Isolation and identification of bacterial pollutants from the Berg and Plankenburg Rivers in the Western Cape, South Africa

Short communication Isolation and identification of bacterial pollutants from the Berg and Plankenburg Rivers in the Western Cape, South Africa AN Paulse1, VA Jackson1, S Khan1 and W Khan2, 3* Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville 7535, South Africa 2 Department of Agricultural and Food Sciences, Faculty of Applied Science, Cape Peninsula University of Technology, Cape Town 8000, South Africa 3 Department of Microbiology, Faculty of Science, Stellenbosch University, Stellenbosch 7600, South Africa 1 Abstract Bacterial species present in the Berg and Plankenburg Rivers (Western Cape, South Africa) were isolated from water and biofilm samples and population shifts between sampling sites were phylogenetically identified. Deoxyribonucleic acid (DNA) extraction of representative isolates was performed and amplified using 2 different primer sets. Various Enterobacteriaceae species were present at all of the sites, confirming faecal contamination. Phylogenetic analyses also showed that, in general, Gram-negative micro-organisms dominated at all of the sites sampled in both the Berg and Plankenburg river systems. Pathogens and opportunistic pathogens, such as Pseudomonas aeruginosa, Staphylococcus sp., and Bacillus cereus, were isolated from the Berg River. Similarly, in the Plankenburg River system, Aeromonas sp., Acinetobacter sp., Stenotrophomonas sp. and Yersinia enterocolitica were also isolated. This raises major health concerns as human population densities along both rivers are high, thus resulting in increased human exposure to these organisms. Keywords: Enterobacteriaceae, faecal contamination, informal settlement, river water, waterborne illnesses Introduction South Africa’s water resources service various industrial, agricultural and domestic sectors, which in turn may contribute to the point or non-point source contamination of these valuable water sources. In many areas of South Africa, and worldwide, informal housing schemes are often established along the banks of river systems. Due to a lack of adequate sanitation and waste removal facilities in the informal settlements, as well as poor management and disposal of sewage, the stormwater drainage pipes leading directly to the rivers are often used as a means of disposal of human and animal waste (SARDC, 2002). The most common bacterial pollutants isolated from water sources include Escherichia coli (E. coli), Vibrio cholerae, Campylobacter, Salmonella, Shigella and Aeromonas hydrophila (Lightfoot, 2003). In addition, the occurrence of biofilms or encrustations in water sources, which harbour various types of micro-organisms (Banning et al., 2003), allows for the multiplication of pathogens such as Pseudomonas, Mycobacter, Campylobacter, Klebsiella, Aeromonas, Legionella spp., Helico bacter pylori and Salmonella typhimurium (Gião et al., 2008). In South Africa, the presence of E. coli pathogens in sewage-contaminated river water and E. coli O157:H7 in sewage (Müller et al., 2001), was confirmed in an assessment conducted in 2003. Pathogens such as Salmonella, Shigella, Vibrio cholerae and coliphages have also been isolated from the final effluent of wastewater treatment facilities in the Eastern Cape, South Africa (Momba et al., 2006). * To whom all correspondence should be addressed.  +27 21 808 5804; fax: +27 21 808 5846; e-mail: Received 27 October 2009; accepted in revised form 2 October 2012. http://dx.doi.org/10.4314/wsa.v38i5.23 Available on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 38 No. 5 October 2012 ISSN 1816-7950 (On-line) = Water SA Vol. 38 No. 5 October 2012 The aim of this study was to identify predominant bacterial species isolated from water and biofilm samples collected from 2 sites along the Berg and Plankenburg Rivers, in the Western Cape, South Africa. Population shifts between sites were also identified using 16S rRNA PCR and DNA sequencing. Phylogenetic analyses were performed by aligning grouped DNA sequences with the Infernal Secondary Structure Based Aligner within the Ribosomal Database Project (RDP) system. Materials and methods Sampling sites A previous study identified 4 sampling sites along the Berg River (Paulse et al., 2007) and 4 sites along the Plankenburg River (Paulse et al., 2009). Based on the results obtained from these studies, deoxyribonucleic acid analysis was performed on samples (water and biofilm) collected from Sites A and B2 (Berg River) and from Sites A and B (Plankenburg River). Sampling One-litre water samples were collected in sterile Nalgenepolypropylene bottles (Cole-Palmer Instrument Company) at each sampling site, once a month, and stored on ice to maintain the lowest possible temperature. Materials such as stones, glass, leaves, etc., were also collected from each sampling site and stored in sterile whirlpack bags for subsequent biofilm isolation. Sonication of biofilm samples Glass, stones and leaves collected from the river were sonicated for 10 min in 60 mℓ sterile dH2O using a UMC 5 819 A B 1 2 3 A B 1 2 3 4 5 6 7 8 5 6 7 8 9 10 11 12 13 14 15 16 17 18 13 14 15 16 17 18 900 600 400 300 4 9 10 11 12 900 600 400 300 Figure 1a Polymerase chain reaction analysis of extracted DNA samples (BB1-138 to 155) (with Primer Set 1: forward (RW01) primer; reverse (DG74) primer) isolated from Site B in the Berg River. Lanes 1–18: Samples 138 to 155 (BB1); Lane A: Marker (MassRuler™ DNA Ladder Mix, #SM0403 (Fermentas)); Lane B: Negative control. Figure 1b Polymerase chain reaction analysis of extracted DNA samples (BB2-138 to 155) (with Primer Set 2: forward (RDR080) primer; reverse (DG74) primer) isolated from Site B in the Berg River. Lanes 1–18: Samples 138 to 155 (BB2); Lane A: Marker (MassRuler™ DNA Ladder Mix, #SM0403 (Fermentas)); Lane B: Negative control. ultrasonication bath (Instrulab Inc.). The sonication step was repeated at least 3 times, depending on the amount of material collected from each sampling site. The bacterial suspension obtained was used for further microbiological analysis. amplification. The PCR procedure included an initial denaturation step of 5 min at 95°C, followed by 30 cycles of amplification (25 s at 95°C, 25 s 55°C and 1 min at 72°C). The final extension step was performed at 72°C for 10 min. DNA extraction and agarose gel electrophoresis 16S ribosomal RNA sequencing Cultures from planktonic and sessile samples (sonicated bacterial suspension) obtained from the sampling sites were spreadplated onto Nutrient Agar (NA) (Merck, Biolab Diagnostics) after serial dilutions (10 -1 to 10 -7) were performed. Plates were incubated for 3–4 days at 37°C. Thereafter, distinct visible cells (colony forming units (CFU)) were identified based on morphological differences and re-streaked onto NA plates for isolation of pure cultures (approximately 824 isolates were selected for further a (...truncated)