In a series of field and watershed scale studies, the genetic diversity of Escherichia coli in secondary habitats (e.g. soils and sediments) of an agricultural watershed was assessed in order to examine the dynamics of E. coli inhabiting these matrices and to determine their contribution to waterborne populations. Using replicated field plots, persistent subpopulations of E. coli were observed to be significantly affected by hillslope position due to inherent differences in soil texture and moisture content. The dynamics of E. coli populating tile drainage effluent in a working cultivated field were monitored and it was observed that putatively naturalized E. coli dominated the effluent after approximately 55 days following manure amendments. The contribution of tile drainage effluents to the waterborne E. coli population in an adjacent stream was exponentially related to tile discharge rates, regardless of whether the effluent was populated by manure-associated or naturalized E. coli strains. Streambed E. coli populations differed according to stream geomorphological features, with strains responding to sediment texture and water velocity distributions among the features. In a temporal study of sediment E. coli, population turnover was observed to be affected by sediment redistribution in highenergy stream reaches and was stabilized by immigration from adjacent catchment sources in low-energy stream reaches. Reach-specific connectivity between sediment and waterborne E. coli populations was observed in this watershed. Reach- and catchmentscale hyporheic processes are speculated to be occurring, which may be in part influenced by strain-dependent attachment behaviour of E. coli strains in disjoint stream reaches influenced by different catchment sources of E. coli. The attachment of waterborne E. coli to suspended particles was observed to be associated with land use, water quality and suspended particle variables. The relationship of land use type to particle attachment reinforces the hypothesis that strain-specificity in attachment behaviour can affect the transport of E. coli in fluvial systems. This work provides evidence that putatively naturalized strains in cultivated fields can contribute a large part to waterborne E. coli, and that reach-specific hydrological factors need to be considered when relating sediment- to waterborne E. coli in fluvial systems.

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

In low-resource contexts, household-level point-of-use water treatment (POUWT) techniques are the final, and sometimes only, barrier against waterborne illnesses, and in these and other water-related applications, health risks can be quantified using one of two methods. Firstly, Escherichia coli (or other indicator organism) counts can be used to monitor water and determine adherence to a health-based limit (i.e. compliance monitoring). Secondly, E. coli can be used to conduct a quantitative microbial risk assessment (QMRA), indicating the level of protection conferred by a given POUWT device by spiking test water with E. coli to ascertain a reduction efficacy relative to that target organism, a process referred to as challenge testing, which is typically carried out in a laboratory context. Although both methods are well established, both have scope for improvement for effective field application in low-resource contexts. Regarding compliance monitoring, I assessed the performance of a new low-cost field kit for E. coli enumeration, which was designed by others. I also assessed the feasibility of re-using some disposable materials, in terms of sterility and mechanical wear. The use of the new low-cost field kit was successful during the fieldwork campaign; however, re-using disposable materials introduced a relatively high occurrence of false positive results during E. coli enumeration. Use of the new low-cost field kit can reduce financial barriers, thus enabling greater water quality testing coverage. Regarding challenge testing, the aim of this study was to adapt current protocols to assess the household performance (as opposed to laboratory performance) of POUWT techniques. I developed a conceptual framework to conduct Field Challenge Tests (FCT’s) on POUWT techniques, using a probiotic health supplement containing E. coli as the challenge organism. I successfully carried out a FCT in Malawi with limited resources, verifying FCT viability. Applications of such FCT’s include quality control practices for manufactured devices, guiding QMRA and recommendations by public health organizations regarding POU device selection, and assessing the impact of user training programmes regarding POUWT techniques. / Graduate

Water quality in the Vaalharts region in the Northern Cape Province, South Africa, decreased over the past few years and there was a need for the microbiological and physico-chemical assessment. This problem was identified through discussions with Vaalharts Water (Vaalharts Water User Association) in 2010 when the issue of the impact of deteriorating water quality on drinking water production was raised. It was thus important to investigate concerns of the water users association pertaining to water quality issues. The aim of this study was to assess the physico-chemical and microbiological quality of household water in the Vaalharts irrigation scheme. The main residential areas were Hartswater, Pampierstad, Jan Kempdorp and Warrenton. Faecal coliforms were detected in the raw water of all the drinking water distribution systems during 2011 and 2012. No faecal coliforms were detected in the household water during 2011. This was a very positive result, because not only did the household water comply with the SANS 241 (2011) standard (0 CFU/100ml), but the purification processes were successful by removing all the E. coli’s from the raw water. However, during March 2012 faecal coliforms were detected in the household water of Jan Kempdorp (191CFU/100ml). This could be due to point pollution and possible breakage of faecal coliforms in the distribution system. Low amounts of total coliforms were detected in the raw water of some of the drinking water distribution systems. This could be due to high amounts of other colonies (pink and purple) growing on the m-Endo agar which suppress the growth of the metallic green sheen (total coliform) colonies. The total coliform numbers complied with the SANS 241 (2011) standard of ≤10 CFU/100ml at most of the distribution systems, except for Hartswater during July 2011 (14CFU/100ml) and Warrenton during March 2012 (256 CFU/100ml). Heterotrophic plate count bacteria were very high in the household water of some of the distribution systems during 2011 and 2012 which exceeded the SANS 241 (2011) standard of ≤1000 CFU/ml. A large number of pigmented (yellow, orange, pink) and non-pigmented (white) colonies were isolated on R2A agar. This can be an indication of some failure in treatment processes. Other microbiological parameters that were tested such as faceal streptococci, Clostridia, Pseudomonas aeruginosa and fungi did not indicate any danger, but there were high levels of total anaerobic bacteria in the raw water during 2011 and 2012. A high level of anaerobic bacteria was detected in the household water of Hartswater during July 2011. Clostridia were also present in the household water of some of the distribution systems during 2011 and 2012. Sequencing results of the mdh, lacZ and uidA genes indicated that one of the isolates was identified as Enterobacter cloacae and the other isolates were E. coli. Four of the isolates were identified as Escherichia coli O104:H4. This is a pathogenic strain and raised concern. The physicochemical parameters that were measured complied with the SANS 241 (2011) standards during 2011 and 2012, but some of the parameters increased gradually from 2011 to 2012. Statistical analysis indicated that physico-chemical parameters had an influence on microbiological parameters and that deteriorating raw water may have an impact on drinking water quality. Another concern currently is that there is no SANS 241 (2011) for faecal streptococci, Clostridia, Pseudomonas aeruginosa, fungi and anaerobic bacteria. These are all opportunistic pathogenic bacteria and consuming water with high levels of these bacteria may cause health problems. This study indicated good progress in the treatment processes of the distribution systems over the two years. This may be due to the feedback given to Vaalharts Water during this study regarding the water quality of the residential areas. The physico-chemical and microbiological results of the present study indicated possible biofilm formation in the distribution systems. This may have impacts on the drinking water quality of the distribution systems. It was also evident that deteriorating raw water sources may have an impact on drinking water production. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Water quality in the Vaalharts region in the Northern Cape Province, South Africa, decreased over the past few years and there was a need for the microbiological and physico-chemical assessment. This problem was identified through discussions with Vaalharts Water (Vaalharts Water User Association) in 2010 when the issue of the impact of deteriorating water quality on drinking water production was raised. It was thus important to investigate concerns of the water users association pertaining to water quality issues. The aim of this study was to assess the physico-chemical and microbiological quality of household water in the Vaalharts irrigation scheme. The main residential areas were Hartswater, Pampierstad, Jan Kempdorp and Warrenton. Faecal coliforms were detected in the raw water of all the drinking water distribution systems during 2011 and 2012. No faecal coliforms were detected in the household water during 2011. This was a very positive result, because not only did the household water comply with the SANS 241 (2011) standard (0 CFU/100ml), but the purification processes were successful by removing all the E. coli’s from the raw water. However, during March 2012 faecal coliforms were detected in the household water of Jan Kempdorp (191CFU/100ml). This could be due to point pollution and possible breakage of faecal coliforms in the distribution system. Low amounts of total coliforms were detected in the raw water of some of the drinking water distribution systems. This could be due to high amounts of other colonies (pink and purple) growing on the m-Endo agar which suppress the growth of the metallic green sheen (total coliform) colonies. The total coliform numbers complied with the SANS 241 (2011) standard of ≤10 CFU/100ml at most of the distribution systems, except for Hartswater during July 2011 (14CFU/100ml) and Warrenton during March 2012 (256 CFU/100ml). Heterotrophic plate count bacteria were very high in the household water of some of the distribution systems during 2011 and 2012 which exceeded the SANS 241 (2011) standard of ≤1000 CFU/ml. A large number of pigmented (yellow, orange, pink) and non-pigmented (white) colonies were isolated on R2A agar. This can be an indication of some failure in treatment processes. Other microbiological parameters that were tested such as faceal streptococci, Clostridia, Pseudomonas aeruginosa and fungi did not indicate any danger, but there were high levels of total anaerobic bacteria in the raw water during 2011 and 2012. A high level of anaerobic bacteria was detected in the household water of Hartswater during July 2011. Clostridia were also present in the household water of some of the distribution systems during 2011 and 2012. Sequencing results of the mdh, lacZ and uidA genes indicated that one of the isolates was identified as Enterobacter cloacae and the other isolates were E. coli. Four of the isolates were identified as Escherichia coli O104:H4. This is a pathogenic strain and raised concern. The physicochemical parameters that were measured complied with the SANS 241 (2011) standards during 2011 and 2012, but some of the parameters increased gradually from 2011 to 2012. Statistical analysis indicated that physico-chemical parameters had an influence on microbiological parameters and that deteriorating raw water may have an impact on drinking water quality. Another concern currently is that there is no SANS 241 (2011) for faecal streptococci, Clostridia, Pseudomonas aeruginosa, fungi and anaerobic bacteria. These are all opportunistic pathogenic bacteria and consuming water with high levels of these bacteria may cause health problems. This study indicated good progress in the treatment processes of the distribution systems over the two years. This may be due to the feedback given to Vaalharts Water during this study regarding the water quality of the residential areas. The physico-chemical and microbiological results of the present study indicated possible biofilm formation in the distribution systems. This may have impacts on the drinking water quality of the distribution systems. It was also evident that deteriorating raw water sources may have an impact on drinking water production. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

No controle da qualidade da água, parâmetros microbiológicos são avaliados e devem estar de acordo com os limites estipulados em portarias e resoluções. Todas as metodologias de monitoramento microbiano demandam o cultivo dos organismos-alvo. Em muitos casos, as cepas isoladas devem ser analisadas por teses fenotípicos para determinação da espécie. Por meio de inóculos de E. coli, demonstrou-se que a variação na técnica de plaqueamento se deve especialmente à falta de consistência entre as diluições. Além disso, para a análise de réplicas, comarando-se as médias de dois métodos de diluição, foi demonstrado que ser utilizado apenas uma série de diluição derivada de um único inóculo. Utilizando-se culturas puras de E. coli, E. faecalis e P. aeruginosa, a recuperação foi similar entre os meios seletivos respectivos (m-Endo, m-EI, m-PA-C) e meio não seletivo (Tripticase Soy Agar). A utilização da técnica de membrana filtrante resultou em contagens significativamente maiores para E.coli e E. faecalis em comparação ao método de spread plate. A microbiota natural presente na água potável (torneira) não influenciou significativamente as contagens de E. coli, E. faecalis e P. aeruginosa em meios seletivos. Entretanto, na água mineral engarrafada inoculada artificialmente com P. aeruginosa, a contagem foi significativamente maior na réplica estéril. Na análise de água marinha, e na réplica não estéril inoculada com E. faecalis, a contagem foi signifivativamente menor e as células de P. aeruginosa produziram colônias atípicas. Analisando-se amostras do meio ambiente (biofilmes de estação de tratamento de água, lodo de esgoto e água de córrego contaminado), 45 colônias típicas isoladas em meios de cultura seletivos foram identificadas por meio do sequenciamento do gene 16S rRNA, testes bioquímicos e MALDI-TOF. Os resultados de sequenciamento do gene 16SrRNA tiveram baixa correlação com a identificação dos organismos por testes bioquímicos (46,6% em nível de gênero e 20% em nível de espécie) e MALDI-TOF (60% em nível de gênero e 48,8% em nível de espécie). Para as cepas identificadas como E. coli e Enterococcus spp., a correlação entre o sequenciamento e MALDI-TOF foi de 75% e 62%, respectivamente. Uma vez que a quantificação de micro-organismo por membrana filtrante depende do micro-organismo em análise e do tipo de água, é necessário que os laboratórios realizem testes de padronização antes de implementar essa metodologia. Os resultados demonstram que os métodos convencionais utilizados não são adequados e suficientes para a análise da qualidade da água e, portanto, novas metodologias devem ser empregadas. Idealmente devem ser utilizadas técnicas baseadas em características fenotípicas, genômica e proteômica cujos resultados são complementares e fornecem uma identificação mais acurada. / All over the world regulatory agencies specify microbiological water quality parameters to guarantee water safety. Conventional microbiological water quality analysis is based on the cultivation of the target organisms. In many analysis protocols, phenotypic assays of isolated strains are mandated for species determination. Reference samples are required for quality assessment and quality control of analytical protocols. Using E. coli as a model organism, it was demonstrated here that the variability of plate counts of reference cultures is caused mainly by the spread of counts in individual serial dilutions. Besides, comparing the means obtained by two dilution approaches, it was demonstrated that in the analysis of replicates it is possible to use only a set of dilutions derived for a unique inoculum. Recovery of pure cultures of E. coli, E. faecalis and P. aeruginosa was similar on selective culture media (m-Endo, m-EI, m-PA-C) and with the non-selective medium (Tripticase Soy Agar). The membrane filter technique yielded significantly higher counts for E.coli and E. faecalis in comparison to spread plate method. The autochthonous microbiota of potable water (tap water) did not influence the counts of E. coli, E. faecalis and P. aeruginosa on selective culture media. However, the sterile replica of mineral water spiked with P. aeruginosa showed higher counts for these bacteria. For marine water analysis, the non-sterile replica spiked with E. faecalis yielded low counts and P. aeruginosa produced an atypical colony. Both, sample-induced variation in target strain recovery and colony appearance on culture plates indicates the requirement for method evaluation tests on particular sample matrices before implementing routine microbiological analysis by culture-based methods in the laboratory. 45 typical colonies obtained from environmental samples in selective culture media (biofilms from activated sludge, drinking water treatment plants and water from creek contaminated with raw sewage) were analyzed by 16S rRNA gene sequencing, biochemical phenotypic assays and MALDI-TOF. Sequencing showed low correlation with phenotypic assays (46,6% at the genus level and 20% at the species level) and MALDI-TOF (60% at the genus level and 48,8% at the species level). On the other hand, the strains identified as E. coli an Enterococcus spp. by 16S rDNA gene sequencing demonstrated a high correlation with MALDI-TOF (75% and 62%, respectively). Since the results showed that conventional methods aren´t suitable and sufficient to assess water quality, new technologies should be employed. Ideally, techniques should be based on phenotypic, genomic and proteomic features since the results are complementary and provide a more accurate identification.