Methods for the detection and isolation of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) are an important cause of hae morrhagic colitis and the diarrhoea-associated form of the haemolytic uraem ic syndrome. Of the numerous serotypes of E. coli that have been shown to p roduce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non-motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, ra pid microbiological diagnosis of individual patients enables the prompt not ification of outbreaks and implementation of control measures to prevent mo re cases. Most human infections caused by STEC have been acquired by the co nsumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person-to-pers on contacts. To identify the reservoirs of STEC and the routes of transmiss ion to man, sensitive methods are needed as these pathogens may only be pre sent in food, environmental and faecal samples in small numbers. In additio n, sensitive and rapid detection methods are necessary for the food industr y to ensure a safe supply of foods. Sensitive methods are also needed for s urveillance programmes in risk assessment studies, and for studies on survi val and growth of STEC strains. Cultural methods for the enrichment, isolat ion and confirmation of O157 STEC are still evolving. Several selective enr ichment media have been described, of which modified tryptone soy broth wit h novobiocin and modified E. coli broth with novobiocin, seem to be the mos t appropriate. These media are minimally-selective broths that give a somew hat limited differential specificity favouring isolation of O157 STEC, as o pposed to other Gram-negative bacteria, in the sample. An incubation temper ature of 41-42 degrees C further enhances selectivity. The occurrence of he at-, freeze-, acid- or salt-stressed STEC in foods means that it is importa nt to be able to detect cells that are in a stressed state, as STEC general ly have a very low infectious dose, and injured cells mostly retain their p athogenic properties. For the isolation of stressed O157 STEC, pre-enrichme nt in a non-selective broth is necessary. The most widely used plating medi um for the isolation of typical sorbitol-non-fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT-S MAC). As some STEC strains are sensitive for tellurite and/or are sorbitol- fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) followin g selective enrichment, and subsequent spread-plating of the concentrated t arget cells onto CT-SMAC agar, appears to be the most sensitive and cost-ef fective method for the isolation of E. coli O157 from raw foods. IMS increa ses sensitivity by concentrating E. coli O157 relative to background microf lora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time-consuming, l abour-intensive and hence, costly, rapid immunological detection systems ha ve been developed which significantly reduce the analysis time. These metho ds include enzyme-linked immunosorbent assays (ELISAs), colony immunoblot a ssays, direct immunofluorescent filter techniques, and several immunocaptur e techniques. Both polyclonal and monoclonal antibodies specific for the O and H antigens are used for these methods. Many of these test systems are a ble to detect less than one O157 STEC cell g(-1) of raw meat after overnigh t enrichment. Presumptive results are available after just one day, but nee d to be completed with the isolation of the organisms. The primary use of these procedures is therefore to identify food and faeca l samples that possibly contain O157 STEC.