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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.

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.

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.

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

At present, there is no comprehensive soil quality assessment practice for soil stockpiles in the South African coal mining industry. Soil microorganisms and enzymes are suitable indicators for soil quality monitoring. Therefore, this study investigated the microbial community and enzyme (beta-glucosidase and urease) activities in soil stockpiles of opencast coal mines in the coal-rich region of South Africa. Soil stockpiles of three opencast coal mines were sampled at depths of 0–20 cm (‘topsoil’) and >20 cm (‘subsoil’) across three seasons. Beta-glucosidase and urease activities were mostly higher in soil stockpiles than in unmined soils and were significantly influenced (P < 0.05) by the interaction of site and seasonal factors. However, analyses of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of partial 16S rRNA gene and internally transcribed spacer 2 (ITS2) sequences revealed higher microbial diversity in unmined (reference) soils compared to soil stockpiles across all seasons. Redundancy analysis further revealed that microbial communities of topsoil were not significantly (P > 0.05) influenced by soil properties, whereas microbial communities of subsoils were significantly (P < 0.05) influenced by pH, organic carbon, total nitrogen and phosphorus contents. Furthermore, operational taxonomic units (OTUs) belonging to genera of known phytobeneficial species such as Azomonas, Aureobasidium, Phialocephala, Phoma and Sordariomycetes were detected in these soils. Overall, results suggest that the microbial community structure and diversity observed in stockpiles is impaired (compared to the unmined site), although variations in the microbial community structure of soil stockpiles across seasons are site-specific. The impaired microbial community of stockpiles may have negative implications on soil biological processes driven by microbes; especially those that are critical for nutrient cycling and ecosystem sustainability. More importantly, such alteration in soil biodiversity may impair post-mining land use capability of stockpile soils

Bacterial communities in human-impacted rivers and streams are exposed to multiple anthropogenic contaminants, which can eventually lead to biodiversity loss and function. The Wonderfonteinspruit catchment area is impacted by operational and abandoned gold mines, farms, and formal and informal settlements. In this study, we used 16S rRNA gene high-throughput sequencing to characterize bacterial communities in the lower Wonderfonteinspruit and their response to various contaminant sources. The results showed that composition and structure of bacterial communities differed significantly (P

Mafikeng, the capital of the North West Province, receives water from two sources, namely the Molopo eye and the Modimola dam. Once treated, the potable water is mixed and supplied to the city via distribution systems. This study was designed to assess the quality of drinking water in Mafikeng and also to determine whether the water from the two sources has an impact on the mixed water quality. Physico-chemical parameters and bacteriological quality (faecal coliforms (FCs), total coliforms (TCs), heterotrophic bacteria and Peudomonas spp.) was monitored at three drinking water sites weekly for 4 months. The results revealed that the physico-chemical quality of the water was generally acceptable. The pH ranged from 5.7 ± 0.18 to 8.6 ± 0.14, the temperature ranged from 18.3 ± 0.69 to 25.1 ± 0.69 °C and the total dissolved solids (TDS) ranged from 159.9 ± 22.44 to 364.4 ± 12.44 mg/l. These values are within the target water quality range for drinking water as prescribed by WHO, Department of Water Affairs and SANS 241. What is of concern was the microbial quality of the water. FCs, TCs, heterotrophic bacteria and Pseudomonas spp. were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas spp. (>100/100 ml).

Evidence suggests that heterotrophic plate count (HPC) bacteria may be hazardous to humans with weakened health. We investigated the pathogenic potential of HPC bacteria from untreated borehole water, consumed by humans, for: their haemolytic properties, the production of extracellular enzymes such as DNase, proteinase, lipase, lecithinase, hyaluronidase and chondroitinase, the effect simulated gastric fluid has on their survival, as well as the bacteria's antibiotic-susceptible profile. HuTu-80 cells acted as model for the human intestine and were exposed to the HPC isolates to determine their effects on the viability of the cells. Several HPC isolates were α- or β-haemolytic, produced two or more extracellular enzymes, survived the SGF treatment, and showed resistance against selected antibiotics. The isolates were also harmful to the human intestinal cells to varying degrees. A novel pathogen score was calculated for each isolate. Bacillus cereus had the highest pathogen index: the pathogenicity of the other bacteria declined as follows: Aeromonas taiwanensis>Aeromonas hydrophila>Bacillus thuringiensis>Alcaligenes faecalis > Pseudomonas sp. >Bacillus pumilus>Brevibacillus sp. >Bacillus subtilis>Bacillus sp. These results demonstrated that the prevailing standards for HPCs in drinking water may expose humans with compromised immune systems to undue risk.

Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013 Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. Masters