(a) Single-colony PCR ribotyping
(b) Recovery of C. difficile (naturally present in test beef)
(c) Recovery of C. difficile (experimentally inoculated in beef)
Figure 3: Moist heat resistance of C. difficile, naturally present and experimentally inoculated in retail ground beef, to various time-temperatures. Multistrain experiments in inoculated low 3% fat beef matrix. (a) Single-colony PCR ribotyping was used to subtype inoculated, naturally present, and heat-resistant experimentally isolates used (PCR 078, 027 and ATCC-9689). “PCR-”: prototypic strain. Note that other new meat isolates (PCR ribotypes) were naturally present in beef product purchased for the assay and are different from experimental isolates. (b) Single-colony PCR ribotyping based heat inhibition curves of C. difficile (pie charts connected with solid lines, average recovery frequencies) indicate presence of PCR 027 and 078 and other ribotypes naturally present in the meat package purchased for this assay (see panel a), at estimated concentrations of 2-3 log10 CFU/gram of contaminated raw ground beef tested. For comparison purposes, notice effect of pasteurization at 63°C on background indigenous anaerobic beef microbiota CFU isolated from two meat packages at different time points (dashed lines, mean ± SD, triplicate) for two fat concentrations 3 and 30%. (c) Single-colony PCR ribotyping heat inhibition curves of experimentally inoculated C. difficile indicate that PCR ribotype 078 would be more resistant to heat as temperature and time increase.