Proposed Method for Isolation of Escherichia Coli 0157:H7 from Environmental Samples

Article excerpt


Multiple cases of hemorrhagic colitis and hemolytic uremic syndrome (HUS) have been linked to the pathogen Escherichia coli, serogroup 0157:H7, present in meat and poultry products (1,2), raw milk, and community water supplies (3). Foods of bovine origin, especially ground beef, are the most frequently incriminated vehicles of infection. Additionally, there have been two waterborne outbreaks of infection induced by E. coli 0157:H7. The first, in Cabool, Missouri, was associated with the breakdown and associated repairs of an unchlorinated and aging sewerage and water distribution system (4). The second occurred in Portland, Oregon, during the summer of 1991, where a large number of visitors to a public swimming facility were stricken with E. coli 0157:H7, independent of food consumption (5). In a recent study performed in rural areas surrounding Philadelphia, Pennsylvania, abundant wildlife populations were linked to a reservoir contaminated with E. coli 0157:H7 (6). To date, the literature has concentrated on isolation of the organism from food or fecal matrices. Research focusing on the isolation of E. coil 0157:H7 in a water or soil environment, and the persistence in these milieu, is wanting. Coliform bacteria are commonly found in fide water, harbors, and bays, which are often badly polluted. Although E. coli and other lactose-fermenting bacteria do not constitute part of the normal intestinal flora of marine fish, if ingested, such facultative bacteria may survive for considerable periods of time (7). The potential for exposure to the E. coli 0157:H7 serogroup in a marine milieu (i.e. consumption of contaminated fish or shellfish, incidental ingestion of contaminated water through recreational usage of a harbor or bay) may be significant. The purpose of this study was to examine the utility of a procedure, adapted in part from Doyle and Schoeni (1), for detecting E. coli 0157:H7 in a spiked environmental sample. As the procedure is applied to other matrices, it is anticipated that this methodology may be useful for determining presence and survivability of E. coli 0157:H7 in unspiked receiving waters and soils.

Methods and Materials

Using a sterile glass jar and sampling spoons, approximately 1.5 kg of slurry was collected from a bay shore at high tide. Sample pH was 7.5, and salinity was calculated at 3.1% (7). The sample was secured just below the water surface, then divided into three 400 g groups. The first subsample ("high" group) was inoculated and mixed with 4.0 ml of an 18-hour pure culture of 0157:H7 in Trypticase soy broth (TSB)([10.sup.-2] dilution). Culture incubation temperature was 35 [degrees] C. The second subsample ("low" group) was prepared by adding 1 drop (50 microliters) of the same 18-hour culture to a 99 ml phosphate-buffered saline blank and shaken. A 0.1 ml aliquot was then transferred to another 99 ml dilution blank and shaken. A further 0.1 ml aliquot of this dilution was then added to the slurry and homogenized ([10.sup.-8] dilution). The third (unspiked) subsample was utilized as a negative control. Subsamples were cooled immediately to 4 [degrees] C and stored in the dark until tested for E. coli 0157:H7 on day 3 and day 20.

After 3 days and then 20 days of refrigeration, ten-fold serial dilutions of each sample were prepared to a [10.sup.-10] dilution. From each dilution, a 0.1 ml sample was pipetted (in duplicate) to MacConkey (BBL Microbiology Systems, Cockeysville, Md.) and MacConkey-sorbitol (Difco Laboratories, Detroit, Mich.) agar plates. Each aliquot was spread-plated until dry on the agar surface. Plates were inverted and incubated aerobically for 18-24 hours at 35 [degrees] C. The dilution range of interest produced between 50-100 well-isolated CFUs per plate. Colonies which were considered to be presumptive E. coli 0157:H7 were pink-red on MacConkey agar and clear on MacConkey-sorbitol media.

Modified vancomycin Trypticase soy broth (mvTSB) was prepared and dispensed in 96 well microtiter plates. …