Africa remains the most cholera stricken continent in the world as many people lacking access to safe drinking water rely mostly on polluted rivers as their main water sources. However, studies in these countries investigating the presence of
Pathogenic strains of
Although found to be species-specific for all
Between 1973 and 2013, South Africa reported a total of 186463 cholera cases to the World Health Organisation [
Water and sediment samples were collected from the Apies River situated in the Province of Gauteng in South Africa. A full description of the river, its tributaries, the sampling sites, and the main land uses have earlier been published [
Map of the Apies River showing sampling points and wastewater treatment works (WWTW). Sites AP3, AP4, and AP5 are tributaries to the main river.
Briefly, the river has its source in the south of the Gauteng Province and flows northward into the Northwest Province. The portions of the river around the Pretoria CBD (Central Business District) have been canalised due to the development of the area. The portions towards the northern end of the river, as it joins the Pienaars River in the Northwest Province, have not been canalised and thus give easy access to the communities around whose inhabitants use the water for different purposes. As the river flows through rural, urban, and informal settlements around the City of Tshwane, it is used for agricultural (irrigation and animal farms), domestic, and recreational purposes [
A total of 120 samples (60 water and 60 sediment samples) were collected weekly for six weeks between January and February 2014 from ten sites along the Apies River. Water samples were collected in sterile plastic bottles following standard procedures [
Sediment samples were processed as previously described [
The identification of VAGs from the samples was done on a Corbett Life Science Rotor-Gene 6000 Cycler (Qiagen, Hilden, Germany) using three different PCR sets. Set 1 was for identification of the
Primers used for the identification of VAGs of
Target gene | Primer sequence (5′–3′) | Amplicon size |
Reference |
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304 | [ |
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536 | [ |
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947 | [ |
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451 | [ |
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862 | [ |
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216 | [ |
R: CCAAGCTCAAAACCTGAAA | |||
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779 | [ |
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179 | [ |
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A confirmed
The reaction mixture for PCR Set 1 was as follows: 10
The PCR products from PCR Set 1 and Set 2 were separated by a 2.0% agarose gel electrophoresis supplemented with ethidium bromide as stain and run in a Tris-acetate-EDTA buffer. A GeneRuler™ 100 bp (Thermo Fisher Scientific, South Africa) was used as the DNA ladder. Gels were visualised under UV light and images were captured either using a digital camera or using an INGenius Syngene Bio Imaging System (Vacutec, South Africa). Once captured, the gel images were analysed using the GelAnalyzer 2010 software (
All physicochemical parameters were directly measured on-site during sample collection. The dissolved oxygen (DO; mg/L), electrical conductivity (EC;
For a site to be considered positive for the gene of interest, the duplicate water or sediment samples had to be positive for the PCR assay. The occurrence of VAGs in water and sediments were compared for any statistical difference using a Kruskal Wallis test. The nonparametric Spearman’s rank correlation analysis was performed to investigate any relationship between the various VAGs and the physicochemical parameters in both matrices. All tests were performed using SPSS version 20 (Statistical Package for the Social Sciences; IBM Corporation, Armonk, New York, USA) and were considered significant at
A total of 60 sediment and 60 water samples were analysed for the presence of
Of the 120 samples analysed, 68 (37 sediment and 31 water) samples were positive for the species-specific
Distribution of
Sample type | Number of positive samples per gene (%) | |||||||
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Sediment ( |
37 (61.7) | 0 (0) | 11 (18.3) | 0 (0) | 13 (21.7) | 19 (31.7) | 13 (21.7) | 0 (0) |
Water ( |
31 (51.7) | 0 (0) | 12 (20.0) | 2 (3.3) | 8 (13.3) | 30 (50.0) | 9 (15.0) | 0 (0) |
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None of the samples analysed, both water and sediments, was positive for the
The VAGs investigated in this study were not evenly distributed amongst the sampling sites. Some genes like the
Presence/absence of the virulence genes at each of the sampling sites.
Sample type | Sample site |
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Sediment | DAS | + | − | + | − | + | + | + | − |
AP1 | + | − | + | − | + | + | + | − | |
AP2 | + | − | + | − | + | + | + | − | |
AP3 | + | − | − | − | − | + | − | − | |
AP4 | + | − | + | − | − | + | − | − | |
AP5 | + | − | − | − | − | − | − | − | |
AP6 | + | − | + | − | + | + | + | − | |
AP7 | + | − | + | − | − | + | + | − | |
AP8 | + | − | + | − | + | + | + | − | |
AP9 | + | − | + | − | + | + | − | − | |
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Water | DAS | + | − | + | + | − | + | + | − |
AP1 | + | − | + | − | + | + | + | − | |
AP2 | + | − | + | + | + | + | + | − | |
AP3 | + | − | − | − | − | + | − | − | |
AP4 | + | − | + | − | − | + | + | − | |
AP5 | + | − | − | − | − | + | − | − | |
AP6 | + | − | + | − | + | + | + | − | |
AP7 | + | − | + | − | + | + | + | − | |
AP8 | + | − | + | − | + | + | + | − | |
AP9 | + | − | + | − | + | + | + | − |
Results of the molecular profiling of the 68
Distribution of the possible
Genotype | Number of samples |
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9 |
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23 |
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4 |
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3 |
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2 |
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6 |
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8 |
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1 |
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1 |
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2 |
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1 |
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3 |
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4 |
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1 |
The majority of the samples (30 of the 68 positive samples) revealed a single virulence gene. Of the 30 samples with a unique profile, the most detected genotype was the
The water temperature during the entire sampling period ranged between 20.2°C and 28.1°C with mean temperature being 23.7°C. The DO, pH, EC, and turbidity ranged between 3.4 mg/L and 7 mg/L (mean = 5.5 mg/L), 6.6 and 8.2 (mean = 7.6), 187.8
Within the water column, none of the genes studied showed a significant correlation with any of the physicochemical parameters measured. However, in the sediments, there was a significant positive correlation between the occurrence of the
The main aim of the present study was to investigate the presence of
The detection of
The high detection of
Although it is generally accepted that most environmental strains of
It should however be noted that the results presented in the current study are on the presence or absence of the VAGs. The enrichment step probably increased sensitivity with respect to the detection of the VAGs. Although the enrichment could have also probably ensured that the VAGs found were most likely carried in viable cells, the actual quantification of the number of cells carrying the genes could not done. This is because the APW would have favoured the multiplication of the organisms and quantitative information derived in this case would give a false impression of a high concentration of the pathogens in the samples, thus affecting the actual risk. Nevertheless, the detection of the VAGs calls for the need to further investigate the actual concentration of
Environmental factors have been found to influence the growth and survival of microorganisms in the aquatic environment. In the present study, only the
Based on the results obtained from the present study, we conclude that sediments of the Apies River harbour many
Opinions expressed and conclusions arrived at are those of the authors and are not necessarily attributed to the NRF, the WRC, or TUT.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors thank the Water Research Commission (WRC), South Africa (WRC Projects K5/2169 and K5/2147), the National Research Foundation (NRF) of South Africa, and the Tshwane University of Technology (TUT) for their generous financial support.