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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

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.

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

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).

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015 Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. Masters

This study aimed to identify bacterial community structures in the Vaal River using PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) and high-throughput sequencing. The impact of physico-chemical characteristics on bacterial structures was investigated through multivariate analysis. Samples were collected from 4 sampling stations along the Upper Vaal River during winter (June 2009) and summer (December 2010). Physico-chemical analysis was conducted on-site. Additional physico-chemical data were obtained from statutory bodies. DNA was directly isolated from water samples and PCR amplified using universal bacterial primer pairs. PCR products were subjected to DGGE fingerprinting and high-throughput sequencing, followed by Shannon-Weaver diversity calculations, cluster analysis and multivariate analysis. Physico-chemical parameters did not exceed the prescribed South African water quality standards for domestic use, aquatic ecosystems, livestock watering and irrigation. DGGE banding patterns revealed similar bacterial community structures for 3 of the 4 sampling stations. PCA and RDA indicated that pH, water temperature and inorganic nutrient concentrations could be used to explain changes in bacterial community structures. High-throughput sequencing data showed that bacterial assemblages were dominated by common freshwater groups: Cyanobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Other freshwater phyla such as Deltaproteobacteria, Epsilonbacteria, Acidobacteria, Verrucomicrobia, Firmicutes, Fusobacteria, Flavobacteria and Fibrobacteres were found in low proportions. This study provides an overview of the dominant bacterial groups in the Upper Vaal River and the impact of environmental changes on bacterial diversity Water Research Commission (WRC) and North-West University http://www.wrc.org.za/Pages/KH_WaterSA.aspx?dt=5&L0=1&L1=4 http://www.wrc.org.za/Pages/DisplayItem.aspx?ItemID=10288&FromURL=%2fPages%2fKH_WaterSA.aspx%3fdt%3d5%26ms%3d%26d%3dVolume%26e%3d39+No.+3%2c+WISA+2012+Special+Edition+2013%26start%3d1 http://dx.doi.org/10.4314/wsa.v39i3.7