Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.

Isolation of Clostridium difficile from fecal specimens has been facilitated by the development of a selective and differential medium, cefoxitin-cycloserinefructose agar (CCFA). We substituted 0.1% sodium taurocholate for the 2.5% egg yolk in CCFA and compared the growth of 15 isolates of C. difficile on the resulting medium with growth on conventional CCFA. The taurocholate-containing medium (TCCFA) quantitatively recovered vegetative forms of C. difficile in the same numbers as CCFA medium. Recovery of spores was a mean 1.7 log(10) higher on TCCFA than on CCFA. Thirty-six of 60 patient stool specimens growing C. difficile gave a heavier growth on TCCFA than on CCFA, and 9 failed to yield C. difficile on CCFA. TCCFA detected spores of 75 colony-forming units per ml from artificially inoculated fecal specimens when conventional stool culturing techniques were used. Fluorescence of colonies of C. difficile was more intense on TCCFA than on CCFA. TCCFA was simpler to prepare and, overall, was more sensitive than CCFA.     

Comparison of methods for recovery of Clostridium difficile from an environmental surface

Survival of Clostridium difficile in an aerobic environment is possible because of spore formation. When sodium taurocholate is substituted for the egg yolk of a selective medium, cycloserine-cefoxitin-fructose-agar (CCFA), enhanced recovery of C. difficile spores is shown. This selective medium (TCCFA) does not improve recovery of vegetative forms. In this study, dry and saline-moistened swabs, adhesive paddles, and Rodac plates containing CCFA and TCCFA were compared in their ability to recover C. difficile spores from an inoculated surface. Rodac plates grew 20 to 25 times as many spores on TCCFA as on CCFA. Saline-moistened swabs recovered fewer organisms than Rodac plates. Dry swabs and adhesive paddles rarely recovered spores. Prereduction of agar in an anaerobic chamber was not necessary for optimal spore recovery. Optimal growth of vegetative C. difficile required prereduced media. Agar prereduced for 2 h supported the growth of 12 C. difficile isolates as well as agar prereduced for 18 h. Vegetative cells of C. difficile survived for only 15 min in room air. Use of Rodac plates containing TCCFA is preferred for detection of C. difficile spores in the hospital environment.     

Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium difficile from environmental sites.

Cycloserine-cefoxitin-fructose agar (CCFA) and cycloserine-cefoxitin-fructose broth (CCFB) containing either 500 or 250 micrograms of cycloserine per ml were compared for efficacy in the isolation of Clostridium difficile from hospital ward environmental sites. A RODAC imprint technique was used to inoculate prereduced CCFA. Moistened swabs were used to inoculate prereduced CCFB from environmental sites immediately adjacent to the RODAC sample sites. CCFA (6% positive) was significantly more sensitive than CCFB (3% positive; P less than 0.005), regardless of the cycloserine concentration. When the CCFA cycloserine concentration was decreased from 500 to 250 micrograms/ml, the overall rate of positive cultures rose from 4 to 17%. Medium containing 500 micrograms of cycloserine per ml may be too inhibitory to isolate many moderately sensitive strains of C. difficile from environmental sites. Regardless of the cycloserine concentration, the CCFA RODAC imprint technique is superior to the CCFB method.     

Comparison of media for screening of diarrheic stools for the recovery of Clostridium difficile.

Recoveries of Clostridium difficile from stool specimens by using three media, cycloserine-mannitol agar (M-CMA), cycloserine-mannitol-blood agar (M-CMBA), and cycloserine-cefoxitin agar (M-CCA), were compared. Of 321 clinical specimens, 37 yielded C. difficile. Thirty-four were positive on M-CCA, 21 were positive on M-CMA, and 20 were positive on M-CMBA. M-CCA recovered significantly more C. difficile than did M-CMBA or M-CMA.     

New selective medium for isolating Clostridium difficile from faeces.

AIMS: To compare CCFA (cycloserine, cefoxitin fructose agar) with a new selective medium CDMN (containing cysteine hydrochloride, norfloxacin, and moxalactam) for the isolation of Clostridium difficile after direct faecal culture. METHODS: The minimum inhibitory concentration (MIC) of norfloxacin was determined for 64 strains of C difficile, 17 strains of other Clostridium sp, and 66 various isolates of faecal origin, together with MIC determinations of moxalactam against the 81 strains of Clostridium sp and 15 isolates of Bacteroides sp. Using C difficile agar base with 0.5 g/l of cysteine hydrochloride, norfloxacin and moxalactam were incorporated into the medium and compared with CCFA for the isolation of C difficile after direct faecal culture. RESULTS: Norfloxacin (12 mg/l) inhibited the growth of enterobacteriaceae and faecal streptococci; moxalactam (32 mg/l) inhibited the growth of most strains of Bacteroides sp tested, together with Clostridium sp other than C difficile. Using the antibiotics in combination (CDMN), the growth and colonial morphology of 64 strains of C difficile were unaffected. When CDMN medium was compared with CCFA for the isolation of C difficile from 832 faeces from inpatients with diarrhoea, the CDMN agar isolated 20% more strains and reduced the number of contaminating colonies by 30%. CONCLUSIONS: CDMN both improves the isolation rate of C difficile from faecal specimens and reduces the growth of other organisms compared with CCFA.     

Effect of sodium polyanetholesulfonate and gelatin on the recovery of Gardnerella vaginalis from blood culture media.

Sodium polyanetholesulfonate (SPS) is used as a routine supplement to blood culture media to enhance recovery of microorganisms, but it inhibits the growth of Peptostreptococcus anaerobius, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptobacillus moniliformis. Comparative clinical blood culture studies at the University of Colorado Hospital suggested that SPS also inhibits the growth of Gardnerella vaginalis. We inoculated 16 blood culture isolates of G. vaginalis into 11 blood culture media containing SPS or sodium amylosulfate, with and without gelatin. In the absence of gelatin, only brain heart infusion and thiol broths with SPS supported the growth of more than five strains of G. vaginalis, whereas all media except Bactec 6B and 7C and brucella broths recovered most isolates with SPS and gelatin or with sodium amylosulfate alone. We conclude that SPS inhibits the growth of G. vaginalis in blood culture media but that this inhibition is medium dependent and can be overcome by supplementation of most media with gelatin.     

Gas chromatographic identification of Clostridium difficile and detection of cytotoxin from a modified selective medium.

A modification of an existing selective medium for Clostridium difficile is described. Inclusion in the medium of DL nor-leucine and p-hydroxyphenylacetic acid enables identification of C difficile to be made directly from primary isolation plates by gas chromatographic detection of caproic acid and p-cresol. Plugs of agar withdrawn from the selective medium also allow the detection of cytotoxin production in vitro.     

Comparison of the Rodac imprint method to selective enrichment broth for recovery of vancomycin-resistant enterococci and drug-resistant Enterobacteriaceae from environmental surfaces

We compared the Rodac imprint technique to selective enrichment broth for detecting vancomycin-resistant enterococci (VRE) and multidrug-resistant Enterobacteriaceae (MDRE) on surfaces. Rodac plates contained tryptic soy agar with 5% sheep blood, vancomycin (6 microg/ml), ceftazidime (2 microg/ml), amphotericin B (2 microg/ml), and clindamycin (1 microg/ml). Two types of broth were used: brain heart infusion (BHI) and BHI plus vancomycin (6 microg/ml) and ceftazidime (2 microg/ml) (BHIVC). Of the 46 surfaces cultured for VRE, 12 (26%) were positive. Of the 12 VRE-positive surfaces, 11 (92%) grew from Rodac, 8 (67%) grew from BHIVC, and 7 (58%) grew from BHI. A larger study is needed for MDRE, as only 4 of 43 surfaces were MDRE positive. The Rodac imprint technique successfully recovered VRE from environmental surfaces.     

Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions

The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.     

Rapid detection and presumptive identification of Clostridium difficile by p-cresol production on a selective medium.

A modification of a selective medium for Clostridium difficile is described. The ability of Cl difficile to produce p-cresol from p-hydroxy phenyl acetic acid provides a means for the rapid, sensitive detection and presumptive identification of this species in faecal cultures.     

Evaluation of the Clearview Clostridium difficile Toxin A Test and various selective culture media in comparison with the cytotoxin assay for the diagnosis of Clostridium difficile-associated diarrhoea

AIM: Clostridium difficile is the major pathogen associated with nosocomial diarrhoea. We evaluated the performances of a commercially available toxin A enzyme immunoassay (EIA; Clearview C. difficile Toxin A Test), culture and tissue culture cytotoxin assay in the diagnosis of C. difficile-associated diarrhoea. METHODS: Comparative test performance was determined from data obtained from 166 faecal samples. The initial analysis compared the performance of toxin A EIA and culture with that of cytotoxin assay, this being defined as a 'laboratory gold standard'. A second analysis compared the individual performance of the toxin A EIA, culture and cytotoxin assay using a combined clinical and laboratory diagnostic assessment as a 'clinical gold standard'. In a parallel, study three selective culture media were compared. RESULTS: From the initial analysis, the sensitivity and specificity of the methods were, respectively, 84.6 and 65.4% for the toxin A EIA, and 38.5 and 93.5% for culture. From the second analysis, the sensitivity and specificity of the methods were, respectively, 100 and 67.5% for the toxin A EIA, 63.6 and 96.7% for culture and 72.7 and 98.0% for cytotoxin assay. Media containing d-cycloserine 250mg/L and cefoxitin 8mg/L performed best, growing 88.2% of the isolates. CONCLUSION: The toxin A EIA we evaluated had poor specificity in the diagnosis of C. difficile-associated diarrhoea. We conclude that in our laboratory the combination of culture and cytotoxin assay is a preferred approach to the diagnosis of C. difficile-associated diarrhoea.     

Purification and characterization of toxin B from Clostridium difficile.

Toxin B from Clostridium difficile was purified to homogeneity and characterized. Purification of toxin B was achieved by gel filtration, chromatography on two consecutive anion-exchange columns, and chromatography on a high-resolution anion-exchange column in the presence of 50 mM CaCl2. The molecular weight of toxin B was estimated to be 250,000 by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 500,000 by gel filtration. No subunits were apparent when the toxin was reduced and analyzed by SDS-PAGE. The estimated molecular weight of native toxin B indicated that dimers may form in solution. Toxin B was homogeneous by SDS-PAGE, native PAGE, and high-resolution anion-exchange chromatography. No secondary sequences were detected when the amino terminus of the toxin was sequenced, which also indicated that contaminating peptides were absent from the preparation. The amino terminus of toxin B was determined to be NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg. One cytotoxic unit of toxin B was estimated to be 0.2 to 0.8 pg.     

Loss of surface fibronectin from human lung fibroblasts exposed to cytotoxin from Clostridium difficile.

Clostridium difficile cytotoxin caused an irreversible dose- and time-dependent loss of fibronectin from the surfaces of human lung fibroblasts, paralleling the appearance of the cytopathic effect. Fibronectin was not required for the intoxication process. The results lend further support to a transmembrane connective link between fibronectin and the microfilaments.