GENERAL CONCLUSIONS GENERAL CONCLUSIONS GENERAL CONCLUSIONS
5.3 ISOLATION AND IDENTIFICATION OF BACTERIAL POLLUTANTS FROM THE BERG- AND PLANKENBURG RIVERS IN THE WESTERN CAPE, SOUTH
AFRICA
Organic and inorganic waste, as well as industrial and agricultural waste, sewage, human activities, human and animal excreta, are the main sources responsible for the deterioration of the valuable water bodies (Last, 2002). In South Africa, water resources such as rivers, service various industrial, agricultural and domestic sectors, which may also contribute to the point or non-point source contamination of these valuable water sources. Waterborne outbreaks have been associated with microbial agents, such as Salmonella sp., Campylobacter sp. and Escherichia coli amongst others (Gerba, 1996), as well as viruses, protozoa, helminths and fungi. These micro-organisms have led to significant health risks in humans with lowered resistance levels such as infants, the elderly and immunocompromised individuals (Theron & Cloete, 2002).
The aim of this study was to identify the predominant bacterial species, isolated from planktonic and sessile samples, present in the Berg- and Plankenburg Rivers in the Western Cape, South Africa using 16S rRNA PCR and DNA sequencing. For this study only the sites upstream from and at the informal settlements were selected for analysis.
Micro-organisms isolated from water and biofilm samples obtained from the Berg- and Plankenburg Rivers were analysed. All figures mentioned in this section refer to Article three as discussed in Chapter four.
Sampling at the Berg- and Plankenburg River-sites started in June 2004 and continued for a period of 1 year until June 2005. Four sampling sites were identified along the Berg River (Figure 4.1) and included Site A (agricultural farming area); Site B (informal settlement - sites B1 and B2) and Site C (Newton pumping station). Site B2 (Plot 8000) was identified as the site where storm water drainage pipes from the informal
settlement flow directly into the river. The Newton pumping station services the residential area of Newton as well as certain sections of Mbekweni. Sampling sites along the Plankenburg River (Figure 4.2) included Site A (agricultural farming and a residential area from Stellenbosch); Site B (informal settlement); Site C (industrial area) and, Site D (agricultural and industrial area).
Deoxyribonucleic acid (DNA) extractions of isolated micro-organisms were performed using the High Pure PCR Template Preparation Kit as per manufacturer’s instructions (Roche Diagnostics) followed by subsequent 16S rRNA sequence amplification by means of the Polymerase Chain Reaction, sequenced and identified using the Basic Local Alignment Search Tool (Blastn) (Altschul et al., 1997) obtained from the National Centre for Biotechnology Information website. A MassRuler™ DNA Ladder Mix, #SM0403 (Fermentas) was used to compare amplicon size. Subsequent DNA concentrations of PCR products were determined by means of the Qubit™
fluorometer (Invitrogen) using the Quant-iT™ dsDNA BR (Broad-range) Assay kit 2 – 1000 ng as per manufacturer’s instructions (Molecular probes and Invitrogen).
Phylogenetic analysis was performed by aligning grouped DNA sequences with Clustal X (1.81) using default parameters and the Blosum matrix. Unrooted trees were constructed using the neighbour-joining program of MEGA version 4.1 (Molecular Evolutionary Genetics Analysis 4.1), while branching patterns were evaluated by pairing 1000 replicates.
Figure 4.3a indicates representative samples isolated at Site B in the Berg River, which were amplified using primer set 1 (Greisen et al., 1994), while the amplified PCR samples represented in Figure 4.3b were obtained using primer set 2 (Greisen et al., 1994). The unrooted phylogenetic trees illustrated in Figures 4.4 to 4.7 include various species of both the Gram-positive and Gram-negative heterotrophic bacteria. Bacterial species isolated from the planktonic and sessile samples obtained from Site A in the Berg River (Figure 4.4) displayed a wide diversity of micro-organisms. The presence of various members of the Enterobacteriaceae, such as Klebsiella sp., Serratia sp., Enterobacter sp., Shewanella sp. and Aeromonas sp., confirm faecal contamination in the river. In addition, pathogenic micro-organisms including Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Bacillus cereus were also isolated at this site.
Research has shown that Stenotrophomonas sp. are affecting more humans (Gilligan and Whittier, 1999) and the fact that Site B is situated directly next to the informal
settlement, raises concern as exposure to the organism could result in increased infection rates. Staphylococcus epidermidis which form part of the normal flora of the human skin (O’Gara & Humphreys, 2001) was also identified at this site. This organism however, may affect people with altered immune systems and might be responsible for endocarditis and infection in these patients (Prescott et al., 1990). Enterobacteriaceae species isolated at Site B along the Berg River (Figure 4.5) also included Citrobacter freundii and Pseudomonas sp. Compared to Site A, a greater species diversity was observed at Site B. Various bacterial species were introduced at this Site B and included species such as Burkholderia sp., Acidovorax sp., Variovorax koreensis, Thermomonas sp., Lysobacter sp., and Kocuria sp., amongst others. Similarly many species were conserved between the sites for example pathogenic micro-organisms such as Klebsiella sp., Aeromonas sp., Pseudomonas sp., Stenotrophomonas sp. and Bacillus cereus.
From the phylogenetic tree illustrated in Figure 4.6 (Plankenbrug River, Site A), two distinct clusters could be identified with the Gram-negative bacteria comprising the largest. This cluster was divided into five subclusters and several clades. One subcluster included once again, members of the Enterobacteriaceae thereby confirming faecal contamination in the Plankenburg River. Species such as Aeromonas hydrophila, Alcaligenes faecalis, Klebsiella oxytoca and various other species were also identified at this site. Aeromonas sp. and particularly, A. hydrophila is associated with gastroenteritis, cellulitis and other diseases in humans. Bacterial species such as Lysobacter taiwanensis, Thermomonas fusca and Acinetobacter sp. were also identified at this site and are all indicator organisms of faecal contamination. The presence of these faecal indicators also relate to a previous study (Paulse et al., 2008) where significantly (p < 0.05) high faecal coliform and E. coli counts were recorded by means of the most probable number technique (MPN). During this study, faecal coliform and E. coli counts of 3.5 x 106 micro-organisms/100 ml respectively, were observed at Site B in the Plankenburg River. In addition, pathogens such as Bacillus cereus and B. anthracis were also identified at this site. The presence of these two organisms is responsible for major health illness such as food poisoning (B. cereus) and the disease, anthrax (B. anthracis). These organisms also raise concerns as both organisms were conserved between Site A and Site B (Figure 4.7), where increased population numbers are observed, thus resulting in increased exposure to the pathogens and thereby
increased possible infections. Other important bacterial species introduced at Site B included various members of the Enterobacteriaceae group such as Serratia sp., Citrobacter sp., Yersinia ruckeri and Enterobacter sp. thereby again confirming faecal contamination at this site. As previously mentioned this is the point of the river that is closest to the informal settlement. It is also the site where faecal wastewater from nearby sanitary facilities is flushed into the river. Bacterial species from the genus, Yersinia are considered major human pathogens and may be the causative agent of plague in susceptible individuals.