Immunomagnetic separation of <i>Escherichia coli</i> O157:H7 from environmental and wastewater in South Africa

Abstract

Recreational and drinking water supplies polluted with sewage have become an important source of E. coli O157:H7 infection. Immunomagnetic separation (IMS) has been extensively used for the isolation of E. coli O157:H7 from food and stool samples but not for samples such as wastewater. In this study the IMS method was used in combination with the E. coli O157:H7 selective media, immunoassays, biochemical tests and PCR, to assess the prevalence of E. coli O157:H7 in selected sewage and environmental water in South Africa. Environmental and wastewater were seeded with E. coli O157:H7 to determine the sensitivity and selectivity of the enrichment-IMS-selective agar method. Naturally occurring E. coli O157:H7 organisms were recovered from selected samples by means of IMS. The IMS concentrates were plated on three selective E. coli O157:H7 media. E. coli O157:H7 was detected in seeded sewage and river water samples with numbers as low as 1.2 cfu·ml -1 . The IMS procedure was used to investigate the prevalence of E. coli O157:H7 in randomly selected sewage and river water samples in South Africa. A total of 91 sewage- and 40 river water samples were tested and 17.6% and 20% yielded suspected E. coli O157:H7 colonies on CT-SMAC agar medium respectively. PCR was used to confirm the presence of genes coding for Shiga toxin-1 (Stx1), Shiga toxin-2 (Stx2), enterocyte attaching and effacing genes (eaeA) and enterohaemolysin (hly). Standard immunoassay kits specific for the O157 and H7 antigen and a biochemical indole test were used for further E. coli O157:H7 confirmation. Three colonies from one sewage sample (1.1 % of all sewage samples) agglutinated with anti-E. coli O157 and H7 antiserum and contained the genes coding for Stx2, eaeA and hly. None of the colonies isolated from the river water samples were positive for E. coli O157:H7. CT-SMAC proved to have limited E. coli O157:H7 selective capabilities from samples such as sewage with high bacterial counts. Seeded sample experiments indicated that IMS is a suitable method for isolating E. coli O157:H7 from samples with high bacterial interference and low numbers of E. coli O157:H7. Evidence has been presented that the enrichment-IMS-selective agar procedure substantially increased the sensitivity of E. coli O157:H7 isolation compared to direct plating of test samples onto selective agar generally practised in the past.