Sensitivity of Direct Plating for Detection of High Levels of E. coli O157:H7 in Bovine Fecal Samples

Abstract

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10(9) colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10(-1) to 10(-4)) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37 degrees C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37 degrees C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 microg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 x 10(4) cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.