Determination of cytotoxicity and invasiveness of heterotrophic plate count bacteria isolated from drinking water

Evidence has been presented that some heterotrophic bacteria often detected in drinking water supplies possess features associated with pathogenicity. This suggests that even the low numbers of heterotrophic bacteria considered acceptable by drinking water specifications may constitute a health risk, particularly to immunocompromised consumers. In this study, 339 bacteria were isolated at random from routine heterotrophic plate count (HPC) tests on selected drinking water supplies in South Africa. In a first screen for potentially pathogenic properties, 188 of the isolates (55.5%) displayed a- or b-haemolysis on blood agar. Further analysis of the haemolytic isolates for enzymes associated with virulence revealed the presence of chondroitinase (5.3%), coagulase (16.0%), DNase (60.6%), elastase (33.0%), fibrinolysin (53.7%), gelatinase (62.2%), hyaluronidase (21.3%), lecithinase (47.9%), lipase (54.8%) and proteinase (64.4%) of the isolates. No fluorescein or pyocyanin was detected in any of the isolates. Among the haemolytic isolates 68.6% were resistant to oxacillin (1 μg), 59.6% to penicillin G (2 units), 47.3% to penicillin G (10 units), 53.7% to ampicillin (10 μg) and 42.6% to ampicillin (25 μg). Cytotoxicity, invasiveness and adherence properties of the haemolytic isolates was determined on HEp-2 and Caco2 cell lines. Among the haemolytic isolates 96% were cytolytic on the HEp-2 cell line. All the haemolytic isolates adhered to HEp-2 and Caco₂ cells but gram-negative isolates tended to adhere in larger numbers than gram-positive isolates. HEp-2 cells were invaded by 42% of the haemolytic isolates. Heterotrophic bacteria, which most frequently revealed the above features associated with pathogenicity included species of the following genera: Aeromonas, Acinetobacter, Aureobacterium, Bacillus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, Tsukamurella and Vibrio. The results obtained in this study support earlier indications that bacteria detected by routine heterotrophic plate counts on drinking water supplies may include bacteria associated with potentially pathogenic properties. The extent to which these bacteria in drinking water supplies may constitute a health risk remains to be investigated.