Theses and Dissertations

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Determination of the impact of Antiretroviral therapy in the proportion and genetic diversity of diarrheal associated gut microbiota among HIV infected population
(2023-10-05) Musetsho, Phumudzo Pertunia; Mavhandu - Ramarumo, L. G.; Bessong, P.O.
Background: Human gut microbiota are microorganisms that reside in the gastrointestinal tract of humans. Gut microbiota provide various functions in the gut including protection from invasion of pathogenic microbes, shaping host immunity and substrate metabolism. HIV which targets the CD4+ T cells in the gut associated lymphoid tissue (GALT), causes gut microbiota alteration due to disruption of the gut barrier. This result in microbial translocation and pathogen invasion which lead to decreased diversity and proportion of gut microbial and increase in pathobionts and pathogens. As a result, HIV infected patients suffer from diarrhoea due to compromised gut microbiota. Antiretroviral therapy (ART) suppresses viral replication and lead to an undetectable viral load, thus restoring the immune system. Previous studies show that ART does not entirely restore the depleted gut microbiota, hence this study aims at determining the change in proportion and genetic diversity of diarrheal associated gut microbiota in HIV infected patients. Hypothesis: Antiretroviral therapy causes changes in the proportion and diversity of diarrheal causing gut microbiota Objective: The objective of the study was to determine the impact of ART in the proportion and genetic diversity of gut microbiota among HIV infected patients. Methodology: Paired blood and stool samples were collected from 17 HIV infected and 11 HIV uninfected individuals (controls). Samples were collected during baseline (before ART initiation); and samples were collected after every three months thereafter, for a total of 12 months. Blood samples were used for CD4 and viral load measurements using BD FACSPresto machine (BD Biosciences) and HIV Qualitative PCR, respectively. Stool samples were used for extraction of total nucleic acid (TNA) using a modified Qiagen QIAamp Fast DNA Stool Mini Kit. Purification of TNA was done using Ampure XP bead. DNA library preparation kit (Illumina) was used for DNA library preparation. Illumina miniseq sequencing was used for sequencing and the obtained sequence reads were analysed using Geneious prime software for trimming and filtering low sequence reads and to determine the genetic diversity of diarrheal causing gut microorganisms. DRAGEN metagenomics was used for taxonomical classification to generate the proportion of diarrheal causing gut microorganism. GraphPad prism was used to generate graphs and for statistical analysis to generate the association between CD4+ T Cell/viral load and diarrheal causing gut microbiota. Results: Escherichia coli (82.33%), Bacteroides fragilis(2.46%), Shigella spp.(1.96%), Salmonella enterica (1.07%), Clostridioides difficile(0.99%), Campylobacter jejuni (0.20%) were the most prevalent detected microoganisms from HIV infected individuals at prior ART initiation. Among HIV negative individuals, Escherichia coli (62.52%), Bacteroides fragilis(18.43%), Shigella spp.(0.40%), Salmonella enterica (3.05%), Clostridioides difficile(15.06%), Campylobacter jejuni (0.54%) were found. The proportion of Escherichia coli (90.77%), Clostridioides difficile (2.20%), Shigella spp. (2.73%),and Salmonella enterica (1.28%) had increased with treatment at three months post treatment in HIV infected individuals. At six months post ART, there was an increase in the proportion of Bacteroides fragilis (88.65%) and Vibrio cholera (0.01%) when compared to HIV positive samples at three months post-treatment. Escherichia coli (9.74%), Cryptosporidium parvum (1.74%), and shigella spp. (0.35%) increased with treatment from six months to nine months post-treatment. Escherichia coli (58.58%), Shigella spp. (0.81%) and Clostridioides difficile (24.77%) had increased with ART at twelve months post treatment. The proportion of diarrheal causing gut microbiota were fluctuating throughout the intake of ART at different time points. There was no significant correlation between CD4/viral load and diarrheal causing microorganisms, as a result ART did not have an effect in the proportion of diarrheal causing gut microorganisms. Genetic diversity of diarrheal causing gut microbiota was higher in HIV positive individuals prior ART than in HIV negative individuals except for Salmonella typhi. Among HIV infected individuals, genetic diversity of most diarrheal causing gut microbiota was higher at three months post treatment. The fluctuation of diarrheal causing gut microbiota’s genetic diversity from six months to twelve months post treatment may be due to inconsistent change in number of viral loads and CD4 counts at different time points of ART. However, this was statistically insignificant. As a result, the change in the genetic diversity of diarrheal causing gut microorganisms was not due to ART. Conclusion: ART does not eradicate diarrheal causing gut microbiota. However, ART encourages a low genetic diversity of diarrheal causing gut microbiota.
Embargo
Investigating the therapeutic effects of silver and gold nano particles capped with selected medicinal plants against tuberculosis
(2023-10-05) Motene, Matshoene Violet; Traore, A. N.; Potgieter, N.; Mavumengwane, V.
Background: Tuberculosis (TB) is implausibly still considered as one of the leading causes of death, in the 21st century. Despite the curative treatments and measures of control in the communities, TB plays a significant role in human infectious disease. Studies have shown that there is an emergence of major drug-resistant TB. Aim: This study was aimed at evaluating the biological activities of silver and gold nanoparticles capped with selected medicinal plant extracts against Mycobacterium tuberculosis. Methodology: P. africanum and Z. mucronata barks and leaves were collected in the Vhembe district of Limpopo. Plant phytochemical constituents were extracted using distilled water and absolute methanol. The silver nanoparticles (AgNP) and gold nanoparticles (AuNP) were capped using crude extracts through the process of green synthesis; and were characterized by Ultraviolet-Visible spectrophotometry (UV-VIS), and transmission electron microscopy (TEM). Metabolites present in the plant extracts were profiled using liquid chromatography mass spectrometry (LC-MS). Cytotoxicity activity of plant extracts and nanoparticles were determined by MTS assay on HEK293 cells. The anti-inflammatory activity was determined through the nitric acid synthase (NOS) inhibitory test. The antimycobacterial activity was determined using microbroth dilution. Results: Following extraction by maceration, methanol was able to yield more extracts compared to distilled water due to their differences in polarity. The selected plants were found to contain numerous antioxidant significant for anti-inflammatory and cytotoxic activity. The metabolites play a role in the formation of the nanoparticles. Plant extracts and gold nanoparticles had little impact on the cell lines, thus were concluded to be non-toxic. Whilst silver nanoparticles exhibited toxic activity on the cell line at both concentrations, hence were considered toxic to human. Silver nanoparticles of ZML and ZHL, and gold nanoparticles and plant extracts of PML exhibited anti-inflammatory activity at 100 μg/mL, whereas PML was able to decrease nitrite concentration at both concentrations. Conclusion: Adoption of TB strategies recommended by World Health Organization (WHO) to reduce TB deaths and incidence rate by 90% and 80%, respectively, (less than 20 TB cases per 100 000 population).
Embargo
Development of Liquid Chromatography Mass Spectrometry technique for evaluation and differentiation of plant derived isomers
(2023-10-05) Mbedzi, Dakalo Terrence; Madala, N. E.; Mathomu,L. M.; Mhlongo, M. I.
Cinnamic acid containing molecules have been shown to possess unprecedented pharmacological activities against various physiological disorders. Most of the plants containing these compounds have been shown to exhibit multiple bioactivities, notably against various diseases which are currently without a cure, for example human immunodeficiency virus (HIV) associated ailments. As in other plants, most of the pharmacologically relevant metabolites are produced in minute concentrations, and, as such, alternative means to produce high levels of these compounds are imperative. Herein, UV-induced photochemical conversion of cinnamic acid containing molecules is presented as a feasible means to diversify the number of bioactive metabolites in plant extracts. Naturally, UV rays from sunlight were shown to result in the formation of geometrical isomers. The presence of these isomers has resulted in analytical challenge, which has resulted in erroneous identification and isolation of an active isomer. Virtual screening and docking studies provide insights into the potential interactions between molecules and their targets, and can be useful in predicting the biological activity of compounds. This study further shown that synergistic isomerism could be beneficial during inflammation conditions, and it suggests that certain isomers of a compound may work together to enhance the desired therapeutic effect. Herein, liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) method capable of distinguishing between these isomers was developed.
Embargo
Assessment of knowledge, attitude and practices of small household farmers towards heartwater disease and molecular characterization of Ehrlichia ruminantium in Limpopo province, South Africa
(2023-10-05) Mathebula, Doris; Samie, A.; Sigidi, M. T.
Background: Heartwater is a disease spread by ticks that is brought on by the obligatory intracellular bacterium Ehrlichia ruminantium. Heartwater disease is one of the major obstacles to livestock production as it affects many livestock animals including domesticated animals such as goats, cattle, and sheep as well as wild ruminants. In Southern Africa, it is spread by Amblyomma hebraeum ticks, and in the rest of Sub-Saharan Africa, it is spread by A. variegatum ticks. In this study, epidemiological and molecular features of Ehrlichia ruminantium in Mopani and Vhembe Districts, from ticks isolated from cattle, goats and sheep were investigated. Method: A total of 121 small household farmers from different villages in the Vhembe and Mopani Districts were recruited in the study after they have signed an informed consent. The participants were interviewed using a questionnaire to collect data related to their knowledge, attitude and practices towards heartwater disease. Ticks were collected from cattle, goats, and sheep belonging to 48 households, yielding a total of 244 ticks. Genomic Deoxyribonucleic acid was extracted from the ticks and analysed using real-time qPCR assay targeting a 226bp fragment of the PCS20 gene. Finally, a number of samples were sent for sequencing to identify different strains circulating in the region. Neighbor-joining method was used to infer phylogenetic positions on the basis of 16S rRNA gene. Results: According to the findings of the questionnaire evaluation, only about 23.1% of the participants had some knowledge of heartwater disease. Furthermore, the highest proportion of the study population (76%) associated heartwater with air-borne transmission and 77.7% of the participants failed to identify the season in which heartwater commonly occurs. About 69% of the respondents associated ticks with animal diseases while 49.6 % correctly highlighted that, ticks are disease carriers. Farmers had a positive attitude towards control and treatment of heartwater by stating that they would use prescribed medicine 23.9%, vaccines 7.4% or consulting a specialist 2.5%. Few farmers indicated that they use homemade mixtures (0.8%), dipping and spraying (0.8%) to manage animal diseases. The most preferred method of tick control used by farmers was spraying of acaricide treatment 63.6%. On account of the poor animal services reported among the visited rural communities, some farmers opted for removing ticks by hand 28.9% as their supplementary tick control method. Majority of the farmers fed their livestock at the bush 68.6% which was the most contributing factor to increased tick infestation as reported by farmers. Several plants used as medicine to treat various animal diseases were mentioned. These included: Melia azedarach, Albizia adianthifolia, Gymnosporia senegalensis, Dicerocarryum senecioides etc. The type of disease affecting the livestock mentioned by the participants was diarrhoea (43.0%) which is among the list of heartwater symptoms. The study demonstrates that respondents had inadequate knowledge about heartwater disease. Animal services need to be upgraded in order to help the farmers improve the quality of their produce. The results of the PCR test showed that 56.2% of the household farms had E. ruminantium infection. The distribution of E. ruminantium by source of ticks and animal type revealed that cattle are more prone to tick distribution 43.5% as compared to other animal sources of ticks. Nzhelele municipality had the highest prevalence of E. ruminantium (37.0%) compared to the other municipalities. Prevalence of E. ruminantium by gender of farmers was found to be high from males 36.7% than females 25.0%. Farmers with household income > R20000 had the highest prevalence of E. ruminantium (50.0%) than farmers with household income < R500, (14.3%). The age of the farmers 15 – 20 years old revealed the highest prevalence 46.7 % of E. ruminantium infection. However, there were no infection among the ticks obtained from animals belonging to farmers above 60 years old. Farmers who had tertiary level of education showed the highest prevalence 46.8% probably because of limited time to attend to their livestock. Many clades were identified by phylogenetic analysis of the E. ruminantium PCS20 genotypes. Conclusion: This study showed that there is need to further educate small farmers on heartwater disease. It also showed that indigenous knowledge still contributes significantly to the management of animal diseases in most rural communities. The application of real time PCR showed a high prevalence of E. ruminantium infected A. hebraeum ticks from livestock and should be considered in the continuous monitoring of the animal population in order to avoid heartwater disease outbreak in the communities which could be detrimental for the local economy. Furthermore, future vaccine development against E. ruminantium should consider the diversity observed in the present study. That could be helpful in managing heartwater disease in the areas that were investigated.
Embargo
Cytotoxicity and anti-mycobacterial activities of sclerocarya birrea and Dodonae viscosa anguistifolia against MTB strain
(2023-10-05) Marole, Tsireledzo; Traore, A. N.; Potgieter, N.; Mavumengwane, V.
Background: Tuberculosis is a major public health concern with over 2 billion people currently infected, 8.6 million cases per year and more than 1.3 million deaths annually. Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB) which mainly affects the lungs causing pulmonary TB. In South Africa, the use of the Directly Observed Treatment Short Course (DOTS) strategy is applied where patients have to travel to primary Health care facilities (DOTS clinics) to get their medication. The rising prevalence of multidrug resistant and extensive drug resistant TB throughout the world highlights the critical need for novel anti-tuberculosis compounds/drugs. Objective: The aim of this study was to determine the cytotoxicity and antimycobacterial activities of Sand Olive and Marula. Methodology: The leaves (L) and barks (B) of Dodonaea Viscosa Anguistifolia (DVA or D) and S. birrea (S) plants were collected in the Vhembe district, Limpopo. Separate macerations in methanol (C) and distilled water (H) were done to obtain a total of 8 crude extracts (SBC, SBH, SLC. SLH, DBC, DBH, DLC, DLH). Phytochemical analysis was conducted on all extracts using thin layer chromatography (TLC), the profiling of metabolites was achieved with liquid chromatography-Mass spectrometry (LCMS) method. The antimycobacterial activity against M. smegmatis was determined using the microbroth dilution assay. The cytotoxicity of the plant extracts was assessed using the MTT assay. Results: The percentage yield ranged from 21.07% (DBC) to 4.4% (DBH). The phytochemical screening showed the presence of saponins, cardiac glycosides, steroids, terpenoids and tannins. The BEA solvent system revealed more bands than the CEF solvent system while the EMW solvent system was the least efficient. LCMS method showed sufficient revolving power to separate isomeric forms of several compounds. From the extracts of DVA and S. birrea, 508 chemicals were present in all the chromatograms assessed, but only 40 compounds were putatively identified and comprised of flavonoids, phenolics, terpenoids, and saponins. Of the 8 plant extracts that were tested 5 (DLH (50μg/ml (83.5%), (100μg/ml (85.9%)), DBH (50μg/ml (70.9%), (100μg/ml (60.3%)), DBC (50μg/ml (76.6%), 100μg/ml (83.9%) SLH (50μg/ml (66.8%), (100μg/ml (66.1%) and SLC (50μg/ml (79.9%), (100μg/ml (77.1%)) were found to have moderate cytotoxic effects on Vero cells at both treatment concentration while 3 (DLC (50μg/ml (91.5%), (100μg/ml (105%), SBH (50μg/ml (98.7%), (100μg/ml (106.8%) and SBC (50μg/ml (105.3%), (100μg/ml (118.2)) did increase viability of Vero cells. Amongst all the samples screened for antimycobacterial activity, only 3 plant extracts (SBC, DBC and DBH) inhibited the growth of M. smegmatis. Conclusion: The results of the study demonstrate the potential use of DVA and S. birrea as alternative treatment for tuberculosis. The bark of the plants may contain active compounds with anti-TB activities needing further investigation.
Embargo
Microbial analysis of street vended ready-to-eat meat around Thohoyandou Area, Vhembe District, Limpopo Province, RSA
(2023-10-05) Raedani, Tshimangadzo Jeanette; Musie, E. M.; Afsatou, Traore
Background: Street-vended meats are meats that are prepared and sold by vendors on the street. Different types of street vended meats are chicken, pork, beef etc. Street vendors are an integral part of urban economies around the world, offering easy access to a wide range of goods and services in public spaces. Despite these benefits, meat has been well-known as a potential channel for spreading food-borne diseases due to its high-water activity, high protein content, and approximately neutral pH, which create favourable conditions for the multiplication and survival of pathogenic bacteria. Street foods are frequently associated with food-borne illnesses due to their exposure to contamination hence this reduces the quality of these meat. Meat sold by the street vendors could be the source of infectious pathogens and previous studies showed that there is high number of pathogenic bacteria found on meat. The aim of the study was to determine the microbial quality and safety of street vended ready-to-eat meat sold around Thohoyandou Area. Method: A total of 168 samples of street-vended meats consisting of chicken (n=84) and beef (n=84), were collected from the local street vendors around Thohoyandou area. Samples were selected using simple random sampling and purchased meat samples were transferred from vendor packaging into sterile lunch boxes at the point of purchase. The packed samples were placed in a cooler box and immediately transported to the Department of Food Microbiology laboratory, University of Venda for further analysis. Ten grams (10g) of chicken or beef samples were then transferred into a zip lock bags containing 90 ml of peptone buffered water and then cultured in different plate’s containing the selective media: MSA (Oxoid Ltd) was used to culture Staphylococcus aureus, Eosin Methylene Blue (EMB) for E. coli 0157, xylose lysine deoxycholate (XLD) agar, (Oxoid Ltd) for Salmonella, and Sorbitol McConkey (Oxoid Ltd) for Shigella, and then plates were incubated for 24 hours at 37oC. The presumptive colonies were then sub-cultured on Nutrient agar for purification and the plates were incubated at 37oC for 24 hours. The microorganisms were identified using Gram staining and biochemical tests (Catalase, API 20E and Klingler iron Agar Test, and Vitek 2 system). The antibiotic susceptibility was done to determine susceptibility of the microorganisms using antimicrobial Agent such as Ampicillin, Chloramphenicol, Penicillin, Neomycin, Tetracycline, Streptomycin and Amoxicillin). The molecular 5 characterization was done to determine different pathotypes of E. coli using multiplex PCR. Results: Out of 168 samples tested, 32 (19%) were found to be positive for Staphylococcus spp with highest percentage found in cooked chicken meat. The most prevalent staphylococcus species identified in this study were S. xylosus (13.2%) and S. saprophyticus (10.5%). The prevalence of E. coli was found to be 29 (19.3 %) in which highest percentage was found in fried chicken. The antibiotic susceptibility profile of E. coli isolated showed that 100% were Resistant to Ampicillin (AMP), Tetracycline (T) and penicillin (PG) and 100% were susceptible to Neomycin (N). Staphylococcus spp. isolates showed 100% resistance to Ampicillin (AMP) and 100% susceptible to Neomycin(N). The virulence genes ranged from 13,33% to 86,67% with asta, stx1, and eae being the most prevalent. The pathotypes that were detected in this study were EPEC, EHEC, ETEC, EAEC, and EIEC and majority of the isolates were positive for mixed pathotypes (contamination). Conclusion: This current study demonstrated that the microbial quality and safety of street vended meat is inadequate and therefore not acceptable for safe consumption. Therefore, it is essential to monitor the presence of microbes in meat and the detection of these organisms in all beef and chicken meats investigated serves as a warning of foodborne diseases that could be associated with poor personal hygiene, and poor food preparation.
Open Access
Assessment of microbial quality and safety of ground beef/product sold in differrent retailers around Thohoyandou Area, Vhembe District, Limpopo
(2023-05-19) Mukosi, Fulufhelo Valentine; Musie, E. M.; Traore, A. N.
Title: Assessment of microbial quality and safety of ground beef/product obtained from selected retailers around Thohoyandou area; Vhembe district; Limpopo. Background: It has been proven that animal products are easily contaminated with microorganisms, and this supports microbial growth if not properly handled, processed, and preserved. Ground beef and its wholesale products are becoming popular because of the demand for rapid meal preparation and services, especially in the fast-food industry. Despite the control measures in place, foodborne infections continue to be an immense problem, with millions of cases occurring annually worldwide. In South Africa, illnesses and deaths related to food consumption continue to be reported. In addition to the misery caused, the financial loss associated with meat spoilage and illnesses is enormous. Therefore, this study aimed to assess ground beef's microbial quality and safety in different retailers around Thohoyandou area, Vhembe District. Methodology: A total of 160 ground beef/product samples was randomly purchased from various retailers in Thohoyandou and transported on ice to the University of Venda microbiology laboratory for analysis. The potential microbes were cultured in enrichment media (peptone buffered water) for 5 minutes in room temperature. The culture was then sub-cultured in different plates containing selective media (e.g., EMB for E. coli, SS for salmonella and Shigella, MSA for Staphylococcus spp.) using the spread plate technique. Isolates were then identified by the Gram staining technique and biochemical tests such as Catalase, Urease, Citrate, Kligler Iron Agar, and VITEK system. Moreover, the antibiogram activity of isolated pathogens was screened against medically used and commercially available antibiotics. Furthermore, the DNA of the isolates was extracted, and multiplex PCR was conducted to determine different virulence genes and pathotypes. Hemolysin test was done in blood agar plates to identify virulence characteristics of E. coli isolates. Results: Out of 160 samples analyzed, E. coli was detected in 80 (50%), Staphylococcus spp. in 117 (73.12%), Salmonella in 60 (37.5%), and Shigella species in 108 (67.5%). Most Enterobacteriaceae (E. coli, Salmonella and Shigella) isolates were resistant to Ampicillin and Cefoxitin. Staphylococcus isolates showed high resistance to Cefoxitin (93.33%) and Oxacillin (93.33%). Out of 30 E. coli isolates subjected to mPCR assay, 23 isolates were of different pathotypes with EPEC (53.33%) being the most prevalent pathotype. Asta with 73.33% was the dominant virulence gene obtained. Thirty (30) E. coli isolates were tested for hemolysin activity and Alpha hemolytic activity was observed in 76.6% isolates, while beta hemolytic activity observed in 10% isolates. Some of the isolates presented non-hemolytic strains (13.3%). Conclusion: It was concluded that ground beef/products from established retailers were contaminated with pathogenic bacteria, and microbial quality was thus inadequate.
Open Access
Characterization of cervicovaginal HPV virome and bacteriome in HIV -i infected women in Northern South Africa
(2023-05-19) Ratshilindela, Mpho; Bessong, Pascal Obong; Tebit, Denis
BACKGROUND: The presence of a highly diverse bacterial species in the cervicovaginal niche is linked to a higher risk of contracting human immunodeficiency virus (HIV), persistent infection with human papillomavirus (HPV) and consequently cervical cancer. It is unknown how cervicovaginal bacterial species associate and interact with other viruses. This study hypothesized that HIV/HPV co-infected women have an increased diversity of cervicovaginal virome and bacteriome. OBJECTIVES: The study’s primary objective was to characterize cervicovaginal HPV virome and bacteriome in HIV-infected women from selected health care facilities in Northern South Africa. Specific objectives were to determine the presence of HPV virome in HIV-infected women and HIV-noninfected women, to determine the presence of bacteriome in HIV-infected women and HIV-noninfected women, and to determine the relational occurrence of virome and bacteriome according to HIV status. METHODOLOGY: Cervical swabs from 50 HIV/HPV co-infected women; 50 HIV-infected, HPV-noninfected women; and from 50 HPV-infected, HIV-noninfected women were used to extract total deoxyribonucleic acid (DNA). To determine HPV virome, total DNA was enriched by rolling circle amplification (RCA) using an illustra TempliPhi amplification kit. To determine bacteriome, a two-round polymerase chain reaction (PCR) was employed targeting a bacterial 16S rRNA gene. Amplification products obtained from RCA and PCR were purified and sequenced by Next Generation Sequencing (NGS) using a MiniSeq platform (Illumina). The quality of the sequences was validated using the FastQC program. Sequences of good quality were assigned to viral family and genera using the Dragen metagenomics online tool with BaseSpace Sequence Hub. Bacterial sequences were assigned and categorised into vaginal community state types (CSTs) using the Dragen metagenomics online tool with BaseSpace Sequence Hub. The relational occurrence of virome and bacteriome according to HIV status was assessed through Chi-square statistical analysis available in GraphPad Prism version 9.3.1. Differences in occurrence were expressed as probability (P)-values. RESULTS: A diverse group of viral families was observed among HIV/HPV co-infected women. Papillomaviridae (14/34; 41%) was the most prevalent (P<0.0001), followed by Herpesviridae and Phycodnaviridae (12/34; 35%) each, Poxviridae (10/34; 29%), Mimiviridae (7/34; 20%), Maiseilleviridae (3/34; 9%) and Anelloviridae (2/34; 6%) in order of decreasing prevalence. A highly diverse group of bacteriophages was observed, with Myoviridae (31/34, 91%) being the most prevalent (P<0.0001). Among HPV-infected HIV-noninfected women, v Papillomaviridae (8/26; 31%) was the most prevalent (P<0.0001), followed by Anelloviridae (4/26; 15%), Phycodnaviridae (4/26; 15%), Poxviridae and Herpesviridae (2/26; 8%) each in order of decreasing prevalence. Myoviridae (6/26; 23%) was the most prevalent bacteriophage family detected (P=0.0005). Among HIV-infected HPV-noninfected women, a small group of viral families was observed, with Myoviridae (3/11,27%) and Siphoviridae (3/11, 27%) being the most prevalent viral families detected (P=0.0005 each). HPV 16 was the most common high risk (hr) HPV type in HIV-infected women and HIV-noninfected women of this study. This genotype co-existed with other hrHPV or probable hr types including HPV 54, HPV 53, and HPV 26. Overall, hrHPV types were more prevalent in HIV-infected women than in HIV-noninfected women, although this difference was not statistically significant (P=0.2832). When the occurrence of hrHPV types were considered individually, HPV 54 occurred more significantly in HIV-noninfected women than in HIV-infected women (P<0.0003). Bacteriome revealed the presence of community state types (CSTs) one, two, three, four and five among study participants. Among HIV/HPV co-infected women, CST one (L. crispatus), CST three (L. iners) and CST four (high bacterial diversity) were observed, with CST four being the most prevalent. Among HPV-infected, HIV-noninfected women, CST one, three, four and five (L. jensenni) were observed, with CST four, also being the most prevalent. Among HIV-infected, HPV noninfected women, CST three and four were observed. CST three and four were associated with viral infections. Gardnerella vaginalis (75%), Pretovella spp (54%), Atopobium spp (50%), Bifidobacterium spp (27%), Porphyromonas spp (53%), Pseudomonas spp (50%), Faecalibacterium prausnitzii (47%) and Bacteroides spp (35%) were the most prevalent anaerobic bacterial species detected in CST four. A diverse group of bacterial families occurred more significantly among HIV-infected women as compared to HIV-noninfected women for NGS data of RCA products (P<0.0001) and NGS data of 16S amplification products (P<0.0001). CONCLUSION: In conclusion, this study showed a higher diversity of cervicovaginal virome and bacteriome in women who were HIV/HPV co-infected than in those singly infected with HIV or HPV. The relationship between viral infections and a high diversity of bacterial species (CST four) observed herein may be a useful indicator of the individual’s disease state, indicating the likelihood of developing cervical lesions and cervical cancer. As a result, additional research is necessary to uncover the association between viral infections and CST four with disease state. Vaccine development and antiviral research should also target compounds that boost the cervicovaginal environment and maintain vaginal homeostasis.
Open Access
Examining institutional (policy) and administrative framework for water and sanitation (WSS) services in Zimbabwean cities to inform development of a new framework
(2022-11-10) Taonameso, Solomon; Potgieter, N.; Traore, A. N.; Mudau, S. L.
The lack of access to water supply and sanitation (WSS) services in developing countries has caused various waterborne diseases and related millions of deaths. The challenges have compelled various countries to take note of the need to incorporate both technical and policy aspects of WSS services in order to promote the delivery of these services. This research examines the current institutional (policy) and administrative framework for water services in Zimbabwean cities in order to find insights that will inform the development of a new framework. The study examines the causes of urban water conundrums by identifying qualitative gaps between the ZNWP and its implementation using both an empirical and a secondary-based study; assesses the causes of urban water conundrums by focusing on quantitative gaps between the ZNWP and its implementation in the provision of water supply services using both an empirical and a secondary-based study; evaluates the current service level on water supply and sanitation, risk assessment and audit water safety plans; and also evaluates the existing paradigm, institutional and administrative framework for WSS services in urban areas to inform the development of a new management framework. Water policy implementation gaps are examined using a four-part capacity framework that includes institutional, technical/human, financial and social capacities. The framework for assessment of service level is informed by the human rights principles on water and sanitation provision that incorporates the human rights normative and cross-cutting criteria. Data for the study is collected from a literature review, semi-structured interviews with households and other stakeholders that include water service authorities and other institutions that were systematically selected. Additional data is collected from water sampling using the Compartment Bag Test (CBT), field observations across communities in Masvingo and Harare city councils with residential (suburb) categories used as units of analysis. Both descriptive and inferential and risk assessment. The Statistical Package for Social Sciences (IBM SPSS) version 26 is used to analyse quantitative data and a thematic approach used to analyse qualitative data. Themes are drawn phenomenon under study and data coded and put into categories based on the research aims. The study also identifies the elements of implementation of the ZNWP that constrain the provision of services in urban areas in the City of Masvingo and Harare and these are: financial, institutional, technical/human and social capacities. Other constraints that include political al meddling in city council duties by the central government, financial and technical/human resource capacity shortages that impact on urban local authorities (ULAs)’s capacity to deliver on WSS services are also considered in the study. The results show that financial capacity is needed to support the ZNWP programmes that include the provision of drinking water and recruitment of skilled staff among others. The notes the need for adequate political support to implement the ZNWP programmes, adequate financial support to be offered to ULAS by central government to assist in capital intensive programmes investments such as WSS service augmentation, and for transparency , communication, education and awareness to be enforced at all levels in ULAs. The study recommends that there be an urgent development of water safety plans including education on household water treatment and safe storage (HWTS), and for the establishment of an emergency response plan by ULAs to safeguard public health and protect the right to safe drinking water for all. Consequently, the study proposes a new institutional and administrative framework that includes modifications of the old framework to reduce or eliminate the identified gaps, incorporates an independent WSS regulator who must enforce regulation of WSS services, and consists of a component of urban WASH that should be overseen by the Ministry of Health and Child Welfare’s Department of Health in light of recent episodes of drinking water related diseases which are mostly the result of poor hygiene including poor storage drinking at household level. The new institutional and administrative framework should also ensure an active role of water users by including civil society organisations (CSOs) such as the rate payers’ associations as legal entities in the management of WSS services.
Embargo
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.
Open Access
Molecular characterization of human sapoviruses circulation in the rural communities of Limpopo Province, South Africa
(2022-11-10) Magwalivha, Mpho; Potgieter, N.; Traore-Hoffman, A. N.
Background: Viral diarrhoea is a common cause of mortality among children less than five years of age in developing countries. Sapovirus (SV), one of the enteric viruses has been reported to be associated with viral diarrhoea worldwide. Reported studies on SVs in selected provinces of South Africa (SA) have been published based on the patients admitted in the hospitals located in the urban areas. There is a need for continuous epidemiological studies of SVs from the rural based regions within SA, especially from outpatients reporting in rural health care centers. Objective: To determine the genotypes, characterize, and analyze a capsid protein of the detected human Sapovirus strains associated with diarrhoea in children less than 5 years of age from rural communities in SA, and also compare the detected SV strains in this study with other strains reported elsewhere around the world. Method: A review article on the prevalence of human SV in developing countries was compiled to support the rationale of this study. To find the SV genotypes, characterizing, and comparing them with previously reported SV strains, an investigation on the “Prevalence and genetic characterisation of human sapovirus from children in the rural areas of Vhembe district” was conducted. A total of 284 stool samples were collected from children under 5 years of age suffering with diarrhoea (n=228) and without diarrhoea (n=56). Samples were screened for SV using real-time PCR. Sapovirus positive samples were further analysed for genogrouping by a One-Step Ahead RT- ix PCR, and SV Strains were genotyped using Sanger sequencing. A polyprotein (partial capsid protein) was successfully amplified using One-Step RT-PCR from 25% (10/40) positive samples, and further sequenced using Sanger method. Results: From a review report, 6.5% prevalence rate for SV in the low and middle income countries was determined, with significance difference of SV prevalent rate seen between low income and middle income countries. This study reports 14.1% (40/284) SV detection from stool samples [16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples]. Genogroup-I was found as the most prevalent strain comprising 68.75% (11/16), followed by SV-GII 18.75% (3/16), and SV-GIV 6.25% (1/16), with GI and GII detected in 6.25% (1/16) of the sample. Significant correlation between SV positive cases and water sources was noted (Chapter 4). A partial VP1 was successfully sequenced from 10/16 amplicons, and results showed genotype GI.1 to be the most prevalent (60%; 6/10), followed by 20% of SV-GII.1, and 10% of each SV-GI.3 and SV-GII.3 (Chapter 5). The relatedness of strains detected from non-human host with the detected strains from this study was noted with a concern (Chapters 4 and 5). Conclusion: The presence of SV, and substantial evidence of SV associated with diarrhoeal disease in low income regions was determined. This study defined human SV strains in rural communities from Vhembe district, and therefore outpatients in rural settings are possibly at a risk of the burden of diarrhoeal disease triggered by enteric-viruses among other pathogens. However, reports on SV as an emerging diarrhoeal causative agents in the developing regions are limited. Investigations on the analysis and surveillance of human SV strains in rural settings (at a community or household level) is essential to assess burden of diseases.
Open Access
Identification of acyl transferases responsible for the diverse hydroxycinnamoyl chemical space in bidens pilosa
(2022-11-10) Mathatha, Khuliso; Madala, N. E.
For decades, plants have been the backbone of complicated traditional herbal medicine system. Plants have been used by people and animals as a source of nutrients as well as medicine. Production of secondary metabolites by these plants is a characteristic that makes them attractive to both animals and humans. In plants, secondary metabolites play a role in defence mechanism and assist the plant to adapt to their immediate environment. Secondary metabolites are known to possess anti-diabetic, anti-malaria, anti-inflammatory and alleviate complications associated with obesity and cardiovascular diseases. Plants from the Asteraceae family are known to contain metabolites with nutraceutical properties. Plants such as Helianthus annuus, Lactuca sativa, Chicorium intybus and Bidens pilosa are some of the edible examples within the Asteraceae family known to exhibit interesting nutraceutical properties. B. pilosa adapt to almost every environmental condition, which makes it to be found in all parts of the world. As such, this plant has been used to manage and treat illnesses affecting humankind. Unique to this plant is the existence of large contingency of structurally diverse chlorogenic acids. B. pilosa is known to produce different structural hierarchies of chlorogenic acids (i.e., mono-, di-, tri- acyls). The biosynthetic pathway to produce the diverse array of chlorogenic acids in B. pilosa is not yet elucidated. It is known from other plants like Helianthus annuus that the production of chlorogenic acids is coded by hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase gene (HQT) and hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT). Apart from the chlorogenic acids (quinic acid acyls), B. pilosa is known to also produce acyls of tartaric acid, also characterised by existence of structurally diverse isomers thereof. From other plants, the tartaric acid esters are coded by the hydroxycinnamoyl-CoA: tartaric hydroxycinnamoyl transferase (HTT) gene and, surprisingly, the gene encoding the tartaric acid esters/acyls from B. pilosa is also not known. It is therefore imperative that a study aimed at establishing/identification of the gene elements which are responsible for the diversification of chlorogenic acids and related compounds (such as tartaric acid acyls) in B. pilosa is conducted. To achieve this, a Single Molecule Real Time (SMRT) sequencing approach was used to establish the full-length gene transcripts, in attempt to identify the acyl transferase Executive summary xi responsible for chlorogenic acids production in B. pilosa. The SMRT sequencing technique has brought a lot of improvement from the Sanger and Next generation sequencing such as generation of long reads which overcomes the challenges of sequence assembly synonymous with the short reads achieved by the former two sequencing approaches. Moreover, this technique allows detection of isoforms of a specific gene, caused by either inherited genetic code or alternative splicing events. From the SMRT sequencing results, three HQT genes and one HCT gene responsible for production of wide array of chlorogenic acids and two HTT genes responsible for production of tartaric acid esters were identified through series of bioinformatics analyses of the sequences obtained through SMRT sequencing. All the identified genes contained the conserved regions that are found in already published acyltransferases, with the highly conserved DFGWG motif present in all transcripts identified herein. The second motif, the HXXXD motif showed a single amino acid variation from gene to gene, with HQT1 and HQT2 showing HTLSD motif and HQT3 having HTLAD motif, all of which are synonymous with the plants from the Asteraceae family. In HTT genes, the second motif identified in B. pilosa has never been recorded in literature. HTT1 and HTT2 showed to have HRVLD and HRVAD motif respectively. From these sequences, the open reading frames (ORFs) were computed, and these sequences can be used to design primers that can be used to amplify these genes in the future. Through multi sequence alignment and phylogenetic trees, the identified genes were also found to have similarities with the genes of other plants from the Asteraceae family. In conclusion, the SMRT sequencing approach enabled identification of acyltransferases genes that plays a role in the biosynthesis of CGAs in B. pilosa. Bioinformatics tools were shown to be sufficient to annotate and characterise these genes. Through LC-MS analyses of randomly collected B. pilosa plants, the CGAs content of this plant were revisited, and these plants were found to produce structurally diverse CGAs compounds, suggesting that the identified genes are functional. Future studies should aim to clone these transcripts in plant systems that do not produce CGAs in attempt to enhance their nutraceutical attributes.
Open Access
The role of molecular chaperones in Escherichia coli cells subjected to gold nanoparticles induced stress
(2022-07-15) Mathomu, Lutendo Michael; Shonhai, Addmore; Zininga, Tawanda
Colloidal suspensions of gold particles of nanometre (nm) sizes are termed gold nanoparticles (AuNPs). Although stable, AuNPs have been reported to be toxic to E. coli cells by collapsing the bacterial cell membranes and promoting protein misfolding. An understanding of biodistribution in drug delivery and the effects AuNPs have on the function and structure of proteins such as heat shock proteins is important. Heat shock proteins facilitate protein folding and are particularly important during cellular stress. At high concentrations, AuNPs are thought to promote protein aggregation. Heat shock proteins are thought to alleviate cell stress induced by AuNPs. This study explored the role of heat shock proteins in conferring cytoprotection to E. coli against the effects of AuNPs. Citrate-AuNPs were synthesized and their integrity was validated at 520 nm by ultraviolet-visible-near infrared spectroscopy (UV-Vis-NIR). Crystallinity was confirmed by X-Ray diffraction (XRD), while dynamic light scattering (DLS) estimated the size distribution at 13 nm. Furthermore, transmission electron microscopy (TEM) and scanning electron microscope (SEM) revealed the spherical shape and crystal lattice surface morphology of citrate-AuNPs respectively. A complementation assay was conducted using cells deficient of DnaK function (E. coli ΔdnaK52). E. coli ΔdnaK52 was transformed with a recombinant dnaK before examining both DnaK deficient and transformed cells using TEM. E. coli O157:H7 was exposed to citrate-AuNPs (0 – 50 μg/ml) and allowed to grow at 37 oC before protein expression was analysed using electrophoresis followed by LC-MS analysis. This led to the identification of highly expressed proteins such as DnaK, GAPDH, ClpX, DnaJ, and GroEL. Subsequent co-affinity assay revealed possible interaction protein partners of DnaK. These were identified as ClpB, HtpG, GroEL, DnaJ, and SurA proteins. Furthermore, circular dichroism and fluorescence spectroscopy established that recombinant DnaK is stable at citrate-AuNPs concentrations less than 10 μg/ml and the protein was unstable at concentrations beyond 10 μg/ml citrate-AuNPs. In addition, the ATPase activity of recombinant DnaK increased in the presence of citrate-AuNPs at 2.5 μg/ml. The ability of DnaK to suppress aggregation of MDH in vitro was abrogated by the presence of >10 μg/ml citrate-AuNPs. The findings suggest that at low concentrations (<10 μg/ml) citrate-AuNPs seem to stabilize proteins and similarly at elevated concentrations (>10 μg/ml), citrate-AuNPs destabilizes protein conformation and function. Altogether the findings suggest that DnaK in cooperation with its network partners is implicated in E. coli cytoprotection against citrate-AuNPs toxicity.
Open Access
In cellulo and biophysical exploration of the role of GGMP residues of Hsp70
(2022-07-15) Dongola, Tendamudzimu Harmfree; Shonhai, A.,; Zininga, T.
Hsp70 is a prominent molecular chaperone. Structurally, Hsp70 is composed of two domains, C-terminus substrate binding domain (SBD) and nucleotide binding domain (NBD). The NBD of Hsp70 is responsible for its ATPase activity. Some Hsp70s of parasites particularly those of apicomplexa are marked by GGMP residues. For example, Plasmodium falciparum Hsp70-1 (PfHsp70-1) which occurs in the cytosol and nucleus harbors seven GGMP repeats on the C terminus upstream of the EEVD motif. The function of GGMP residues of Hsp70 is largely unclear but they were recently reported to be involved in substrate and co-chaperone binding. Therefore, the main aim of this study was to investigate the role of the GGMP residues of Hsp70s using E. coli Hsp70 (DnaK) and chimeric protein, KPf as models. Chimeric protein KPf is made up of NBD of DnaK and SBD of PfHsp70-1. E. coli DnaK lacks the GGMP residues that are present in PfHsp70-1. DnaK-G (DnaK modified to include GGMP residues) was created to elucidate the function of these residues. Exogenously expressed KPf and DnaK are known to reverse the thermosensitivity of E. coli dnaK756 cells. The native DnaK of E. coli dnaK756 cells is functionally compromised making this strain heat sensitive. KPf617-647 and KPfΔG mutants were previously created by conservative substitution and deleting the GGMP residues of KPf, respectively. Circular dichroism and tryptophan fluorescence highlighted that the insertion of GGMP residues did not drastically change the secondary and tertiary structure of DnaK. Cytoprotection of E. coli dnaK756 cells could not be recovered when cells were heterologous expressing DnaK-G. DnaK recovered the cytoprotection of the same cells as expected. Furthermore, mutations on GGMP residues adversely impacted the chaperone function of KPf with respect to cytoprotection of E. coli dnaK756 cells. On the other hand, the absence of the GGMP residues results in KPf losing its chaperone function. This suggests that the SBD of PfHsp70-1 requires the GGMP residues to function, whereas E. coli DnaK does not require these residues. It was further noted that insertion of the GGMP residues into DnaK led to toxicity in E. coli dnaK103 cells under permissive growth temperature. A proteomic study conducted to establish the functional deficiencies of DnaK-G established that the presence of the GGMP residues compromised the capability of DnaK-G to bind some substrates. Altogether the findings suggest that GGMP repeats account for the specialized function of PfHsp70-1 in distinction to that of E. coli DnaK.
Open Access
Phytocompound profiling and assessment of antioxidant antibacterial, anti-inflammatory, and cytotoxic activities of Momordica balsamina leaf extracts
(2021-11) Mabasa, Xitsakiso Euphodia; Sigidi, M. T.; Musie, E. M.; Mathomu, L. M.
Background: The use of medicinal herbs has raised considerable interest worldwide attributed to their health-promoting effects. Momordica balsamina (M. balsamina) is a medicinal herb that has long been used to treat various ailments. Therefore, this study aims to profile the phytocompounds and assess antioxidant, antibacterial, anti-inflammatory, and cytotoxic activities of M. balsamina leaf extracts. Methodology: Methanol and water were used as extraction solvents. Profiling of phytochemical constituents of M. balsamina extracts was done using TLC, phytochemical screening tests, UV-Vis, FTIR, and UHPLC-qTOF-MS analysis. Biological activities were assessed using in vitro bioactivity screening (cytotoxicity and anti-inflammatory) assays, an antioxidant assay using free radical scavenging (DPPH) activity, and determination of minimum inhibitory concentration using the serial micro broth dilution technique. Results: Phytochemical screening revealed the presence of cardiac glycosides, flavonoids, saponins, tannins, and terpenoids in both extracts. The UV-VIS profile revealed various absorption bands ranging from 200 – 750 nm indicating the presence of flavonoids, phenolic compounds, tannins, terpenoids, carotenoids, chlorophyll, and alkaloids. FTIR spectra confirmed the presence of alkaloids, flavonoids, terpenes, anthraquinones, and phenolic compounds. The UHPLC-qTOF-MS detected flavonoid aglyclones such as quercetin, isorhamnetin, and kaempferol as well as dicaffeoylquinic, feruloyl isocitric and pseudolaroside A acids in the methanolic extract. Based on our knowledge, this is the first report on the presence of pseudolaroside A and feruloyl isocitric acid in M. balsamina leaves. UHPLC-qTOF-MS could not identify the compounds in the water extract. Both extracts had antioxidant potential and exhibited no antibacterial activity on gut-associated bacteria. In vitro cytotoxicity results showed that extracts were non-toxic against human colorectal adenocarcinoma (HT29 and Caco2), Vero, and RAW 264.7 cells. Methanolic extract showed anti-inflammatory activity on RAW 264.7 cells and water extract exhibited no activity. Conclusion: M. balsamina leaves contain plethora secondary metabolites with no cytotoxic potential and may be used as antioxidant and anti-inflammatory agents.
Open Access
Development of liquid chromatography mass spectrometric fingerprinting based method for HIV-1 integrase detection
(2021-04) Vele, Tikedzani Geraldine; Madala, N. E.; Mathomu, L. M.
Human Immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS) is a relatively modern disease that has caused extensive morbidity, mortality and suffering worldwide. HIV/AIDS is associated with different enzymes that are responsible for viral replication and one of such enzyme is the HIV-1 integrase. Integrase is responsible for the incorporation of viral DNA into the host cell, the enzyme is emerging as a novel target for intervention by chemotherapeutics, thus making the scientific investigation of HIV-1 integrase an important field of research. Currently, Enzyme-Linked Immunosorbet Assay (ELISA) based method are used for diagnosis of HIV infections across the world. However, other methods which are capable of detecting HIV-1 proteins with high sensitivity and specificity are imperative. Mass Spectrometry (MS) is known to offer unprecedented sensitivity and specificity during protein identification and, as such, its use for medical use is of greater interest. Therefore, this study sought to develop an MS method which can be applied for detection and characterization of HIV-1 integrase. The main objective of the current study was to identify and detect HIV-1 integrase using Multiple Reaction Monitoring (MRM). Detection of HIV-1 integrase using MRM will provide a method which may serve as a basis for diagnosis HIV/AIDS infection. Using Nested PCR and Sanger sequencing, pET15b and integrase construct were successfully confirmed. To this end, recombinant HIV-1 integrase was expressed in E. coli BL21 (DE3) cells. The recombinant HIV-1 integrase was successfully purified using nickel-affinity chromatography. Using electrospray ionization (ESI) based MS method, it was observed that integrase produces peptides masses that matches with theoretical masses, however some of the peptide masses differs due to multiple charge state. In addition, using precursor ion 600.84 for MRM, it was noted that the precursor ion produce three product ions acquired during ionization in the ESI chamber. Furthermore, the peak intensity of the product ions were noted to increase as the collision energy increases. These findings make the precursor ion 600.84 and its product ions a fingerprint that can be used as a tool integrase detection. As a proof of concept, the current method can be further developed by means of incorporating other MS ions originating from HIV-1 proteins, thereby increasing the likelihood of a future diagnosis.
Open Access
Investigation of biochemical strategies leading to metabolome complexity of two closely related Coccinia species through LC-QTOF-MS-based metabolite fingerprinting
(2021-04) Nengovhela, Ndamulel; Madala, N. E.; Mhlongo, M. I.; Mathomu, L. M.
Medicinal plants play an important role in both health and the livelihood of people. These plants have been used for medicinal purposes since time memorial, and they are deemed safer than their synthetic counterparts. One such plant is Coccinia grandis (Ivy Gourd), which has been used in traditional medicine as a household remedy for various diseases. Coccinia grandis have been reported to possess antidiabetic properties. This is owing to the presence of secondary metabolites which are naturally produced by plants for adaptation to their immediate environment. Secondary metabolites are a diverse group of chemical compounds which include flavonoids, glycosides and hydroxycinnamic acids. These biologically active compounds chemically and structurally complex and their characterization pose undisputed analytical challenges. Mass spectrometry techniques have however, over the years been developed as feasible technique to assist in the characterisation and structural elucidation of plant metabolites. Liquid Chromatography Mass Spectrometry (LC-MS) is the most widely and preferred analytical platform due to its high sensitivity, resolution and detection specificity. Moreover, MS is able to produce accurate mass, unfragmented and fragmented data, which is important for compound characterization and structural elucidation. As such, in the current study LC-MS based metabolomics approach was used to screen for phytochemicals from commercially cultivated C. grandis and its widely growing relative, Coccinia rehmannii which has no reported medical use in literature. To establish the chemo-taxonomic relationship between these two species, multivariate statistical modelling revealed differential metabolites distribution pattern between them. The results revealed that these two closely related plant species possess a wide variety of important secondary metabolites such as flavonoids and hydroxycinnamic acids. Some interesting subtle differences were also noted, for instance the flavonoids composition of these plants were to have different glycosylation patterns (Chapter 3). Here, it was noted that flavonoids attached to di- or tri- saccharides are prone to isomerization, a phenomenon termed glyco-isomerization. Apart from positional migration of sugar moieties, acylation of sugars by other biologically active molecules such as derivatives of cinnamic acids (i.e. caffeic and coumaric acid) was also noted ix as a contributor towards glyco-isomerization. Here, C. rehmannii was found to attach these cinnamic acid derivatives to its flavonoids, a phenomenon which was absent in C. grandis. It was then concluded that C. rehmannii produce more flavonoid molecules than C. grandis. Apart from flavonoids composition, hydroxycinnamoyl conjugation was revealed to be a discriminatory chemical factor between these two closely related Coccinia species. Through UHPLC-qTOF-MS/MS metabolite fingerprinting, C. grandis was found to produce different chlorogenic acids, thus cinnamic acids conjugated to a quinic acid (Chapter 4). Ironically, only few chlorogenic acids were found in C. rehmannii. The findings revealed that the two closely related species utilise Hydroxycinnamic acid (HCA) conjugations as another strategy to diversify their metabolite composition, a phenomenon which has not been suggested in chemo taxonomical studies. Furthermore, the current study revealed that these two plants use both glyco-isomerization and conjugation as an evolutionary strategy to maximise their metabolome complexity. This was further highlighted in (Chapter 5) where LC-MS analyses of C. grandis revealed multiple peaks sharing same precursor ion (with molecular formula C27H30O16) and associated fragmentation patterns. Further analyses revealed all six peaks to be Rutin, a most common and highly pharmacologically acclaimed flavonoid molecule. As demonstrated herein, these results also had analytical implications, such that subsequent developments of other specific methods such as multiple reaction monitoring (MRM) could not distinguish these compounds. In conclusion, the two Coccinia species contain a wide range of phytochemicals, dominated by flavonoids and hydroxycinnamic acids derivatives. This study further demonstrated LC-MS as an effective tool in comparing metabolite profiles between C. rehmannii and C. grandis certified by the varying amount and composition of flavonoids and hydroxycinnamic acids derivatives between the two species. Two biochemical phenomenon known as glyco-isomerization and conjugation were shown to be well co-ordinated strategies used by these plants to diversify their metabolite composition. LC-MS in combination with multivariate data models was effective in the investigation of metabolites distribution patterns between the two species.
Open Access
Characterization of Plasmodium falciparum PFF1010c and screening of pyrimidine-quinoline hybrids as inhibitors of HsP70-HsP40 functional partnerships of the Malaria parasite
(2021-05) Mudau, Pertunia Thendo; Shonhai, A.; Zininga, T.
With nearly half the world’s population at risk of malaria infection, 229 million new cases were recorded in 2019 with Sub-Saharan Africa accounting for most of these cases. In 2020, 384 000 malaria deaths were reported in the WHO African region. Plasmodium falciparum is the most virulent species responsible for over 90 % of all malaria infections. The P. falciparum life cycle occurs through multiple stages in humans and mosquitoes. Many advances have been made in the fight against malaria through vector control and treatment against infection by the parasite. However, resistance of P. falciparum to antimalarial medicines/treatment has hindered global efforts to control and eliminate malaria. A multidimensional approach to fighting malaria by targeting is essential protein machinery is needed. Heat shock proteins (Hsp) are molecular chaperones that are upregulated in response to stress and have long been projected as antimalarial drug targets. P. falciparum Hsp70-1 (PfHsp70-1; PF3D7_0818900) is an essential heat shock protein involved in the folding of newly synthesized and misfolded polypeptides and is also implicated in protein trafficking. PfHsp40 (PF3D7_1437900.1) is a type I Hsp40 co-chaperone of PfHsp70-1 that presents substrates to PfHsp70-1 and stimulates its ATPase activity, while PFF1010c (PF3D7_0620700.1) is a type IV Hsp40 found in the gametocytes that is yet to be characterized. The current study explored pyrimidine-quinoline hybrid (PQH1-4) compounds as possible inhibitors of PfHsp70-1 interaction with PfHsp40. Furthermore, this study sought to characterize the structure and function of PFF1010c. Notably, PFF1010c possess SVN residues in place of the HPD motif located in the so-called J domain of this Hsp40 co-chaperone. For this reason, PFF1010c is an atypical Hsp40 which may lack the capability to interact with PfHsp70-1. To characterise the role of PFF1010c, and consequently, its SVN motif, the SVN motif of PFF1010c was switched with the HPD motif of PfHsp40. The structure-function features of the mutants were characterized relative to the wild type Hsp40s. Bioinformatics based analysis was employed to predict the fold of the mutants relative to the wild type Hsp40s. Recombinant proteins were expressed in E. coli cells and purified using nickel affinity chromatography. Fluorescence spectroscopy was used to map out the tertiary structure of the Hsp40 proteins. It was noted that the HPD motif confers thermostability to the proteins. Possible interaction of PFF1010c with PfHsp70-1 was evaluated using ATPase assay and SPR analysis. Furthermore, the same study was repeated for the motif switch mutants. PFF1010c was found to interact with PfHsp70-1, though the interaction would not be as conventional as the one between PfHsp40 and PfHsp70-1 because PFF1010c does not have the HPD motif. SPR analysis was also employed to investigate binding of the pyrimidine-quinoline hybrid compounds to PfHsp70-1. Compounds PQH1 and PQH4 were found to have high binding affinities for PfHsp70-1. The compounds also inhibited binding of PfHsp40 to PfHsp70-1. The study provides the first evidence that PfHsp70-1 interacts with a type IV Hsp40, where PfHsp70-1 and PFF1010c could be a druggable complex to reduce transmission of malaria from humans to mosquito. The pyrimidine-quinoline hybrid compounds would play a role in the proliferation of the malaria parasite by inhibiting the chaperone activities of PfHsp70-1 and PfHsp40.
Open Access
Expression of TN5 transposase for next generation sequencing protocol of HIV ad selected oncoviruses
(2021-03) Mathobo, Phindulo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno Grace
Objective: The development of HIV drug resistance is a significant challenge in maintaining suppressed viral load in the management of patients. Next generation sequencing (NGS) is a more sensitive approach to determine the burden of HIV drug resistance. We aimed to describe the uptake of NGS in HIV drug resistance studies in Africa. Methodology: Electronic search was done for studies published between 2005 and 2019, from three databases: Pubmed, Web of Science and Google scholar. The search approach was carried out according to PRISMA guidelines. Studies included in the analysis were those that reported the use of NGS on HIV drug resistance using samples from Africa or in which the studies were done in Africa. Only articles published in English were included in the analysis. Results: Four thousand one hundred and eighty articles were identified according to the search criteria. Out of these, 238 studies met the inclusion criteria and were included in the analysis. Thirty studies (12.6%) used NGS, 194 (81.5%) used Sanger sequencing, and 14 (5.9%) used both techniques. Evidence of in-country application of NGS was observed in six studies (13.6%), all from South Africa. In other studies, NGS was either done outside of Africa using samples obtained from Africa or the country in which NGS was done was not indicated. From 2005 to 2012, only one study was reported on HIV drug resistance using NGS; however, 44 studies were published by 2019. Out of the 54 African countries, investigators from 13 countries (24.1%) published data on HIV drug resistance using NGS, as at end of 2019. Conclusion: There is an uptake in the application of NGS in HIV drug resistance studies in Africa, albeit in a slower pace, with investigators from about a quarter of African countries applying the technology for this purpose.
Open Access
Strain improvement and characterization of antibiotic producing microorganisms from soil
(2021-06-23) Khwathisi, Adivhaho; Samie, A.; Traore, A. N.
Since the discovery of Antibiotics in the 20th century, the idea of searching for antimicrobial compounds from natural sources came into existence. However natural products from microbial origin (especially soil microorganisms) have grasped a great devotion over the course of several decades. Recently, bacterial resistance have been observed against antibiotics of all classes, however it appears that the emergence of antimicrobial resistance is inevitable to almost every new drug.This necessitates carrying out studies that will generate new effective antibiotics. The present study is an attempt to identify, characterize and improve strains of bacteria for the ability to produce antibiotics. About 12 soil samples were screened for antibiotic-producing bacteria against 4 pathogenic microorganisms including Escherichia coli, Klebsiella pneumoniae Staphylococcus aureus and B. subtilis. After preliminary screening, active isolates with secondary metabolites showing activity were selected for secondary screening by agar well diffusion method to identify antibiotic potency. VITEK 2 system was used for rapid identification of the active isolates. The amplification of the 16s rRNA by PCR followed by sequencing and sequence analysis was used for the molecular identification of these strains. Optimization of chemical and physical culture conditions was carried out by manipulation of fermentation parameter such as pH, Temperature and incubation period. The results revealed 7 strains of antibiotic producing organisms. 4 bacterial strains demonstrated convincing growth inhibitory properties against pathogenic test organisms. Of these, were identified as Gram-positive cocci and 1 was identified as Gram-negative of the group of Rods and most of the isolates were active against the Gram positive than gram negative pathogens. Phylogenetic analysis of amplified 16S rRNA gene showed the isolates shared sequence identities of 99.65% with known Staphylococcus and Pseudomonas species. TSH2 and TSP3 clustered together with a sequence identity of 99.68% with Staphylococcus sciuri. Isolate TSP1 sequence had a sequence identity of 100% with Pseudomonas formosensis strain CC-CY503. The production of antimicrobial substances started on the 4th day and went on increasing till it reached a maximum peak on the 7th day. The optimum growth conditions were pH 7.5, temperature at 35°C, and incubation period in 7 days. In conclusion, the results of the present study indicate that soil contain great diversity of antibiotic producing organisms and the production of antimicrobial substances can be improved by manipulating the growth conditions.