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

Abstract

Heterotrophic plate counts (HPCs) are commonly used to assess the general microbiological quality of drinking water. Drinking water quality specifications world-wide recommend HPC limits from 100 to 500 cfu.m1-1. However, a number of recent studies revealed evidence that commonly used indicator bacteria may not be as harmless as generally accepted. It appears that immuno-compromised individuals, which represent increasing components of many consumer populations, are particularly at risk. This would include the very young and very old, patients with diseases such as AIDS, and patients on therapy after organ transplantations and cancer treatment. Since, epidemiological and animal infectivity studies are complex and difficult to control, attempts have been made by researchers to examine HPCs directly in order to assess health risks. These analyses included: cytotoxicity, invasiveness, enzyme analyses, antibiotic susceptibility and identification. In this study, 339 bacterial colonies were isolated at random from selected drinking water supplies in South Africa using heterotrophic plate count tests. In a first step to screen for potentially pathogenic properties, 188 (55.5%) of the isolates showed α- and β-haemolysis on human- and horse-blood agar media. Subsequent analysis of the haemolytic isolates for enzymatic properties associated with pathogenicity revealed the presence of chondroitinase in 5.3% of the isolates, coagulase in 16.0%, DNase in 60.6%, elastase in 33 .0%, fibrinolysin in 53.7%, gelatinase in 62.2%, hyaluronidase in 21. 3 %, lecithinase in 47.9%, lipase in 54.8%, and proteinase in 64.4%. Fluorescein and pyocyanin were not produced by any of the isolates. The Kirby-Bauer quality controlled disc diffusion method was applied in the demonstration of antibiotic resistance by the HPC isolates. Among the haemolytic isolates 77.7% were resistant to oxacillin (1 µg), 59.6% to penicillin G (2 units), 47.3% to penicillin G (10 units), 54.3% to ampicillin (10 µg) and 43.1% to ampicillin (25 µg). Cell culture studies revealed that 96% of haemolytic isolates were cytotoxic to HEp-2 cells and 98.9% of the 181 cytotoxic isolates adhered to HEp-2 or Caco-2 cells. Gram-negative isolates tended to adhere in larger numbers than gram-positive isolates. The average index of adherence for Gram-negative bacteria was 20-30 bacteria per HEp-2 cell, compared to 3-7 for Gram-positive bacteria. HEp-2 cells were invaded by 43.6% and Caco-2 cells by 49.7% of the 181 cytotoxic isolates. The invasion index on HEp-2 cells was 1.9xlO-1 to 8.9xl0-6, compared to 7.7xl0-2 to 8.3xlO-6 on Caco-2 cells. The most commonly isolated genera showing potentially pathogenic features were: Aeromonas, Acinetobacter, Aureobacterium, Bacillus, Chryseobacterium, Corynebacterium, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, Tsukamurella and Vibrio. All these genera are known to contain opportunistic pathogens. Our results support earlier findings on potentially pathogenic features of bacteria detected by heterotrophic plate counts on drinking water. These findings seem to be in agreement with some epidemiological studies, which indicated an association between HPCs of drinking water and the incidence of gastroenteritis in consumers. However, the extent of the health risk concerned needs to be defined in detail for meaningful revision of quality guidelines for HPCs in drinking water.