Evaluation of Cycloserine-Cefoxitin Fructose Agar (CCFA), CCFA with Horse Blood and Taurocholate,and Cycloserine-Cefoxitin Mannitol Broth with Taurocholate and Lysozyme for Recovery of Clostridium difficile Isolates from Fecal Samples

Cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate (CCFA-HT), and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared for recovery of Clostridium difficile from 120 stool specimens. Compared to CCFA, CCFA-HT enhanced C. difficile growth and improved recovery by 4%. In a separate study, 9% (8/91) of stool samples previously C. difficile negative on plate medium were C. difficile positive when cultured in CCMB-TAL.     

Selective and differential medium for isolation of Clostridium difficile.

Clostridium difficile is a recognized cause of pseudomembranous (antimicrobial agent-associated) colitis and may be one of the causes of antimicrobial agent-induced diarrhea. A selective and differential agar medium that contains cycloserine, cefoxitin, fructose, and egg yolk (CCFA) was developed to facilitate the isolation of C. difficile from fecal specimens. Quantitative cultures of 16 stock strains of C. difficile on this medium (and on a medium containing cycloserine, fructose, and egg yolk) yielded counts equivalent to those obtained on blood agar; other media selective for clostridia, including Clostrisel agar, reinforced clostridial agar plus 0.2% para-cresol, and egg yolk-neomycin agar (the latter was inoculated with cultures subjected to prior heat shocking), were also tested and found to be inhibitory to the growth of C. difficile. Of 28 fecal or colostomy effluent specimens cultured on the above media, 14 yielded C. difficile. CCFA was found to be the most sensitive and selective of these media for the recovery of C. difficile. Colonies of C. difficile growing on CCFA had distinctive morphological and fluorescent properties which were sufficient for presumptive identification. CCFA should provide a rapid method for the screening of fecal specimens from patients with antimicrobial agent-associated diarrhea or colitis for C. difficile.     

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin

A procedure was devised for routine examination of feces for Clostridium difficile with selective enrichment broth culture containing increased levels of carbohydrates and antibiotics to detect cytotoxin and volatile acids in broths inoculated with fecal samples. C. difficile was detected and identified with a rapidity comparable to that of conventional culture on selective cycloserine-cefoxitin fructose agar. Detection rates for C. difficile in inoculated broths (111/401 or 27%) were significantly higher than for culture on cycloserine-cefoxitin fructose agar (47/401 or 11%, P greater than 0.001). All fecal samples containing C. difficile and cytotoxin were correctly identified by the procedure. Isocaproic acid peak heights greater than 2 mm in selective enrichment broths inoculated with fecal samples indicated that C. difficile was present in the fecal sample examined. Of the positive specimens examined, 58% (64/111) produced peak heights greater than 10 mm. Peak heights less than 2 mm were not associated with C. difficile in the fecal sample. The investigated procedure provided a reliable alternative to the routine processing of feces for detecting C. difficile and associated cytotoxin in feces. Inoculated broths with isocaproic acid peak heights greater than 2 mm, after 24 to 48 h of incubation, and in which cytotoxin was detected, were subcultured to blood agar to obtain isolates of the organism as required. Broths which showed isocaproic acid peak heights less than 2 mm, and in which cytotoxin was not detected, were discarded as negative for C. difficile. The procedure was deemed potentially useful for epidemiological surveys of C. difficile.     

Effective and Reduced-Cost Modified Selective Medium for Isolation of Clostridium difficile

Both for epidemiologic studies and for diagnostic testing, there is a need for effective, economical, and readily available selective media for the culture of Clostridium difficile. We have developed a reduced-cost substitute for cycloserine-cefoxitin-fructose agar (CCFA), which is an effective but expensive selective medium for C. difficile. The modified medium, called C. difficile brucella agar (CDBA), includes an enriched brucella base as a substitute for proteose peptone no. 2, and the concentration of sodium taurocholate has been reduced from 0.1% to 0.05%. To compare the sensitivities and selectivities of CDBA and CCFA, cultures for C. difficile were performed using stool samples from patients with C. difficile-associated disease. CDBA was as sensitive as CCFA for the recovery of C. difficile, with a similar frequency of breakthrough growth of stool microflora (25% versus 31%, respectively). A liquid formulation of the modified medium, termed C. difficile brucella broth (CDBB), stimulated rapid germination and outgrowth of C. difficile spores, at a rate comparable to that in cycloserine-cefoxitin-fructose broth. Our results suggest that CDBA and CDBB are sensitive, selective, and reduced-cost media for the recovery of C. difficile from stool samples.     

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.     

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.     

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.     

Role of culture and toxin detection in laboratory testing for diagnosis ofClostridium difficile-associated diarrhea

Two variations of an egg yolk agar base medium containing cycloserine, cefoxitin, and fructose (CCFA), one with 250 g and the other with 500 g of cycloserine/ml of agar medium were compared to study the effect of the cycloserine concentration on recovery ofClostridium difficile from stool samples. In addition, the role of prior anaerobic reduction of these media in the detection ofClostridium difficile-associated diarrhea (CDAD) was tested. Each medium was studied over a two-month period, with outcome compared between the testing periods and to historical data from our institution. Clinical correlation of test results was performed. The use of the originally described formulation of CCFA with 500 g of cycloserine/ml of agar combined with 4 h of anaerobic reduction prior to specimen inoculation increased the rate of isolation of toxigenicClostridium difficile from clinical specimens from 6 to 17% (p < 0.001). Combining direct detection of stool toxin and properly performed culture for toxigenicClostridium difficile enhances the potential for diagnosis of CDAD. For optimal performance the culture medium should contain the originally proposed cycloserine concentration of 500 g/ml of agar and should be anaerobically reduced at least 4 h prior to specimen inoculation.     

Comparison of five cultural procedures for isolation of Clostridium difficile from stools.

Several procedures have been described for the culture of Clostridium difficile from stool specimens. The goal of this study was to determine the effectiveness of five of these methods for the isolation of C. difficile from feces of patients suspected of having C. difficile-associated illness. A total of 564 stool specimens were cultured by using heat shock, ethanol treatment (ET), and direct plating on Carr-Scarborough cycloserine-cefoxitin-fructose agar (CCFA) with horse blood (C/S medium), BBL CCFA medium, and Remel C. difficile agar. Cytotoxin assays were performed on all specimens. A total of 113 specimens (20%) were positive for C. difficile by one or more methods. The numbers of positive cultures by using heat shock, ET, and direct plating on C/S medium, BBL CCFA medium, and Remel C. difficile agar were 79 (70%), 89 (79%), 91 (81%), 79 (70%), and 52 (46%), respectively. We concluded that ET and direct plating on C/S medium were the most effective procedures for isolating C. difficile from stool specimens and found significant variation in the performance of modified CCFA from different manufacturers.     

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 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.     

Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile.

The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA). Fifty stored isolates of C. difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C. difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive. Other Clostridium species that were PRO Disc positive could be differentiated from C. difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining.     

Evaluation of Brilliance MRSA 2 Agar for Detection of Methicillin-Resistant Staphylococcus aureus in Clinical Samples

We compared 2 chromogenic media (Oxoid Brilliance MRSA 2 agar [Thermo Fisher Scientific] and MRSA-ID [bioMérieux]) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in 1,368 hospital samples. For both media, broth enrichment was essential to obtain satisfactory diagnostic performance. Although with direct cultures only, the diagnostic performance (particularly sensitivity) of Brilliance MRSA 2 agar appears better than that of MRSA-ID, no difference in sensitivity or specificity between the media was detected after inclusion of an enrichment step.     

New semisolid agar for the detection of motile salmonellae.

A semisolid selective motility agar based on selenite broth and designated semisolid selenite fecal (SSF) agar was developed for recovery of motile salmonellae from large numbers of rodent fecal specimens. The medium is easily prepared and inoculated; results are readily interpreted. When combined with selenite or gram-negative broth enrichment, SSF agar yielded significantly better Salmonella isolation from experimentally infected rodents than a standard battery of media. Only 14 non-Salmonella isolates from 1,002 Salmonella-free rodent fecal specimens migrated on SSF agar.     

Agar media that indicate acid production from sorbitol by oral microorganisms.

Two varieties of agar medium (Trypticase [BBL Microbiology Systems]-serum-sorbitol-bromcresol purple agar [TSSB] and Trypticase-blood-sorbitol-CaCO3 agar [TBSCa]) indicating microbial acid production from sorbitol were tested. The media were devised for use in studies on the prevalence of sorbitol-fermenting human oral microorganisms incubated in an anaerobic or microaerophilic atmosphere containing 5 to 6% CO2. TSSB contains bromcresol purple as the pH indicator and NaHCO3 as the main buffering salt. TBSCa contains CaCO3 as both the buffering salt and the indicator of acid production. The growth yield of pure cultures of oral microorganisms on TBSCa was shown to equal that on blood agar incubated under similar conditions. TSSB inhibited the growth of several bacteria to various extents. The recovery of sorbitol-fermenting microorganisms from oral specimens was the greatest when the specimens were assayed with TBSCa. The poorer results obtained with TSSB were mainly due to the decoloration of the pH indicator in this medium and the presence of greater numbers of sorbitol false-positive colonies.     

Comparison of agar dilution, tube dilution, and broth microdilution susceptibility tests for determination of ramoplanin MICs.

Standard broth microdilution (with and without bovine serum albumin [BSA] supplementation), tube dilution, and agar dilution susceptibility tests were compared for determining ramoplanin MICs. With a data base of 246 clinical isolates of gram-positive bacteria from 33 U.S. sites, it was shown that (i) agar and tube dilution susceptibility tests gave essentially the same results (93.9% of the test results were within 1 doubling dilution of equivalence), (ii) broth microdilution susceptibility tests gave results up to 5 doubling dilutions higher than agar or tube assays, and (iii) this data skewing could be reversed by BSA supplementation (final concentration, 0.02%) of the broth microdilution test medium.     

Comparison of compact colony-forming activity and paracoagulation activity of strains ofStaphylococcus aureus in serum and plasmas of various animals

Using 20 strains ofStaphylococcus aureus isolated from clinical specimens, the compact colony-forming activity (CCFA) in serum-soft agar (SSA) in sera from various animals and the paracoagulation (PC) activity of the compact colony-forming active substance (CCFAS) extracted from these strains were investigated. The results of this comparative study revealed that the CCFA and PC ofS. aureus for sera from various animals in SSA were different, not only among different strains but also in the same strains. In addition, the effect of galactose and calcium ions on the PC activity of these strains in experiments employing human fibrinogen permitted the recognition of these groups ofS. aureus strains. In one group, PC activity was decreased by galactose but unaffected by calcium ions, in the second group PC activity was unaffected by galactose but increased by calcium ions, while in the third group it was unaffected by both. These results suggest the possiblity of heterogeneity of CCFA among different strains ofS. aureus.     

Improved growth of Campylobacter pylori in a biphasic system.

The recovery of Campylobacter pylori from clinical specimens is difficult, even when done with an optimal medium, atmosphere, and temperature. The growth of this organism was investigated by comparing a biphasic system with broth culture. The effects of gyration, inoculum, and pH were studied. Brucella agar and broth supplemented with 2.5% fetal bovine serum were used. Growth in the biphasic system was an average of 2 log units (7 X 10(8) versus 5 X 10(6) CFU/ml) greater than that in the broth system (P less than 0.01), and this occurred 12 to 24 h sooner in the biphasic system. When gyration was added, an average of 1 log unit of growth improvement was seen in comparable systems. Improved growth was also seen with low inoculum levels, in which stationary-phase cells in the broth system reached 10(5) CFU/ml compared with 10(7) CFU/ml in the biphasic system. At the three pH ranges studied, growth was best at pH 8 to 9 (6 X 10(9) CFU/ml), averaging 2 log units greater growth than that at pH 6 to 7 and 4 log units greater growth than that at pH 4.5 to 5.5 (P less than 0.01). The improved recovery of the organism for low inoculum levels in a biphasic system may be important for long-term storage and clinical isolation.     

Evaluation of Current Methods for Detection of Staphylococci with Reduced Susceptibility to Glycopeptides

The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant Staphylococcus aureus (MRSA) strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 microg of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 microg of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was > or =8 mg of vancomycin per liter and > or =8 microg teicoplanin per ml or > or =12 microg of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-)glycopeptide-intermediate S. aureus compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (n = 48) and MRSA (n = 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.     

Comparison of the semisolid agar antifungal susceptibility test with the NCCLS M38-P broth microdilution test for screening of filamentous fungi

Antifungal susceptibility testing of pathogenic molds is being developed. A simple screening semisolid agar antifungal susceptibility (SAAS) test accurately measures susceptibilities of yeasts. The performance of the SAAS screening test for filamentous fungi was assessed by comparing MICs of four antifungals (amphotericin B [AMB], AMB lipid complex [ABEL], itraconazole [ITZ], and posaconazole [POS]) for 54 clinical mold isolates with the results of the National Committee for Clinical Laboratory Standards (NCCLS) proposed broth microdilution method (M38-P). The SAAS test utilized inocula stabbed into tubes of 0.5% semisolid heart infusion agar. In both tests MICs were read after incubation at 35 degrees C for 48 h. The isolates tested were Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, other Aspergillus spp., Fusarium spp., Penicillium sp., Mucor sp., Scedosporium prolificans, Trichophyton sp., and an unidentified dematiaceous mold. Concordance of test results was determined as the percent agreement of MICs +/- 1 dilution. The overall agreement between the tests for each drug was as follows: AMB, 94%; ABEL, 83%; ITZ, 94%; POS, 94%. For the Aspergillus spp., all but one were susceptible to ITZ by SAAS test; all were susceptible to POS (MIC range, 0.25 to 4 micro g/ml). Three of six non-Aspergillus molds that were resistant to AMB and ABEL by SAAS (MIC >/= 2 micro g/ml) were also resistant by the NCCLS test. The SAAS test compared favorably to the NCCLS broth microdilution test for molds, and most of the clinical isolates tested were susceptible to all four drugs.