The patient had three prior diarrhoeal illnesses. Her past medical history was notable for biliary atresia diagnosed when she was 3 months of age. A liver transplant was performed when she was 7 months old, and she has been taking immunosuppressive medication (prednisone/tacrolimus/mycophenolate sodium) since that time. No proton pump inhibitor was prescribed.
At our hospital, an initial screen for   C. difficile   toxins is performed for all patients with diarrhoea who have risk factors. A stool sample was examined on the third day of the onset of diarrhoea that was positive for   C. difficile   toxin A/B (we used ProSpecT   Clostridium difficile   Toxin A/B Microplate Remel). We cultured the sample on selective CCFA media (cefoxitin-cycloserine-fructose agar, Oxoid) as well as on fastidious anaerobe broth (FAB), over 5 and 15 days, respectively. We incubated the samples in anaerobic conditions (90 % N2, 10 % CO2). The FAB-positive broth was inoculated onto another CCFA media plate. Characteristic   C. difficile   colonies appeared on CCFA. These were tinged with yellow colour, and presented a ground-glass appearance characterized by circular elements with slightly filamentous edges. These were flat to low with rounded elevations. We inoculated the colonies onto   Brucella   agar with 5 % lysed sheep blood and vitamin K (5 mg ml-1) to identify them further and to perform molecular tests.
The   C. difficile   isolates were confirmed using the RapID ANAII system (Remel) and by PCR amplification of the tpi gene (  C. difficile   housekeeping gene).
Using a previously reported method, we obtained positive results for amplification assays for tcdA and tcdB gene fragments of PaLoc. By contrast, we obtained negative results for the binary toxin gene cdtB as well as the putative regulatory gene tcdC. We conducted molecular strain typing by amplification of the 16S-23S rRNA intergenic spacer sequences and pulsed-field gel electrophoresis (PFGE) with SmaI genomic DNA digestion, as described previously. PCR ribotypes were determined by subjecting data to the database WEBRIBO (http://webribo.ages.at). We compared DNA fragment patterns to those in the database of the National Microbiology Laboratory, Canada, using BioNumerics software (version 5.1. Applied Maths). We designated the isolate as PCR ribotype 046 and the new pulsotype as 1174 .
We determined the antibiotic susceptibility profile using the E-test (bioMerieux) on   Brucella   agar supplemented with vitamin K (5 mg ml-1) and 5 % defibrinated sheep blood. The isolate was resistant to levofloxacin (4 microg ml-1) and susceptible to vancomycin (1.5 microg ml-1), metronidazole (0.38 microg ml-1), rifampicin (<0.002 microg ml-1), moxifloxacin (1.0 microg ml-1) and clindamycin (1.0 microg ml-1). Resistance breakpoints were set in agreement with the guidelines of the Clinical Laboratory Standard Institute (CLSI) (M11-A8). For levofloxacin, we used the moxifloxacin breakpoint; for vancomycin, we used the EUCAST guidelines (http://www.eucast.org/clinical_breakpoints/); and for rifampicin, we used the breakpoint given by O'Conner et al..
The diarrhoea ceased on the fourth or fifth day after the initiation of therapy with metronidazole (7.5 mg kg-1 three times a day for 14 days). No probiotic therapy was used.