Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. BU is the third most common mycobacterial disease after tuberculosis and leprosy, but in Ghana and Cote d’ Ivoire, it is the second. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin which makes the infection painless. However, some patients have complained of painful lesions and delay healing. Painful ulcers and delay healing experienced by some patients may be due to secondary bacterial infections. Main Objective: To identify secondary microbial infections of BU patients, their genetic diversity as well as determine the levels of antibiotics resistance of these microorganisms. Methodology: The study was conducted at Biochemistry, Cell and Molecular Biology, University of Ghana. Subjects were recruited from Amansie Central District, Ashanti Region. Swabs of 51 BU patients were taken and immediately frozen for transport into the laboratory. Microscopy was performed using Ziehl-Neelsen and Gram staining techniques. The samples were also cultured on Luria Bertani, MacConkey, Mannitol, BPA and Sabouraud dextrose agar to identify bacteria and fungi. Antibiotic susceptibility tests were performed on selected bacteria species. DNA was extracted from the samples, after which Polymerase Chain Reaction (PCR) was performed using universal (16S rRNA), MSHA/PA (16S rRNA for mycobacterial) and IS2404 (insertion sequence specific to mycolactone producers) primers to find the different strains of organisms. Finally sequencing was performed on the DNA amplicons that were randomly selected to identify the kinds of microorganisms causing secondary infection. Results: All the samples were positive for bacteria. However 49 and 40 positives were obtained from PCR products using the primers MSHA/PA, and IS2404 respectively, thus 40 BU patients were identified out of the total 51 patient samples. Majority of the bacteria identified after sequencing with universal primers for bacteria were Staphylococcus spp (aureus including MRSA, saprophyticus, and lentus), Alcaligene spp (aquatilis and faecalis), Pseudomonas spp (aeruginosa, stutzeri and koreensis) and bacilli cereus group of bacteria. Interestingly, 60% of the sequencing result for mycobacteria detected the presence of Corynebacterium spp (aurimucosum, diphtheria and striatum). Other bacteria identified were Brevibacterium iodinum and Rhodococcus erythropolis. Majority of these bacteria live in muddy areas and dirty water. The selected bacteria were less susceptible to rifampicin, clarithromycin and amikacin. Conclusion: Other bacteria beside M. ulcerans colonize and proliferate on BU lesions. The selected bacteria were less susceptible to clarithromycin and amikacin and rifampicin. The pains and healing delay experienced by some BU patients could be the result of these bacteria colonizing and proliferating on the ulcer or lesions.