Improved detection of Clostridium difficile in animals by using enrichment culture followed by LightCycler real-time PCR

The performance of our previously published TaqMan real-time PCR (TMrtPCR) for the detection of Clostridium difficile directly from animal faeces was found to be inadequate due to TMrtPCR false negative results. Therefore, we developed a new internally controlled LightCycler real-time PCR (LC rtPCR) capable of detecting variant strains in diarrhoeic and subclinical animals by using two hybridisation probes instead of one hydrolysis probe used in TMrtPCR. While LC rtPCR did not provide better results than TMrtPCR, improved detection of C. difficile in samples with low number of bacteria was introduced, using a pre-enrichment step followed by LC rtPCR. Hence, 40 (100%) rectal swabs were sampled and subjected to direct LC rtPCR, culture without enrichment and enrichment culture; after 24, 48 and 72 h, and seven days of incubation, DNA was extracted and amplified with LC rtPCR. Only one (2.5%) sample was culture positive/LC rtPCR positive without the enrichment step, while 33 (82.5%) samples were culture positive after seven days of enrichment. Only one day of enrichment evidently increases the number of culture positive (15; 37.5%) and LC rtPCR positive (28; 70%) samples. Results of this study demonstrate that one day enrichment culture for C. difficile followed by LC rtPCR assay targeting multiple genes can be applied as accurate and rapid screening test, especially in samples with low number of C. difficile, as no culture positive/LC rtPCR negative samples were observed.