Abstract
M.Tech.
With the recent cholera outbreak in Zimbabwe and the outbreak taking a sub-regional dimension with cholera cases being reported from neighbouring countries like Botswana and South Africa, there was a need to monitor drinking water from environmental water sources as well as household water-storage containers at the point-of-use in rural communities. Although conventional culture-based microbiological methods for the identification of Vibrio species from environmental water samples are reliable, they require several days to complete (Khan and Cerniglia, 1994). Culture dependent and culture independent methods for the detection of Vibrio cholerae and Vibrio parahaemolyticus from water samples were optimised during the current study. With these methods, the occurrence and distribution of V. cholerae and V. parahaemolyticus in source waters as well as in household container stored-waters at the point-of-use in the Nwanedi Catchment, was determined.
The culture based approach analyses involved the enrichment of water samples in alkaline peptone water (APW) for 18 hours at 37°C followed by culture on selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Typical colonies on TCBS agar were confirmed using the API 20NE as well as the two multiplex polymerase chain reactions (m-PCR). The culture independent PCR approach was done by filtering 100 ml of the water sample onto polycarbonate membranes followed by DNA extraction from the bacteria captured on the membranes using an adaptation of the in-house DNA extraction method used in the laboratory. This DNA was used as template for the m-PCR’s. For the culture based PCR detection, 100 ml water was filtered onto nitrocellulose membranes followed by 18 hours enrichment in APW. DNA was then extracted from the enrichment broth and subsequently used as template for the m-PCR’s. All water samples were analysed with all three methods to compare the results and determine the most effective method for the detection of the two-selected Vibrio species present in water samples.
PCR analyses were performed using two m-PCR assays targeting the SodB (V. cholerae species), FlaE (V. parahaemolyticus species) and 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and the V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). The 16S rRNA primers were included in the Multiplex PCR’s as an internal control. The m-PCR assays were 100% specific for total and toxigenic V. cholerae and total V. parahaemolyticus when using target bacteria and various other non-target
bacteria. The m-PCR assays when coupled with an 18 hours enrichment step could detect as few as 4-10 V. cholerae and V. parahaemolyticus cells in pure cultures as well as in spiked environmental water samples.
Fifty water-storage containers and 56 environmental water samples (river, spring and borehole) from rural households in the Vhembe district of the Limpopo Province of South Africa were tested for the presence of selected Vibrio’s, using (1) the standard culture based approach, (2) PCR detection without enrichment and (3) PCR with a brief pre-enrichment. Container water samples were collected before [referred to as free volume (FV) of water] and after dislodging of the biofilm [referred to as dislodged biofilm (BD)] from the inner sidewalls of containers.
Of the samples analysed with the standard cultured based technique combined with colony confirmation using m-PCR 1, 34 (12.8%) tested positive for the presence of V. cholerae (SodB gene), 2 (1.3%) for the presence of V. parahaemolyticus (FlaE gene) and all the samples tested positive for the 16S rRNA gene. In contrast, only 1 (0.6%) tested positive for the presence of V. cholerae and 0 (0%) for the presence of V. parahaemolyticus when the isolates were confirmed with API 20NE. With the culture dependant PCR method, 65 (41.7%) of the samples tested positive for the presence of V. cholerae, 3 (1.9%) for the presence of V. parahaemolyticus and all the samples tested positive for the 16S rRNA gene. Seventeen (10.9%) of the samples tested positive for the presence of V. cholerae (SodB) and 16S rRNA genes, 0 (0%) for the presence of V. parahaemolyticus (FlaE gene) with the culture independent direct PCR detection protocol. All the samples that tested positive for V. cholerae with any of the three methods were tested for the presence of toxigenic V. cholerae species with the second multiplex PCR. Six of the source water samples tested positive for V. cholerae O1 as well as the cholera toxin genes.
Of the 56 source water samples, 14 (25%) were positive for V. cholerae and 0 (0%) were positive for V. parahaemolyticus with one or all of the methods. Six (10.7%) of the V. cholerae positive samples tested positive for V. cholerae O1 rfb gene, and ctxA gene (cholera toxin). Thirty (60%) of the 50 FV and 28 (56%) of the DB water samples tested positive for V. cholerae, and 3 (6%) of the FV and 0 (0%) of the DB samples tested positive for V. parahaemolyticus with one or all of the methods. None of the positive V. cholerae samples tested positive for the presence of toxigenic V. cholerae.
The results presented suggest that the use of culture-based techniques alone is inadequate for detection of selected Vibrio’s in the environmental water samples and that such techniques are not enough to guarantee satisfactory protection of human health. The combination of filtration, enrichment, DNA extraction and m-PCR method provide a sensitive and specific method for the detection of V. cholerae and V. parahaemolyticus in environmental water samples. This method proved to be the most effective for detection and identification of selected Vibrio’s when compared to the culture based method and PCR without enrichment method. The inclusion of an enrichment period allows for the detection of culturable bacteria which is crucial as PCR detection does not give indications on the viability of the detected material. The enrichment period will also dilute any inhibitors for the m-PCR’s that may be present.
Detection of V. cholerae and V. parahaemolyticus in the source water used by the population and in the water-storage containers indicates possible seeding of containers with Vibrio species from the source water. Furthermore, the detection of these organisms in DB samples indicates that these organisms attach to containers’ inner sidewalls, forming biofilms, further sustaining their occurrence and proliferation. The detection of V. cholerae and V. parahaemolyticus in household water-storage containers certainly places the consumers at risk of infection of diseases caused by these organisms.