Ann Lab Med.  2015 Jul;35(4):404-409. 10.3343/alm.2015.35.4.404.

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

• Park KS
• Ki CS
• Lee NY

• KMID: 2045862
• DOI: http://doi.org/10.3343/alm.2015.35.4.404

Abstract

BACKGROUND
ChromID C. difficile agar (CDIF; bioMerieux, 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 (bioMerieux) 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.

Keyword

Clostridium difficile; Culture; Isolation

MeSH Terms

Agar
Clostridium difficile*
Hot Temperature
Humans
Prospective Studies
Shock
Tertiary Care Centers
Agar

Figure

• 

  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).

Reference

1. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol. 2010; 31:431–455. PMID: 20307191.

2. George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979; 9:214–219. PMID: 429542.

3. Carroll KC. Tests for the diagnosis of Clostridium difficile infection: the next generation. Anaerobe. 2011; 17:170–174. PMID: 21376826.

4. Foster NF, Riley TV. Improved recovery of Clostridium difficile spores with the incorporation of synthetic taurocholate in cycloserine-cefoxitin-fructose agar (CCFA). Pathology. 2012; 44:354–356. PMID: 22531346.

5. Wilcox MH, Fawley WN, Parnell P. Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery of Clostridium difficile. J Hosp Infect. 2000; 44:65–69. PMID: 10633056.

6. Borriello SP, Honour P. Simplified procedure for the routine isolation of Clostridium difficile from faeces. J Clin Pathol. 1981; 34:1124–1127. PMID: 7031097.

7. Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol. 1992; 30:514–516. PMID: 1537928.

8. Perry JD, Asir K, Halimi D, Orenga S, Dale J, Payne M, et al. Evaluation of a chromogenic culture medium for isolation of Clostridium difficile within 24 hours. J Clin Microbiol. 2010; 48:3852–3858. PMID: 20739493.

9. Eckert C, Burghoffer B, Lalande V, Barbut F. Evaluation of the chromogenic agar chromID C. difficile. J Clin Microbiol. 2013; 51:1002–1004. PMID: 23269743.

10. Fedorko DP, Williams EC. Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile. J Clin Microbiol. 1997; 35:1258–1259. PMID: 9114419.

11. Lemee L, Dhalluin A, Testelin S, Mattrat MA, Maillard K, Lemeland JF, et al. Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol. 2004; 42:5710–5714. PMID: 15583303.

12. Terhes G, Urban E, Soki J, Hamid KA, Nagy E. Community-acquired Clostridium difficile diarrhea caused by binary toxin, toxin A, and toxin B gene-positive isolates in Hungary. J Clin Microbiol. 2004; 42:4316–4318. PMID: 15365032.

13. Carson KC, Boseiwaqa LV, Thean SK, Foster NF, Riley TV. Isolation of Clostridium difficile from faecal specimens--a comparison of chromID C. difficile agar and cycloserine-cefoxitin-fructose agar. J Med Microbiol. 2013; 62:1423–1427. PMID: 23579394.

14. Boseiwaqa LV, Foster NF, Thean SK, Squire MM, Riley TV, Carson KC. Comparison of ChromID C. difficile agar and cycloserine-cefoxitin-fructose agar for the recovery of Clostridium difficile. Pathology. 2013; 45:495–500. PMID: 23846295.

15. Han SB, Chang J, Shin SH, Park KG, Lee GD, Park YG, et al. Performance of chromID Clostridium difficile agar compared with BBL C. difficile selective agar for detection of C. difficile in stool specimens. Ann Lab Med. 2014; 34:376–379. PMID: 25187891.