Potential pathogenicity of heterotrophic plate count bacteria isolated from untreated drinking water

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria.Master