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 ?-haemolytic. Among the 31 ?-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 ?-haemolytic isolates were resistant to
oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to
vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to
chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and
16.1% to erythromycin 15 ?g. 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. / Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/4380 |
| Date | January 2010 |
| Creators | Venter, Leandra |
| Publisher | North-West University |
| Source Sets | North-West University |
| Detected Language | English |
| Type | Thesis |
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