Presence of potentially pathogenic heterotrophic plate count (HPC) bacteria occurring in a drinking water distribution system in the North-West Province, South Africa
There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were β-haemolytic. Among the 31 β-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the β-haemolytic isolates were resistant to oxytetracycline 30 µg, trimethoprim 2.5 µg and penicillin G10 units, 96.8% were resistant to vancomycin 30 µg and ampicillin 10 µg, 93.5% to kanamycin 30 µg, 74.2% to chloramphenicol 30 µg, 54.8% to ciprofloxacin 5 µg, 22.6% to streptomycin 300 µg and 16.1% to erythromycin 15 µg. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water.