Department of Biochemistry and Microbiology

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Browsing Department of Biochemistry and Microbiology by Subject "579.3149"

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    Molecular characterization of entamoeba species and impact of host genetic polymorphisms on amoebiasis
    (2022-11-10) Davhana, Ndivhudzannyi Caroline; Samie, A.; Bessong, P. O.; ElBakri, A. M. K.
    Background: Enteric infections constitute a serious public health problem globally, especially in low and middle-income countries, particularly in areas of poor sanitation, low socio-economic conditions, inadequate water supply and poor hygiene practices. South Africa is one of the developing countries that has been significantly impacted by diarrheal infections, many of which are due to Entamoeba species. It is still not clear why some individuals once infected with E. histolytica develop clinical amoebiasis while others do not show any symptoms. It has been suggested that both the parasite and host factors play a significant role in the outcome of E. histolytica infection. However, this does not explain why only few infected individuals develop symptomatic diseases. This suggests that there are other factors to explain this transition. Tumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammation cytokine which has been considered as one of the pathogenic factors for various diseases. Several studies have reported an association between TNF-α polymorphisms and inflammatory diseases. However, no study has been carried out on the association between polymorphisms in the promoter of the TNF-α gene and high risk of E. histolytica infections. Aim and objective of the study: The overall aim of the study was to determine the molecular characteristics of Entamoeba species in relation to the occurrence of diarrhea among children and determine the impact of host genetic polymorphism on the occurrence of amoebiasis. This aim was addressed by the following primary objectives: a. To investigate the prevalence, distribution and genetic diversity of Entamoeba species and other parasites circulating among children in the Northern part of South Africa. b. To investigate the genetic polymorphism of TNF-α gene promoter region in a cohort of children in Northern South Africa. c. To identify any association between TNF-α promoter gene polymorphism with diarrhea, vomiting, fever, acute lower respiratory infection, gender and malnutrition. d. To identify any potential association that may exist between TNF-α promoter gene polymorphism and parasitic infections. Brief methodology and results: This study was nested within the Madi project and Malnutrition and Enteric Diseases project (MAL-ED) South Africa and received approval from the Health, Safety and Research Ethics Committee of the University of Venda. The stool samples were from Madi project and the blood samples were from the MAL-ED project. A total of 394 stool samples were collected in selected household with children under the age of 5 years who were randomized to receive a silver-impregnated ceramic water, a silver-impregnated ceramic disk, a safe-storage water container, or no intervention, and were followed quarterly for two years from Dzimauli rural communities of South Africa in Limpopo province. All the stool samples were observed under a light microscope for the presence of intestinal parasites. In order to accurately detect Entamoeba species in all faecal samples, polymerase chain reaction (PCR) protocols were performed using genus-specific PCR primers based on small-subunit rRNA gene sequences for E. polecki, E. chattoni, E. dispar, E. histolytica, E. hartmanni and E. coli. Real-time PCR protocol was also used to detect and identify the specific Entamoeba species such as E. histolytica, E. dispar, E. bangladeshi and E. moshkovskii in order to gain information on their accurate prevalence, distribution and genetic diversity in the community. The present study reported an overall prevalence of intestinal parasites including Entamoeba species, Strongyloides stercoralis, Cystoisospora belli and Ascaris lumbricoides as determined by microscopy to be 140 (35.5%), 138 (35.0%), 114 (28.9%) and 78 (19.8%), respectively. The genus-specific PCR was positive for Entamoeba spp. in 140 (35.5%) samples. Real time PCR detected E. histolytica in about 4% of the samples while E. moshkovskii occurred in about 9% of the samples. Results identified by qPCR showed that children in silver-impregnated ceramic water filter group at month 12 had higher E. moshkovskii infection 6 (13.0%), while those in intervention group had lower E. moshkovskii infection 3 (6.8%). None of the participants were infected with Entamoeba bangladeshi. Sequence analysis showed a wide variety of Entamoeba species with E. coli and E. polecki appearing to be the most common organisms followed by E. moshkovskii and E. dispar. Further studies using next generation sequencing technologies are needed to understand the real importance of each of these organisms in the community in terms of the pathogenesis of amoebiasis (Chapter 3). TNF-α is a multi-functional pro-inflammatory cytokine and a primary mediator involved in the early phase of the cytokine cascade and plays an important role in the initiation and regulation of the immune response. The present study aimed to investigate the genetic polymorphism of tumor necrosis factor-α (TNF-α) gene promoter region in a cohort of children in Northern South Africa. A total of 199 blood samples were evaluated from children who were part of the MAL-ED study cohort. The TNF-α gene at positions ‑1031T/C and ‑308G/A were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay and confirmed by DNA sequencing. Out of these 199 participants, 94(47.2%) were males and 105(52.8%) were females. Of all the children, 23(11.6%) had low birth weight. A strong association was noted between the CC homozygous genotype at position -1031 and children with diarrhea (P=0.043, OR=4.167, 95% CT=0.942-18.43); whereas TC heterozygous genotype was significantly common in healthy children with no diarrhea (P=0.019, OR=0.446, 95% CT=0.226- 0.882). The T-allele was significantly common in children with diarrhea (P=0.043, OR=0.240, 95% CT=0.054-1.062). A strong association was also noted between the TT homozygous genotype at position -1031 and children with dehydration (P=0.014, 95% CI= 1.224-12.443). A strong association was noted between the GA heterozygous genotype at position -308 and children with diarrhea (P=0.040, OR=2.579, 95% CT=1.019-6.528); while AA homozygous genotype was significantly common in healthy children with no diarrhea (P=0.012, OR=0.3420, 95% CT=143-0.815). Heterozygous GA genotype was more common among healthy children with no dehydration and the result was statistically insignificant (P = 0.894, X2 = 0.018, OR = 1.095, 95% CT = 0.288-4.168); while a strong association was also noted between the heterozygous GA genotype at position -308 and children with vomiting (P=0.019, OR=2.694, 95% CT=1.160-6.256). The G allele was significantly common in children with vomiting (P=0.010, OR=2.816, 95% CT=1.263-6.279). Our study has for the first time revealed that the -1031(T/C) polymorphism of TNF-α promotor gene is associated with diarrhea, dehydration and acute lower respiratory infection among children at five years of age, while the -308(G/A) polymorphism was associated with diarrhea and vomiting among these children (Chapter 4). Intestinal parasitic diseases are common in developing countries including South Africa and have been documented to be the most common in children under the age of five. The present study aimed to identify any potential association that may exist between TNF-α promoter gene polymorphism and parasitic infections. A total of 199 blood samples was evaluated from children who were part of the MAL-ED study cohort. The DNA was used to investigate polymorphism in the promoter region of the TNF-α gene at position 1031T/C. The polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. The TC genotype at position -1031 was significantly higher in healthy controls children than in children who were infected with Entamoeba species (59.9% vs 29.4%, P = 0.015) and Entamoeba coli (59.1% vs 30.8%, P = 0.046), indicating that TC genotype may be protective against Entamoeba infections and Entamoeba coli infections. The CC genotype at position -1031 was more common among children with parasite and diarrhea and the results was statistically significant (P = 0.04). This study has revealed that the CC genotype may be is a risk factor for symptomatic parasitic infections, while the TC genotype might be protective of Entamoeba infections among children in Dzimauli community (Chapter 5). Overall conclusion: The present study demonstrated new data on the prevalence, distribution and genetic diversity of Entamoeba species and other parasites in Northern South Africa. Several Entamoeba species are circulating in the region and the importance of E. moshkovskii in the pathogenesis of amoebiasis seems to be underestimated. This study also revealed for the first time that the -1031(T/C) polymorphism of TNF-α promotor gene is associated with diarrhea and acute lower respiratory infection, Entamoeba species and Cyclospora infections among the children in Dzimauli community, while - 308(G/A) was associated with vomiting and overall illness. Information generated from this study will be useful for understanding the transmission, source of infection and clinical outcome of infection with parasitic infections. However, larger studies need to be conducted in order to confirm these findings.
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    Molecular characterization of microbial communities in small-scale biodigesters using whole genome shotgun metagenomic approach
    (2025-09-05) Matamela, Tendani; Mathomu, L. M.; Matambo, T.
    Increasing attention has been directed towards the role of microbial communities in anaerobic digestion (AD). During AD, microorganisms degrade organic waste, such as animal excrement, through four key phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis, resulting in digestate and biogas production. Advances in metagenomic techniques, surpassing traditional culture-based methods, have enabled a deeper characterization of the microbial populations involved in this process. However, a knowledge gap remains regarding small-scale biodigesters, particularly those in rural areas. This study employs whole-genome shotgun metagenomics integrating data from metagenomic reads, contigs, and metagenome-assembled genomes (MAGs) can achieve a more comprehensive understanding of microbial taxonomy and functional capabilities in biodigesters. This study investigates the microbial communities in small-scale biodigesters, using taxonomic data from various sources to reveal the functional roles of the microorganisms. The research identified 4 superkingdoms, 115 phyla, 107 classes, 189 orders, 322 families, 738 genera, and 2046 species. Bacteria dominated ranging from 79 to 89%, whereas archaea accounted for 11 to 20% of the community. The eukaryotic microbial’s relative abundance was less than 1%. Both read-based and contig-based classifiers found Proteobacteria, Firmicutes, and Bacteroidetes to be the most abundant bacterial phyla, followed by Actinobacteria and Chloroflexi in cow dung-fed biodigesters. The archaeal community was dominated by Euryarchaeota, with Methanomicrobiales and Methanobacteriales as the predominant orders. Functional analysis reveals that most identified microorganisms contribute indirectly to methanogenesis by playing crucial roles in hydrolysis and acidogenesis through genes involved in the metabolism of organic molecules broken down during the four phases of anaerobic digestion. RAST analysis annotated 11 methanogen-related enzymes and their abundance across different samples. Future research should focus on cultivating and monitoring these identified microbes to determine optimal conditions for maximizing biogas production in laboratory settings, thus advancing environmental bioaugmentation strategies.