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Method Development for Enumeration of E. coli O157:H7 for Ground Beef and Hides, Carcasses, and Feces of Cattle


Methodologies for the assessment of microbial pathogen load, at various steps in the beef
production process, are lacking. In order to quantify the risks associated with the slaughter of
animals that may harbor or shed E. coli O157:H7 or Salmonella spp., accurate estimates of the
prevalence and frequency of distribution of these pathogens and their relative concentration on
hides and in feces is needed. Another aspect of the need for enumeration methods is the
evaluation of intervention methods. The complete picture of pathogen contamination on
carcasses cannot be understood with data only relating to the presence or absence of various
pathogens. Comparisons of in-plant antimicrobial interventions have traditionally been based on
prevalence data alone. Yet, interventions that do not completely eliminate pathogens can still be
very effective if they are significantly reducing the pathogen load on hides or carcasses. Beef
processors need to know the levels of pathogens entering their plants and have the ability to
quantify these levels throughout processing, in order to have greater control of their process. <P>
At present, the majority of pathogen enumeration experiments, which are few in number, are
conducted using the most probable number (MPN) dilution technique. This method is an
indirect measure and provides an estimate of the number of organisms present in a sample. The
method was initially developed for the purpose of determining the number of viable bacteria in a
sample. One of the drawbacks encountered when this method is used for the enumeration of a
particular pathogen is the competition between the target organism to be enumerated and the
background microflora of the sample. If the target organism is out-competed, attempts to
quantify it are inaccurate. <P>
Enumeration methods based on direct plating have the advantage of providing a direct measure
of viable bacterial counts, and can be performed on selective media, so as to decrease the
presence of competing microflora. <P>
The objective of this study was to develop a highly accurate method of enumerating E. coli
O157:H7 at all stages of beef production.

More information

Approach: Hide, carcass, and fecal samples will be collected from commercial beef processing plants. These samples will be spiked with known levels of E. coli O157:H7. The E. coli O157:H7 inoculum will come from a freshly thawed frozen aliquot. After spiking, the samples will be stored at 4 deg C for 16 to 18 h. Previous enumeration methods have failed to produce accurate results on naturally contaminated samples since freshly grown lab strains were used in the inoculation studies. A key component in developing an enumeration method that provides accurate results with field study samples is to correctly simulate the lag phase that naturally-occurring E. coli O157:H7 strains undergo in laboratory enrichment conditions. This affects the final concentration of the target organism as well as the level of background microflora in which one must identify the target. We have developed a method that we believe adequately simulates the growth of naturally-occurring strains. <P>Method selection. A variety of MPN, direct plating, and spiral plating techniques will be evaluated. Various enrichment media and incubation times will be compared for robust growth of E. coli O157:H7 and minimal background flora levels. The MRU enrichment conditions will be used as the standard for comparison since these methods have already been optimized for E. coli O157:H7 detection in prevalence assays. Both culture-based and PCR-based detection technologies will be evaluated. PCR-based assays are much more amenable to high sample throughput, which will be an important criteria for method selection. Sample collection. Hide and carcass samples will be obtained using Speci-Sponges (Nasco, Fort Atkinson, WI.) moistened with 20 ml of buffered peptone water (BPW, Difco Laboratories, Becton Dickinson Microbiology Systems, Sparks, MD.). Sponges will be wrung out in the bag, then removed from the bag, and used to swab the hide or carcass. The hide sample will be collected from a 100-cm2 area over the flank. Ten grams of feces will be obtained from rectal grab and placed into a filtered stomacher bag. Ground beef samples will also be collected from either production facilities or retail establishments.<P> Sample processing. Sponge bags will be inoculated to the appropriate cell concentration, massaged thoroughly, and aliquots will be removed for enumeration. The fecal samples will be inoculated and 90 ml of TSB-phosphate will be added. The samples will be homogenized and aliquots will be removed for enumeration. Ground beef samples will be inoculated and homogenized, then aliquots will be removed for enumeration. E. coli O157 detection. Direct plating and immuno-magnetic separation and plating onto ctSMAC and ntChromagar will be used for culture-based detection of E. coli O157:H7. After the plates are incubated, up to three suspect colonies will be picked and tested by latex agglutination (DrySpot E. coli O157; Oxoid, Basingstoke, England). Various conventional and real-time PCR methods will also be used for detection of E. coli O157:H7.

Koohmaraie, Mohammad
USDA - Agricultural Research Service
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