Abstract
Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock.
Highlights
Clostridium difficile is a major nosocomial pathogen that can cause fatal diarrhoeal disease, placing a huge financial burden on health care systems worldwide [1]
We found that a broth enrichment step reduced sensitivity of isolation and of the solid media tested Cycloserine-cefoxitin egg yolk agar (CCEY) was the most sensitive while ChromID C. difficile had an improved recovery rate
It has been suggested that using a broth enrichment step can increase the sensitivity of isolation for C. difficile [9,15]
Summary
Clostridium difficile is a major nosocomial pathogen that can cause fatal diarrhoeal disease, placing a huge financial burden on health care systems worldwide [1]. There have been recent studies into the isolation of C. difficile from clinical stools which have focused on a small range of different media [2e5] or culture from environmental surfaces [6]. Many of these studies have focused on expensive pre-made agars which may not be affordable in the research setting [3e5]. Their main focus is on the recovery of C. difficile from clinical samples and not the semi-quantification of a known concentration of viable spores [2e5]
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