Clostridium difficile Ribotype 023 Lacks the Ability To Hydrolyze Esculin, Leading to False-Negative Results on Chromogenic Agar

LETTER

Clostridium difficile strains are toxigenic, spore forming, Gram-positive, anaerobic bacteria responsible for the majority of cases of colitis and antibiotic-associated diarrhea in the developed world (1). The use of chromogenic media for the rapid and specific detection of clinically important pathogens such as C. difficile has become widespread. Chromogenic agars rely on the ability of the microbe in question to break down or utilize a particular chromogenic substrate. For example, agar containing esculin, or one of its derivatives, such as 3,4-cyclohexenoesculetin-β-glucoside (CHE-β-glucoside), is used as a differential medium for C. difficile (2). The substrate is hydrolyzed to give glucose and esculetin, which reacts with ferric citrate, giving distinctive black colonies as a presumptive identification for C. difficile (Fig. 1) (3–6).

FIG 1.

C. difficile colonies on chromID C. difficile. (A) Standard black colonies produced by C. difficile on chromID. (B) Colorless colonies produced by C. difficile ribotype 023.

We have observed that some C. difficile strains fail to give a positive esculin hydrolysis test. Using a standard method (esculin broth with 0.07% [wt/vol] agar) (7), we tested representative strains from the five evolutionary clades of C. difficile (8) in triplicate: TL178 (clade 1, ribotype 002), R20291 (clade 2, ribotype 027), CD305 (clade 3, ribotype 023), CF5 (clade 4, ribotype 017), and M120 (clade 5, ribotype 078). Esculin hydrolysis was observed only in strains from clades 1, 2, 4, and 5: no esculin hydrolysis was detected in CD305, the clade 3 isolate.

The ability to hydrolyze esculin depends on possession of the appropriate β-glucosidase, esculinase (9). We hypothesize this may be nonfunctional or lacking in ribotype 023 strains from clade 3, and this may affect isolation and identification of these strains using chromogenic agar.

bioMérieux supplies a chromogenic agar, chromID C. difficile, for the specific detection of C. difficile within 24 h (10): C. difficile growth is characterized by the production of black colonies (Fig. 1) (11). We tested a further 35 clinical isolates of C. difficile ribotype 023. All isolates failed to produce black colonies on chromID C. difficile agar (Fig. 1), even after a 48-h incubation period. The C. difficile isolates included in the study were representative of ribotype 023 strains isolated over a period of 4 years from patients in Northern Ireland. Specimens originated from 8 different hospitals and from community patients throughout the region. One strain came from an unidentified hospital source in Scotland. Isolates were selected at random from these archived 023 strains.

The ribotype and toxigenicity of each isolate were confirmed using a previously published ribotyping method and toxin gene PCR (12). A total of 33 of the 35 isolates were also analyzed using multilocus variable-number tandem-repeat analysis (MLVA) (13). Thirty different MLVA profiles were detected and could be grouped into at least 15 distinct genotypes defined by a summed tandem-repeat difference of 3 or more (14), confirming the strains tested were genetically diverse. All of the isolates in this collection of 023 strains failed to produce black colonies on chromID C. difficile chromogenic agar. In addition, one (non-023) nongroupable isolate from our collection also gave colorless colonies, indicating this phenotype may be seen in other strains.

Ribotype 023 is consistently in the top 10 most common ribotypes found to be causing infection (15), so it is important that diagnostic laboratories using chromID C. difficile medium, or any esculin-based assay, are aware of this and that further confirmatory tests may be required. Ribotype 023 strains do indeed give the typical irregular colony morphology associated with C. difficile on chromID C. difficile agar, but the white/colorless colonies could be overlooked during routine testing. We wish to highlight this trait, as it may affect isolation and identification of this clinically significant ribotype when using chromID C. difficile agar or similar esculin-based products.