Extended Data Fig. 4: Isolation of C. difficile colonies and genotype-specific colony PCR confirms that the inoculating strain is responsible for the blooms observed in mice treated with laxatives.
From: Diarrhoeal events can trigger long-term Clostridium difficile colonization with recurrent blooms
Faecal samples from the two mice that experienced the highest magnitude increase in C. difficile measured via 16S rRNA amplicon sequencing were anaerobically homogenized and two dilutions were plated on cycloserine-cefoxitin fructose agar (CCFA) in duplicate. (A) Only one colony morphology was detected from all samples; ground class texture and diffuse edges are consistent with C. difficile. (B) Colony forming unit (CFU) counts roughly reflect the abundance of C. difficile measured via 16S rRNA sequencing. (C) PCR confirms that all isolates isolated on CCFA plates are the same genotype as the inoculating strain (ATCC BAA1801). The C. difficile specific PCR assay amplifies the rplP locus present in all C. difficile strains, while the assay specific to the BAA1801 genotypic cluster amplifies a gene that is present in BAA1801 and its 19 closest sequenced relatives (differentiated by only 270 SNPs, genome-wide) but no other C. difficile lineage. C. difficile strain ATCC-9689 is a toxinogenic control that can be successfully amplified using the rplP PCR assay but not the assay specific to the BAA1801 genotypic cluster. All 72 isolated colonies from all plated dilutions that grew on the CCFA plates were genotyped with both the rplP and BAA1801 genotypic cluster specific PCR assays once.
