C difficile

From 1^st July 2012, diarrheal faecal samples of patients hospitalized in different affected wards, with a positive toxin test, were sent to the NIH reference laboratory.
The faecal samples were cultured, after ethanol shock, on the chromogenic medium chromID® C. difficile (bioMérieux, Marcy l’Etoile, France), under anaerobic conditions. C. difficile-suspected colonies were obtained from all patients, which were identified with the Rapid ID32 A galleries (bioMérieux), and confirmed by detection of gluD gene, which encodes the glutamate dehydrogenase specific for C. difficile[20 (link)].
The isolates were further characterized by capillary gel electrophoresis-based PCR ribotyping, as well as by PCR detection of enterotoxin A and cytotoxin B (tcdA and tdcB genes, respectively), binary toxin (cdtA and cdtB genes), and the deletion in the tcdC gene [21 (link),22 (link)]. The susceptibility to moxifloxacin was determined by E-Test® (bioMérieux).

Stool samples were cultured on chromID^TMC. difficile (bioMérieux, Durham, NC, United States) growth medium and then incubated at 37 °C in anaerobic conditions for 48 h. C. difficile colonies appear asymmetric and black colored [50 (link)]. Isolated colonies were suspended for generation of 1 McFarland turbidity. The inoculum was sub cultured on Brucella Blood Agar (Becton Dickinson, Heidelberg, Germany), supplemented with hemin and vitamin K, and an Etest strip for moxifloxacin (bioMérieux, Durham, NC, United States) was added. The agar plates were incubated in anaerobic conditions at 37 °C for 24 h. Minimum inhibitory concentration (MIC) determination was performed in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines; C. difficile isolates were considered resistant when MIC to moxifloxacin was above 4 µg/mL [12 (link)].