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MSc (Microbiology), North-West University, Potchefstroom Campus Water is a limited resource, and the quality of raw water has an impact on drinking water production. Regular testing is done to ensure that the quality is compliant with regulations. Heterotrophic bacteria are widely used as indicators of drinking water quality. According to SANS 241 (2015), the amount of heterotrophic plate count (HPC) bacteria in drinking water should not exceed more than 1000 colony forming units (CFU)/ml. There is currently a growing concern about the presence of HPC bacteria in drinking water. Many of these might be potentially pathogenic microorganisms, and they are associated with secondary infections in immuno-compromised individuals. The South African population has a higher potential infection rate of HPC bacteria compared to other countries due to the escalating percentage of patients living with HIV. These potential pathogens have been reported in drinking water systems. Therefore, this current study aimed to determine antibiotic resistant bacteria and potentially pathogenic HPC bacteria in the drinking water. Samples were collected from two water production facilities, one in the North West (NW-D) and the other in Gauteng (GH-T) Province. Physico-chemical parameters measured were free chlorine, TDS, pH, nitrate, nitrite, COD, phosphates and temperature. HPC bacteria were isolated and purified with a culture-based method, identified by Gram stain and 16S rRNA sequencing, and tested for α- or β-haemolysis on blood agar. Further tests were done for the production of extracellular enzymes such as DNase, hyaluronidase, lipase, proteinase, chondroitinase and lecithinase. Total coliforms and faecal coliforms were enumerated on MLG Agar, using standard procedures. The levels of these were only used for water quality purposes. Antibiotic susceptibility of the HPC bacteria and whether antibiotic resistant genes (ARGs) could be associated with antibiotic resistance phenotypes were determined. Environmental DNA (eDNA) was also isolated to determine if ARGs could be detected directly in selected samples. Turbidity and nitrite levels were alarmingly high, and in some cases, both exceeded the SANS 241 (2015) of drinking water. The other physico-chemical parameters were mainly within the recommended levels. All microbiological parameters were detected at levels below the standard in drinking water, however the higher levels of heterotrophic bacteria after treatment should not be ignored. Fifty one percent (51%) of the isolated HPC bacteria displayed α- or β-haemolysis. These haemolytic isolates revealed the production of various enzymes: proteinase (74% from GH-T), DNase (58% from NW-D) for the 2017 sampling run and chondroitinase (58% from NW-D) for 2016. Both DNase and proteinase enzymes were produced by most of the haemolysin producing isolates. Hyaluronidase and lecithinase were the least detected enzymes. Among all the isolates, resistance to individual antibiotics were observed in the following order of decrease: ampicillin (89%), trimethroprim (77%), cephalothin (62%) and penicillin G (53%) in water after treatment and drinking water. There was high a percentage susceptibility, and low intermediate resistance percentage of HPC isolates to neomycin, erythromycin, and streptomycin. Penicillin and ciprofloxacin were detected at low levels by liquid chromatography-mass spectrometry analysis. Isolates were resistant to more than two antibiotics classes. The most prevalent MAR phenotype was AMP-TM-KF-PEN-G-VAN-OT. Genes encoding for macrolide-lincosamide-streptogramin (MLS) (ermF and ermB), were detected. However, ermB was more prevalent in the MAR isolates (50%), while ermF gene was found in only one isolate (3%). The tetM and Intl 1 genes could not be detected in any of the isolates. β-lactam (blaTEM and ampC) genes were also detected in samples from the water treatment system. Moreover, ermB and intl 1 was found in raw water when eDNA was tested. The identified species included Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus licheniformis, Micrococcus luteus, Shinella sp., Chryseobacterium sp., Paenibacillus chitinolyticus, Bacillus anthracis, Bacillus wiedmannii, Bacillus Toyonensis and Bacillus pumilus. The predominant genus was Bacillus, for all the sampling sites in both production facilities. These species are linked to various infections, including wound infections and skin and soft-tissue infections. This study confirmed the presence of potentially pathogenic HPC bacteria in the raw and treated water from two water production facilities. The obtained results showed that ARGs were not completely removed during the drinking water production process, and they can also thus be present in the distribution system. This is a cause for concern, particularly for the immune-compromised individuals. The results show a need for education and awareness programmes in the communities about contaminants entering the water source, and ways to treat water at house level. This will help prevent water-borne diseases but particularly the spread of antibiotic resistance through water systems. Masters

MSc (Environmental Sciences), North-West University, Potchefstroom Campus Increased urbanisation and anthropogenic disturbances have caused water quality of many freshwater systems to deteriorate over the years in South Africa. This is due to domestic, industrial, and agricultural waste being disposed of into surface waters and the surrounding environment. To meet growing water requirements a monitoring program needs to be applied nationally. Government agencies set forth this initiative by creating water management areas to meet integrated water resource management needs. Applying data mining techniques, this study attempts to determine the water quality status by analysing historical and current data of one of these management areas, specifically the Mooi River and Wonderfonteinspruit. It focuses on mining microbiological, physico-chemical and geographic information systems (GIS) data to explore relationships between bacterial communities and physico-chemical changes and the correlation between industrial pollution, agriculture and urbanisation on water quality. The results demonstrated that the Mooi River has water usable for all purposes, while the Wonderfonteinspruit’s water is highly polluted with PO43-, SO42- and NO3-NO2. The Wonderfonteinspruit sites also show high EC values. The bacterial community composition of the Mooi River and Wonderfonteinspruit seemed mostly similar. Bacteriodetes, Proteobacteria, Actinobacteria and Cyanobateria are the four most dominant phyla identified spatially and temporally, but the Wonderfonteinspruit had a higher abundance of Cyanobateria. The major land-use activities that influenced physico-chemical parameters and bacterial communities were identified as mining and agriculture, with erosion also playing a role. Masters

Exposure to antibiotics, biocides, chemical preservatives, and heavy metals in different settings such as wastewater treatment plants (WWTPs) may apply selective pressure resulting in the enrichment of multiple resistant, co- and cross-resistant strains of bacteria. The purpose of this study was to identify and characterize potentially pathogenic triclosan (TCS) - and/or, chloroxylenol (PCMX) tolerant bacteria from sewage and river water in the North-West, Potchefstroom, South Africa. Several potential pathogens were identified, with Aeromonas isolates being most abundant. Clonal relationships between Aeromonas isolates found at various sampling points were elucidated using ERIC-PCR. Selected isolates were characterized for their minimum inhibitory concentrations against the biocides, as well as antibiotic resistance profiles, followed by an evaluation of synergistic and antagonistic interactions between various antimicrobials. Isolates were also screened for the presence of extracellular enzymes associated with virulence. High-performance liquid chromatography revealed the presence of both biocides in the wastewater, but fingerprinting methods did not reveal whether the WWTP is the source from which these organisms enter the environment. Isolates exhibited various levels of resistance to antimicrobials as well as several occurrences of synergy and antagonisms between the biocides and select antibiotics. Several isolates had a very high potential for virulence but further study is required to identify the specific virulence and resistance genes associated with the isolates in question.

PhD (Microbiology), North-West University, Potchefstroom Campus Avian influenza (AI) is an infectious viral disease in birds caused by a type A influenza virus of the family Orthomyxoviridae. These viral outbreaks usually cause significant economic losses to the poultry industry. Conventional strategies for AI detection and control using vaccination both start by growing the viruses and isolating them in embryonated chicken eggs. This entire process takes 6 to 8 months from viral detection to the release of the vaccine for that particular strain. A rapid and cost effective diagnostic method for the early detection of AI viruses, as well as a more effective vaccine that can be produced quickly, are urgently needed as tools for AI outbreak management. Therefore, the aim of this study was to develop a strategy for the point-of-care (POC) detection of AI and to develop a vaccine that is not only effective in the treatment of such outbreaks, but can also be produced quickly enough to manage AI viruses before they spread and cause an outbreak. Loop-mediated isothermal amplification (LAMP), which is a rapid nucleic acid amplification assay that takes 30 to 60 minutes, was used for POC diagnosis of the AI viruses. Three LAMP assays were designed − one for the detection of general AI strains and the other two for the specific detection of the H5 and H7 AI subtypes. The device used to make the detection of these viruses at the POC possible was the Axxin T16 isothermal instrument. This device is ideal for POC assays as it is portable, the results can be detected in real time, and it operates on rechargeable batteries. Using the designed LAMP assays, the identification of the AI samples that were tested was successfully achieved within 30 minutes at the POC without having to transport the samples to a laboratory. The Adenovirus vectors are known as suitable recombinant vaccine vectors because they are capable of infecting a wide variety of cell types, can grow to high titres in vitro, and cannot integrate in the host genome. Due to these features, the Adenovirus was used in this study to design a safe and effective vaccine that can be produced quickly. However, policies of the Department of Agriculture, Land Reform and Rural Development (DALRRD) formerly known as the Department of Agriculture, Forestry and Fisheries (DAFF) prohibit vaccination against AI as conventional vaccines previously interfered with surveillance programmes that did not allow any differentiation between infected and non-infected poultry that had been vaccinated using diffierentiation of infected and vaccinated animal (DIVA) strategies. To prove that the strategy proposed by this study will address the challenges that conventional AI vaccines pose, the Newcastle disease virus (NCDV) was used as a target virus in the design of the Adenovirus vector-based vaccine. The vaccine was designed by inserting the Matrix gene of the NCDV into the Adenovirus vector. The functionality and propagation of the recombinant Adenovirus were performed in 293T human embryonic kidney (HEK) cells. This was followed by testing the recombinant Adenovirus in vitro in chicken embryo fibroblasts (CEFs) that were prepared inhouse. The results demonstrated the successful expression of the recombinant Adenovirus in the CEFs, which suggests that the vaccine design strategy that was developed in this study may be successful for future application as the vaccine showed the desired expression in vitro. These results also suggest that the same strategy may be used for the design of Adenovirus-based vaccines against AI and any other viral diseases in chickens. Even more noteworthy is that the current strategy takes only 5 weeks from viral detection to vaccine production, while the conventional strategy takes up to 6 or 8 months for the entire process to be completed. However, despite these exciting results, clinical trials using live chickens will have to be conducted in future studies to check how safe and effective these vaccines are. This strategy may even be efficacious in solving the COVID-19 crisis that the world is currently facing. Doctoral

MSc (Microbiology), North-West University, Potchefstroom Campus Microplastics as pollutants originate from land-based sources and are persistent and ubiquitous in the aquatic environment. The risks posed by microplastics as vectors and habitats for potentially harmful microbial groups and antibiotic resistance genes (ARG) are relatively unexplored in South Africa. The relationship between microplastics and microorganisms’ reels in the threat of creating superbugs that may proliferate resistance profiles of potentially pathogenic bacteria in the environment. This study aimed to characterize the bacterial community composition (BCC) and quantify the ARG on microplastics from freshwater settings. The first objective was to analyze the process of microbial colonization on microplastics and wood substrates through Scanning Electron Microscopy (SEM) using microcosm experiments of river water and wastewater treatment effluent (WWTe). This was followed by next generation sequencing (NGS) targeting the 16S rRNA gene to characterize the BCC on the substrates and surrounding water (river and WWTe) post-incubation. The third objective was to isolate microplastics and collect water samples from across five sites from the Mooi River, followed by the identification of the microbial consortia by 16S rRNA metabarcoding of the environmental DNA samples. The fourth objective was to investigate the prevalence of the intl1 gene and six AmpC β-lactamase gene groups, including ARG copy numbers (AmpC gene groups) on all microplastics, wood, and water samples. The microcosms’ studies showed that substrates can be colonized within 48 hours after incubation. Moreover, the surface properties of the substrates and the nutrient load in water play a role in the process of colonization. SEM images revealed biofilm development in river water is slower than in nutrient-rich water (WWTe) and from environmental microplastic samples, the images revealed a diverse microbial community of bacterial biofilms and eukaryotic organisms inhabiting the surfaces. Two hundred and six (206) microplastics were isolated from 1800 litres of surface running water following in situ filtrations through 300-75 μm pore size metal sieves at each site. Bacterial diversity indices of both microcosm and environmental samples showed that the BCC on microplastics was less rich, diverse, and even than in the surrounding water. Furthermore, the bacterial profiling showed that microbial phyla dominance fluctuated during the incubation period. The BCC dominance on microplastics from the environment differed from that of the surrounding water at each respective site. LEfSe biomarker analysis showed that the families Aeromonadaceae, Enterobacteriaceae, and Legionella were significantly higher (p < 0.05) on microplastics than in water samples, including some of the genera known to contain potentially pathogenic members which show resistance against antibiotics. Conventional PCR revealed that the intl1 gene was present in WWTe samples from the microcosm experiments and in environmental samples that contained more than five AmpC β-lactamase gene groups. Masters

Highlights • Serratia marcescens 39_H1 could enhance the hydrolysis of lignocellulosic biomass. • Serratia marcescens 39_H1 is a plant growth promoting organism. • Genome analysis showed diverse potential biotechnological application of organism. • This is an original report on the hydrolytic and acidogenic attributes ofSerratia marcescens 39_H1 for biogas production., Here, we report a high quality annotated draft genome of Serratia marcescens 39_H1, a Gram-negative facultative anaerobe that was isolated from an anaerobic digester. The strain exhibited hydrolytic/acidogenic properties by significantly improving methane production when used as a single isolate inoculum during anaerobic digestion of water hyacinth and cow dung. The total genome size of the isolate was 5,106,712 bp which corresponds to an N50 of 267,528 and G + C content of 59.7 %. Genome annotation with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) predicted a total of 4,908 genes of which 4,755 were protein coding genes; there were no plasmids detected. A number of genes associated with hydrolytic/acidogenic activities as well as other metabolic activities were identified and discussed.

Until recently, research has focused on Clostridium perfringens in clinical settings without considering environmental isolates. In this study, environmental genomes were used to investigate possible antibiotic resistance and the presence of virulence traits in C. perfringens strains from raw surface water. In silico assembly of three C. perfringens strains, DNA generated almost complete genomes setting their length ranging from 3.4 to 3.6 Mbp with GC content of 28.18%. An average of 3,175 open reading frames was identified, with the majority associated with carbohydrate and protein metabolisms. The genomes harboured several antibiotic resistance genes for glycopeptides, macrolide–lincosamide–streptogramin B, β-lactam, trimethoprim, tetracycline and aminoglycosides and also the presence of several genes encoding for polypeptides and multidrug resistance efflux pumps and 35 virulence genes. Some of these encode for haemolysins, sialidase, hyaluronidase, collagenase, perfringolysin O and phospholipase C. All three genomes contained sequences indicating phage, antibiotic resistance and pathogenic islands integration sites. A genomic comparison of these three strains confirmed high similarity and shared core genes with clinical C. perfringens strains, highlighting their health security risks. This study provides a genomic insight into the potential pathogenicity of C. perfringens present in the environment and emphasises the importance of monitoring this niche in the future.

Anthropogenic activities in catchments used for drinking water production largely contaminates source waters, and this may impact the quality of the final drinking water product. These contaminants may also affect taxonomic and functional profiles of the bacterial communities in the drinking water. Here, we report an integrated insight into the microbiome and water quality of four water treatment plants (NWC, NWE, WCA and NWG) that supply portable water to communities in South Africa. A new scoring system based on combined significant changes of physicochemical parameters and microbial abundance from raw to treated water was used to evaluate the effectiveness of the treatment plants at water purification. Physicochemical parameters which include total soluble solids, turbidity, pH, nitrites and phosphorus among others, were measured in source, treated, and distributed water. There were general statistically significant (P ≤ 0.05) differences between raw and treated water, demonstrating the effectiveness of the purification process. Illumina sequencing of the 16S rRNA gene was used for taxonomic profiling of the microbial communities and this data was used to infer functional attributes of the communities. Structure and composition of the bacterial communities differed significantly (P < 0.05) among the treatment plants, only NWE and NWG showed no significant differences (P > 0.05), this correlated with the predicted functional profile of the microbial communities obtained from Phylogenetic Investigation of Communities by Reconstruction of Observed States (PICRUSt), as well as the likely pollutants of source water. Bacteroidetes, Chlorobi and Fibrobacteres significantly differed (P < 0.05) between raw and distributed water. PICRUSt inferred a number of pathways involved in the degradation of xenobiotics such as Dichlorodiphenyltrichloroethane, atrazine and polycyclic aromatic hydrocarbons. More worryingly, was the presence of pathways involved in beta-lactam resistance, potential pathogenic Escherichia coli infection, Vibrio cholerae infection, and Shigellosis. Also present in drinking and treated water were OTUs associated with a number of opportunistic pathogens.

Antimicrobial resistance is an imminent threat to the effective prevention and treatment of bacterial infections and alternative antimicrobial strategies are desperately needed. Antimicrobial peptides (AMPs) may be promising alternatives to current antibiotics or act as adjuvants to enhance antibiotic potency. Additionally, the use of biodegradable lipid nanoparticles can enhance the antibacterial activity of antibiotics and antimicrobial peptides. In this study, the interaction of the AMPs, nisin Z and melittin, with conventional antibiotics was investigated on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. The effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also evaluated. Findings revealed that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotic adjuvant EDTA significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram-positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections.

The adaptation of Zulu (Nguni) sheep (Ovis aries) to environmental stress and survival under extensive conditions makes them uniquely important to rural Nguni farmers of South Africa. Here, the faecal bacterial community of five Zulu sheep populations managed under extensive conditions across summer and winter seasons was investigated in order to understand the influence of prevailing seasonal factors. Bacterial operational taxonomic units (OTUs)/species (at 97% 16S rRNA gene similarity) in Zulu sheep faeces were more diverse in winter than in summer at most (80%) sites and varied between seasons at specific sites. Firmicutes was the most abundant phyla in both summer and winter seasons, while the relative abundance of Actinobacteria reduced in 80% of sites from summer to winter. The genera (or family) such as Akkermansia, Eubacterium coprostanoligenes group, Intestinibacter, R-7 group (family Christensenellaceae), Ruminococcus, Ruminoclostridium, Treponema and UCG-005 (family Ruminococcaceae) were relatively more abundant and belonged to a ‘core microbiome’ of Zulu sheep faeces. Between seasons, Acinetobacter, Jeotgalicoccus, Methanobrevibacter, Phascolarctobacterium and Planomicrobium were differentially abundant. Overall, results suggest increased richness and diversity of bacteria from summer to winter which may be related to spatio-seasonal variations in grazing management, forage types and availability. This observation serves as baseline evidence, justifying further controlled studies investigating, amongst other factors, effects of forage type and availability across seasons on ruminal microbiota of Zulu sheep grazing in communally managed rangelands. Significance: Spatio-seasonal dynamics in the bacterial community of Zulu sheep faeces suggest differences in forage type and availability across sites potentially influence faecal bacteria of Zulu sheep. The study provides a basis for further controlled studies investigating the influence of environmental factors on rumen and faecal microbiomes of Zulu sheep. Open data set: https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA356736

North-West University, Potchefstroom Campus MSc (Biochemistry), North-West University, Potchefstroom Campus Some of the core issues faced today in developed and developing countries are global energy protection and better utilisation of natural resources. Anaerobic digestion is currently one of the options available that may assist in this regard and research has been done on a wide variety of substrates for anaerobic digestion. Several phases can be observed during a typical batch setup for anaerobic digestion, with the lag phase being of particular interest as the microbial communities present typically adapts to substrate during this phase. The lag phase is also typically one of the longest phases observed in batch reactor setups. Pig slurry is readily available and poses a threat to environmental, including over-fertilisation and eutrophication. The aim of the current study was to determine whether back inoculation will reduce the observed lag time before biogas production occurs in batch reactors using pig slurry as the primary substrate. Batch reactors were used in this study, during which back inoculation was done and sample were taken at key intervals. Samples taken from the batch reactors were subjected to physical and chemical analysis, as well as molecular analysis of the microbial communities present during the sample times using metabarcoding on the MiSeq next generation sequencing (NGS) platform. From the NGS data community composition and predicted metabolic activities were derived. Low biogas yields were observed in this study and could be attributed to factors such as imbalanced nutrient levels and ratios between the main functional groups. Methanogenesis was not optimal as the abundance of methane producing microorganism was inhibited The major phyla observed in the microbiomes in the current study included Firmicutes, Bacteroidetes, Proteobacteria, Synergistetes, Euryarchaeota, Chloroflexi, Actinobacteria and Atribacteria, and the major families were Clostridiaceae 1, Synergistaceae, Ruminococcaceae, Rikenellaceae, Marinilabiaceae, Porphyromonadaceae, Erysipelotrichiaceae and Methanosarcinaceae. These are typically phyla and families associated with the AD processes. The predicted metabolic activities indicated the highest metabolic activity to be unknown, followed by ammonia oxidation. These issues primarily relate to the small size of the bench-top reactors and the state of the seeding sludge. The back inoculation was successful in reducing the observed lag phase, even though the C:N ratio was not optimal. Masters

PhD (Microbiology), North-West University, Potchefstroom Campus An intensive literature review has revealed that there is a global lack of research on AmpC beta-lactamase antibiotic resistance genes in the aquatic environment. The possibility of the dissemination of AmpC genes within and among human settlements, and the consequent spreading and impact of antibiotic resistance due to their prevalence, are matters of grave concern as these genes pose potential threats not only to the environment, but to human health as well. Moreover, a dire implication of the prevalence of these genes in the environment and their exposure to selective pressures and unprecedented evolution may be that novel resistance mechanisms are introduced in clinical settings that may limit treatment options with beta-lactam antibiotics. This group of antibiotics is one of the most preferred bacterial treatment options in human, veterinary and agricultural settings and it is therefore imperative to manage resistance to any of these antibiotics. Therefore, the overarching aim of this study was to explore AmpC beta-lactamase gene intricacies in aquatic systems. To achieve this aim, various intersecting studies were conducted. The first assessed the prevalence and diversity of AmpC beta-lactamase genes in aquatic systems globally by reviewing to what extent research has been conducted on AmpC genes in aquatic environments. A second study determined the prevalence and genetic diversity of AmpC genes in South African rivers. The need for suitable quantification methods of AmpC genes prompted the third study that evaluated quantification methods of AmpC genes for environmental DNA samples. The final study determined which environmental parameters are associated with the prevalence of clinically relevant AmpC genes in aquatic environments. The systematic review determined that very few studies had explored AmpC genes in aquatic environments in the global context. By utilising experimental investigations, it was determined that clinically relevant AmpC genes are present in South African rivers that run either through densely or more sparsely human populated areas and that these genes are genetically diverse from gene variants found on sequence databases. The preferred method for the quantification of AmpC genes is the conventional quantitative PCR because it is forgiving regarding the unknown nature of environmental samples and is time efficient and financially viable when investigating numerous samples. It was also determined that there are limited significant associations between AmpC genes and environmental parameters and strong significant associations with population density. This suggests that AmpC genes are pre-selected and present in the aquatic environment due to human activities that cause pollution. Therefore, bacteria harbouring these genes are indicators of the presence of these genes in aquatic systems associated with faecal contamination. This study thus demonstrated the need for intensified research into AmpC beta-lactamase genes not only in South Africa, but globally for the sake of environmental and human health. Doctoral

Soil microbial communities are suitable soil ecosystem health indicators due to their sensitivity to management practices and role in soil ecosystem processes. Presently, information on structural and functional differentiation of bacterial communities in post-coal mining reclamation soils of South Africa is sparse. Here, bacterial communities in three post-coal mining reclamation soils were investigated using community-level physiological profiling (CLPP), enzyme activities, and next-generation sequencing of 16S rRNA gene. Inferences were drawn in reference to adjacent unmined soils. CLPP-based species diversity and proportionality did not differ significantly (P > 0.05) whereas activities of β-glucosidase, urease and phosphatases were significantly (P P P > 0.05), suggesting redundancy in bacterial community functions between reclamation and unmined soils. Silt content, bulk density, pH, electrical conductivity, Na and Ca significantly influenced soil bacterial communities. Overall, results indicate that bacterial community structure reflects underlying differences between soil ecosystems, and suggest the restoration of bacterial diversity and functions over chronological age in reclamation soils.

MSc (Microbiology), North-West University, Potchefstroom Campus In the North West Province, surface water is polluted from sources such as surface runoff from agricultural settings, storm-water runoff as well as sewage from urban locations, and mining. This study aimed to evaluate the water quality of the Loopspruit River by analysing the physico-chemical and microbiological aspects of the Loopspruit River. Six objectives were set to achieve this. The first objective focussed to identify sampling sites using Geographic Information Systems (GIS) and aerial photographs to ensure. The second and third objectives set out to determine the water quality of two wet seasons and two dry seasons (2018 to 2019) and to analyse the historic data. The fourth was the isolation and identification of possible faecal associated micro-organisms, including Clostridium, presumptive E. coli and Enterococci species. Objectives five and six were to create predictive physico-chemical and microbiological point source contamination visual representation with historic data and data obtained from this study and to compare the outcomes. These isolated bacterial species (objective 4) were used to create a faecal point source pollution visual representation with their associated land-use contributions that were deposited within the Loopspruit River. Historic data were used to develop a predictive geospatial visual representation of the physico-chemical parameters to illustrate the land-use contributions to possible pollution in the Loopspruit River. The historic and current water quality data were visually represented using GIS software for water quantity. The results visually indicated that high magnesium (±41.30 mg/L) levels are prominent in mining and urban areas and pH levels (±9.49) are high in the dam area - all above normal levels. Antibiotic profiles indicated an increase in Multiple Antibiotic Resistances (MAR) with increased urban activities. Genes associated with antibiotic resistance were also detected. These included the intI1 integrase gene and the FOX AmpC β-lactamase gene. The LC/MS analyses revealed an excess amount of Ampicillin in the Loopspruit River with a risk value of 637.95 where the predicted no-effect concentration is 75. The bacterial diversity showed the highest diversity at less polluted areas whereas, in contrast, more pollution-prone areas showed less bacterial diversity. Dominating at all the sites were Proteobacteria, followed by Bacteroidetes, Cyanobacteria, Actinobacteria and Verrucomicrobia, having a broad variation to the total contribution from sample to sample. Finally, the predicted metagenome analysis revealed a correlation between the physico-chemical parameters and the observed taxonomic units (OTU). The temperature had negative correlations with Patescibacteria, Nanoarchaeaeota and Firmicutes (p

AAfter a cluster of human pneumonia cases in Wuhan City (China) on 7 January 2020, a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), was identified as the causative agent of the disease COVID‐19 (World Health Organization 2020). It quickly became a global pandemic and continues to rapidly spread across the world, having infected almost 4 million confirmed cases and caused more than 265 000 deaths at time of publication, far surpassing the total cases of SARS and Middle East respiratory syndrome (MERS)

North-West University, Potchefstroom Campus MSc (Environmental Sciences), North-West University, Potchefstroom Campus Water sources in the North West Province (NWP) are made up of groundwater, surface water and re-useable effluent. However, major water challenges are experienced in the NWP when quality and quantity are considered. The NWP is regarded a water stressed Province, due to its limited water sources. Communities in the NWP rely greatly on ground and surface water for their water provision. Urban areas such as Mahikeng, Lichtenburg, and Coligny rely solely on groundwater. In these regions, groundwater undergoes partial purification treatments. However, communities in rural areas and informal settlements use groundwater untreated. This Province is also known for its great participation in agriculture because of its rich production in crops such as maize, sorghum and sunflower. This also requires a great quantity of water, hence stressing available water sources even more. The aim of this study was to characterize Clostridium sp., Aeromonas sp. and heterotrophic count bacteria in selected groundwater systems in the NWP. During the course of this study, groundwater was obtained across three seasons: winter, summer and autumn. Groundwater temperature fluctuated between the seasons, with the lowest and highest water temperatures observed during summer autumn, respectively. The pH of the groundwater systems was relatively neutral throughout the seasons. However, parameters such as TDS, salinity as well as CODs were exceptionally high in all seasons. Chemical parameters such as nitrates and phosphates were observed in low concentrations in all seasons. The identification and characterization of Clostridium sp., Aeromonas sp., and HPCs was achieved utilizing culture-based methods. A total of 91 isolates were enumerated and identified using 16S rRNA PCR and sequencing. HPCs were observed in high levels with Bacillus being the most predominant HPC species seen across all three seasons. Other species of concern that were identified include Citrobacter sp. and Escherichia sp. Furthermore, Clostridium sp., and Aeromonas sp. were found present in the groundwater systems during summer, however at very low levels. Three Clostridium species (C. perfringens, C. sordellii and C, tepidum) and a single Aeromonas species (A. hydrophila) were identified in this study. Out of all isolates obtained, 80% were beta-hemolytic while 70% of the HPCs were positive for DNase production. Ninety percent of the Aeromonas species were positive for gelatinase production whilst none of the isolated microorganisms tested positive for lecithinase production. The production of extracellular enzymes in microorganisms is a clear indication that they could be regarded as opportunistic pathogens. The antibiotic resistance patterns of the isolated microorganisms was achieved using the Kirby-Bauer Disk diffusion method. Aeromonas sp., and HPC species were exposed to a total of 11 antibiotics and displayed resistance towards Ampicillin (10?g), Penicillin-G (10?g) and Trimethoprim (5?g). The isolated organisms were also screened for antibiotic resistant genes (ARGs) using endpoint PCR. Additionally, the Aeromonas isolates (4) were screened to determine the presence of 10 virulence genes. Of the five ARGs screened, 50 % isolates harboured ampC and blaTEm. Whilst none of the screened Aeromonas species harboured any of the screened virulence genes. The physico-chemical quality of the groundwater systems of interest clearly illustrated that the parameters were favourable for bacterial growth. Whilst the presence of pathogenic multi-drug resistant organisms such as Clostridium, Aeromonas, Bacillus and Escherichia sp. harbouring ARBs is a cause for concern. Thus, the findings of this study indicate that groundwater systems in the NWP are a potential safety hazard and require special attention If the water is to be used for consumption. Masters

PhD (Environmental Sciences), North-West University, Potchefstroom Campus Source water is becoming a scarce resource in South Africa, especially with the extreme periods of drought that the country has faced the last few years. Continuous pollution of rivers and dams is drastically deteriorating the water quality. This puts more pressure on water purification plants to ensure the water is adequately treated and safe drinking water is produced. However, some water purification plants may not have the infrastructure or financial resources to produce drinking water of acceptable quality. Therefore, there is a need for inexpensive and rapid solutions to produce safe drinking water. In this study, three decision-making tools (Hazard Analysis Critical Control Point (HACCP) concept, Artificial Neural Networks, Evolutionary Algorithms and isolation of VBNC bacteria) were evaluated for the improved monitoring of treatment processes. The HACCP concept was evaluated at three water purification plants (Plant A, Plant B and Plant C). This study demonstrated that monitoring certain parameters after each step in the treatment process was useful to identify process failures. Results for Plant B indicated that due to a failure in the filtration process, unacceptable turbidity levels were present in the drinking water. The second decision-making tool investigated in this study was the application of ANNs and evolutionary algorithms (EAs) at Plant A, Plant B and Plant C. Results from this study indicated that the combination of ANNs and EAs resulted in the accurate prediction of electrical conductivity (EC) in the drinking water of Plant A and Plant C. Additionally, this study indicated the importance of consistent monitoring. A prediction model for Plant B could not be generated due to a lack of historical data. The application of EAs resulted in the formation of accurate predictive rule-sets for Plant A and Plant C. The last decision-making tool was the recovery of Escherichia coli (E. coli) in drinking water. Results of this study indicated that viable-but-non-culturable E. coli was recovered from drinking water. Two of the isolates were identified as E. coli O177 and O157. This is concerning as O177 and O157 are Shiga toxin-producing strains of E. coli and could pose a serious health risk. In addition, this study indicated the development of a new resuscitation method adapted from a current method. Based on all the results obtained, decision-making tools for the improved monitoring of water treatment processes was demonstrated. Doctoral

North-West University, Potchefstroom Campus PhD (Microbiology), North-West University, Potchefstroom Campus At present, there is no comprehensive soil quality assessment practice for the coal mining industry in South Africa. Studies have shown that belowground soil biota are crucial to soil ecosystem functioning and are sensitive bioindicators for soil quality monitoring. Due to the limitations of some previously developed methods for analysing microbial community structure, a more robust approach involving high-throughput culture-independent molecular techniques was utilised to assess post-coal-mining reclamation soils for potential ecosystem recovery, support function and potential microbial bioindicators. Also, the potential impact of soil physicochemical properties in shaping soil biotic communities were assessed. The study was conducted in three stages. Firstly, the potential contribution of soil stockpiles to post-mining reclamation success was assessed by analysing arbuscular mycorrhizal fungal (AMF) spore density and viability as well as microbial community diversity and structure. Overall, results suggest that AMF spore density in stockpiles do not differ significantly (P 0.05) differences in microbial community richness and diversity amongst samples while enzyme activities were highly variable within-sites. The assemblages of the obligate plant symbiont, AMF, were less differentiated when compared to other microbial groups suggesting that AMF assemblages could be less suitable bioindicators of ecosystem recovery. Some genera with soil quality indicator potential such as Acidothermus, Bryobacter and Halingium, as well as plant-growth promotion potentials such as Mesorhizobium, Bradyrhizobium and Microvirga were relatively more abundant across soils, whereas a vast majority of other microbial species and their functions in reclamation soils are still largely unknown. Lastly, the capability of the soil to serve as a habitat to support soil biota association and functions was assessed using an ecotoxicological approach by utilising earthworms as bioindicators. Endpoints such as avoidance behaviour, growth, reproduction and mortality of earthworms were assessed. There was no evidence to suggest that the ecosystem habitat function of stockpile and reclamation soils is significantly limited compared to the Organisation of Economic Cooperation and Development’s artificial control soil. Nevertheless, support functions were highest in unmined soils as determined by the earthworm avoidance behaviour test. Data generated in this study strongly supports that microbial species richness and diversity levels are restored over the years since reclamation, though community composition and structure still differ from the pre-disturbance community. Furthermore, microbial communities of reclamation soil environments are predominantly shaped by pH, phosphorus and nitrogen sources. Overall, bacterial communities are the most responsive and indicative of ecological changes during soil ecosystem restoration. In conclusion, as molecular methods are not without limitations, and because the soil ecosystem environment is governed by an interplay of factors, a comprehensive soil monitoring programme for post-mining reclamation soils in South Africa must comprise a combination of physicochemical properties and microbial community diversity indices as part of a minimum dataset. Furthermore a responsible stockpiling procedure which entails proper excavation and storage of topsoil, as well as the inclusion of microbial inoculants during post-mining reclamation operations, is strongly recommended. Such an approach will help improve coal-mining disturbed soil quality as well as facilitate a quicker ecosystem recovery period. Doctoral

PhD (Microbiology), North-West University, Potchefstroom Campus The detection of methicillin-resistant Staphylococcus aureus (MRSA) in wastewater system from clinical sources has posed a public health challenge in many countries worldwide. Of a greater concern is the discharge of untreated or improperly treated hospital effluents into wastewater, which has been identified as possible hotspot for antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs). MRSA, a distinct form of Staphylococcus aureus (S. aureus) showing resistance to methicillin, is widely reported as a nosocomial pathogen. However, there is paucity of data and limited reporting about its occurrence in non-clinical environments, such as wastewater in Nigeria and South Africa. This present study is on the characterization of S. aureus, MRSA and other staphylococci from clinical and environmental sources in Ile-Ife, Nigeria and Potchefstroom, South Africa. The first part of the thesis emphasized the significance of the genus Staphylococcus and their importance as opportunistic pathogens, causing many diseases and infections in humans. The rationale, aim and objectives of the study were also highlighted. The second part of the study was a systematic review aimed to assess the prevalence of mecA gene in Staphylococcal isolates from clinical and environmental (wastewaters) sources. For this review, 50 peer reviewed full texts (35 clinical and 15 wastewaters) from 19 countries and 5 continents across the globe were selected based on pre-determined criteria. Specifically, there is insufficient information on mecA gene in Staphylococcus spp. from aquatic or environmental wastewaters (30.0%). Further, within the database search period (2000 – 2019), only very few studies (10) on Staphylococcus spp. with reference to mecA gene could be traced and retrieved from developed countries. Data extracted revealed that S. aureus and other Staphylococcus spp. are potential contributors of antibiotic resistance bacterial (ARB) infections from both clinical and environmental settings. In the third part of the study, 76 Staphylococcus spp. were isolated and characterized from hospital and environmental sources in Ile-Ife, Nigeria. These comprised of 40.7% (31/76) coagulase-positive (S. aureus) and (45/76) 59.3% coagulase-negative staphylococci (CoNS) constituting S. sciuri and 3 other rare species - S. xylosus, S. warneri and S. kloosi which have not been widely reported. Furthermore, the study confirmed the presence of resistance and virulence genes of public health importance in the Staph. spp. Sixty-three (63%) of the isolates with these genes originated from clinical samples, while 37% were from environmental sources. Interestingly, the presence of mecA gene was also confirmed in coagulase-negative staphylococci (CoNS) which have been previously considered low-risk or non-pathogenic. This calls for concern since their possession of resistance and virulence genes signal that these species can endanger human health and life. The fourth part of the study also focused on Staphylococcus spp. specifically MRSA and mecA gene detection in a wastewater treatment plant (WWTP) in Potchefstroom, South Africa. In the WWTP, 35 staphylococcal isolates of 7 different species were isolated. These included 3 CoNS which are uncommon: S. cohnii, S. nepalensis and S. arlettae. The study confirmed the presence of MRSA and multidrug resistant (MDR) Staphylococcus spp. at the final effluent point which had undergone chemical treatment by chlorination. It was recommended that a more effective treatment plan or modification procedures should be adopted especially if the water is to be reused. The fifth and final part of the study, reported a high quality draft genomes of 8 CoNS isolates obtained from clinical and environmental settings in Nigeria and South Africa. The isolates were identified as S. lentus (1 from South Africa), S. cohnii (3 from Nigeria and 2 from South Africa) and S. haemolyticus (1 from Nigeria and 1 from South Africa). The isolates possessed an open pan-genome, with a few genetic barriers to horizontal gene transfer. Clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas protein system (CRISPR/Cas) were identified in one of the 8 isolates. This study has generated readily available information on the local antimicrobial resistance patterns of potential bacterial pathogens which could assist in improved assessments of human health risks. Doctoral

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), a major insect pest of maize in sub-Saharan Africa, has developed high levels of non-recessive resistance to Cry1Ab toxin expressed in genetically modified Bt maize. Multiple resistance mechanisms to various Cry toxins have been identified in Lepidoptera, but no study has yet been done to determine the mechanism of Cry1Ab resistance in B. fusca. Therefore, the larval transcriptome of B. fusca was sequenced, de novo assembled and characterized. Differential expression analysis was performed to compare gene expression profiles of Cry toxin challenged and unchallenged neonate larvae to assess the molecular basis of the defence mechanism employed by this insect. Several genes associated with Cry toxin resistance in other lepidopteran pests were detected in B. fusca. Results suggest that differential expression of metabolic and immune-related genes might explain Cry1Ab toxin defence in this pest (supplemental file). Transcript expression profiles of neonates demonstrated that 33.59% and 60.31% of the 131 differentially expressed genes were upregulated and downregulated in the toxin-challenged neonate larvae, respectively. Transcripts were grouped into two subclusters according to the similarity of their expression patterns. Transcripts in subcluster 1 were moderately upregulated in the toxin-challenged neonate larvae, and, conversely, downregulated in the unchallenged neonate larvae. The solute carrier organic anion transporter, which is involved in insecticide detoxification, was upregulated in the toxin-challenged neonate larvae. Conversely, most of the transcripts in subcluster 2 were moderately downregulated in the toxin-challenged neonate larvae, and upregulated for neonates feeding on non-challenged maize. Four unidentified transcripts were extremely down-regulated in the toxin-challenged neonate larvae, and upregulated in the unchallenged neonate larvae. Further studies are recommended to establish if there is a direct correlation between these differentially expressed genes and the observed resistance. Elucidation of such defence mechanisms is crucial for developing insect resistance management strategies to ensure sustainable use of genetically modified maize in Africa. Nevertheless, this is the first study on gene expression profiles of B. fusca strains challenged with Cry toxin. The transcriptome characterized in this study provides a significant resource base for future studies on B. fusca and contributes to understanding some of the gene regulation and signalling networks involved in the defence of B. fusca against Cry1Ab toxin.