Isolation and Identification of Clostridium difficile Using ChromID C. difficile Medium Combined With Gram Staining and PRO Disc Testing: A Proposal for a Simple Culture Process

Abstract

Background: ChromID C. difficile agar (CDIF; bioMérieux, France), a chromogenic medium, allows for the isolation and identification of Clostridium difficile strains within 24 hr regardless of pretreatment of stool specimens with heat or alcohol shock. In the present study, we designed and evaluated a simple procedure for the implementation C. difficile cultures using CDIF medium in a tertiary hospital setting.

Methods: We designed a simple protocol for untreated stool specimens using CDIF medium followed by Gram staining and PRO disc (PRO disc K1532B, Key Scientific Products, USA) testing for the identification of C. difficile in colonies produced on CDIF agar. A total of 1,402 prospectively collected stool specimens from patients with suspected C. difficile infection were tested. The protocol was evaluated by phenotypic or molecular identification of C. difficile using Vitek 2 ANC card (bioMérieux) or 16S rDNA/tpi gene sequencing, respectively.

Results: Of 1,402 stool specimens, 650 isolates were cultured in CDIF. Overall, 235 (36.2%, 235/650) strains could be presumptively identified as C. difficile by using Gram staining and PRO disc testing. Of those, 231 (98.3%, 231/235) isolates were confirmed as true C. difficile by molecular assays.

Conclusions: The use of CDIF combined with Gram staining and PRO disc testing of untreated stool specimens would allow for isolation and accurate identification of C. difficile strains and would be advantageous in reducing the multistep process for C. difficile culture.

Keywords: Clostridium difficile; Culture; Isolation.

Fig. 1. Study design and results for the C. difficile culture using CDIF medium. Our proposed protocol for toxigenic C. difficile culture using CDIF medium depicted a gray-colored rectangle with rounded corners.

Abbreviations: CDIF, chromID C. difficile agar (bioMérieux, Marcy-l'Etoile, France); CDAB, Vidas CDAB assay (bioMérieuxe); +, positive; -, negative; Eq, equivocal; GPB, gram-positive bacilli; PRO, PRO disc K1532B (Key Scientific Products, Round Rock, TX, USA).