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Journal of Clinical Microbiology, October 2005, p. 5341-5343, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5341-5343.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of a Selective Enrichment Broth To Recover Clostridium difficile from Stool Swabs Stored under Different Conditions

Luis G. Arroyo,1* Joyce Rousseau,1 Barbara M. Willey,2 Don E. Low,2 Henry Staempfli,1 Allison McGeer,2 and J. Scott Weese1

Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada,1 Department of Microbiology, Mount Sinai Hospital, Toronto, Ontario, Canada2

Received 17 June 2005/ Accepted 25 July 2005


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ABSTRACT
 
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.


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TEXT
 
Clostridium difficile is an important cause of nosocomial and antimicrobial-associated diarrhea worldwide (1, 15, 12, 18). Outbreaks of C. difficile-associated diarrhea (CDAD) are not uncommon; and recent studies suggest that the incidence, severity, and relapse rate may be increasing (9, 17). Typically, the diagnosis of CDAD involves toxin detection in feces by commercial enzyme immunoassays or, to a lesser extent, cell cytotoxicity assays (2). Bacterial culture is less commonly performed because of its limited clinical value and the difficulties inherent in working with this fastidious anaerobe. As a result, isolates are not routinely available for antimicrobial susceptibility testing, molecular typing, or identification of virulence factors. The lack of C. difficile isolates hampers the investigation of apparent changes in the epidemiology or clinical presentation that may develop over time. The submission of fecal samples to reference laboratories for C. difficile culture could be a more feasible approach. However, concerns regarding the loss of viability during sample handling, submission delays, and suboptimal storage and shipping conditions may decrease the willingness of laboratories to centralize C. difficile culture. A sensitive culture technique minimally affected by suboptimal sample handling and delays from the time of collection to the time of processing would make this feasible.

The purpose of this study was evaluate the use of an enrichment culture protocol for the recovery of C. difficile spores from stool swabs by using different storage conditions and over time.

Stool samples were collected from 100 patients diagnosed with CDAD by clinical signs and the results of an enzyme-linked immunosorbent assay. Samples were stored in fecal containers at 4°C at the diagnostic laboratory and were submitted to the research laboratory within a week of collection. Upon arrival, nine standard culture swabs were plunged into each stool sample and placed into either Amies gel with charcoal or liquid Stuart's medium. One swab was used immediately, while the remaining swabs were randomly assigned to two storage groups: storage at room temperature or storage at 4°C. Swabs stored at room temperature (~25°C) were kept in a box, while the swabs stored at 4°C were stored in a standard refrigerator.

At the initial time point, three culture methods were performed: alcohol shock followed by inoculation onto blood agar (AS-BA), direct plating onto cycloserine-cefoxitin fructose agar (CCFA; Oxoid, Nepean, Ontario, Canada) supplemented with 7% horse blood, and enrichment culture in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate (TCCFB). For AS-BA, ~1 g or 1 ml of feces was mixed with an equal volume of absolute ethanol, gently vortexed, and left at room temperature for 60 min. The supernatant was discarded, and an aliquot of the resulting pellet was inoculated onto Columbia agar base with 5% sheep blood (Oxoid). The inoculated plates were incubated anaerobically at 37°C for 24 to 48 h. Suspicious colonies were subcultured; and C. difficile was identified by colony morphology, Gram stain appearance, and production of L-proline aminopeptidase (ProDisc; Remel, Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) (10).

At the initial time point, one swab was inoculated onto CCFA and the plates were incubated in an anaerobic chamber at 37°C and examined daily for C. difficile growth for up to 5 days. Suspicious colonies were subcultured and identified as described above. Following inoculation of CCFA, the swab was placed into 9.0 ml of TCCFB. After 7 days of incubation at 37°C, 2.0 ml was transferred into a sterile test tube and mixed with an equal amount of absolute ethanol and left at room temperature for 60 min. Samples were centrifuged at 3,800 x g for 10 min, the supernatant was discarded, and the resulting pellet was plated onto blood agar and incubated as described above. One swab from each storage group was randomly selected after 1, 2, 4, and 8 weeks of storage for culture. Swabs were directly inoculated into CCFA and then placed in TCCFB as described above.

For comparison of the three culture techniques at the baseline time, the data were evaluated in the form of a randomized complete block design (RCBD), with the fecal specimens being the blocks and the recovery methods being the treatments. The outcome was analyzed in a binary manner, and Cochran's Q test was applied to the data (8), followed by pairwise multiple-comparisons tests. Exact P values were computed for the latter.

An RCBD was used for evaluation of recovery method, storage temperature, and time. In addition to the main effects, two- and three-way interactions were tested. A generalized linear mixed model (GLIMMIX) (SAS [SAS 8.2, 2003; SAS Institute Inc., Cary, NC]) was used to analyze the data. The results were presented either in terms of proportions or, when two treatments or factor levels were being compared, in terms of odds ratios (ORs) with confidence intervals (CIs).

Clostridium difficile was recovered from 69%, 72%, and 94% of the fecal samples at the baseline time by using the CCFA, AS-BA, and TCCFB methods, respectively. The rate of recovery by TCCFB was significantly higher than those by CCFA (OR, 9.459; CI, 3.438 to 25.88; P < 0.001) and AS-BA (OR, 6.580; CI, 2.54 to 17.21; P < 0.001). There was not a significant difference between CCFA and AS-BA (OR, 0.62; CI, 0.587 to 2.718; P = 0.62). The recovery rate was not affected by either the storage time (P = 0.398) or the storage temperature (P = 0.917). The rate of recovery of C. difficile by the CCFA or TCCFB method remained relatively constant over the 8-week period of study.

The selective enrichment protocol used in this study was a sensitive technique for the recovery of C. difficile, even with prolonged time delays and the storage of small volumes of stool on standard culture swabs at room temperature. Additionally, swabs are easier to handle, store, and ship than larger diagnostic specimens.

The enrichment broth was chosen to provide the optimal recovery of C. difficile spores, on the basis of the premise that spores would be the main form of C. difficile to survive suboptimal handling conditions. Bile salts such as sodium cholate and taurocholate have previously been shown to enhance the recovery of C. difficile by facilitating spore germination (3, 6, 20) and may be particularly useful when fecal specimens are handled under suboptimal anaerobic conditions, such as those expected from the conditions used in this study (21). The results are not surprising, since several studies have shown that methods that use enrichment broths are significantly superior for the recovery of C. difficile from stool samples (5, 13, 16, 19). However, the capacity to recover C. difficile from swabs without being largely affected by storage time or temperature adds useful information (11). One of the main advantages of this technique is the ease of sample collection and submission. Anaerobic transportation media were not used; rather, stool samples were collected on standard culture swabs. Further, the lack of a significant decline in recovery over time or with storage at room temperature indicates that laboratories could store samples on swabs and submit them in batches without the need for special storage conditions, thereby decreasing the costs associated with sample handling.

The isolation of C. difficile from CCFA yielded results similar to those previously reported elsewhere (7, 14). However, it was surprising that there was no decline in the rate of C. difficile recovery over time (5, 20). It is possible that CCFA is more effective at spore recovery than is typically assumed. Interestingly, the cultures on CCFA plates tested after 4 and 8 weeks of storage had subjectively less contaminant overgrowth than cultures of fresher stool samples. It is possible that the competing organisms in the stored samples had less viability and that storage allowed the better growth or easier recognition of C. difficile on CCFA.

Alcohol shock of fecal samples has been reported to be a sensitive technique for the recovery of C. difficile from stool samples; however, it was inferior to enrichment culture in this study (4, 14). Alcohol shock culture was not evaluated over time because of the small volume of feces present on the stool swabs and the known negative effects of low fecal volumes on the recovery rate with alcohol shock (7).

This study indicates that a simple method of specimen storage and processing can result in the excellent recovery of C. difficile and make periodic culture or submission of samples to a central laboratory a feasible method of obtaining isolates for epidemiological purposes.


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ACKNOWLEDGMENTS
 
We thank William Sears for assistance with statistical analysis.

All work on this project was performed at the Ontario Veterinary College.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 823-8840. Fax: (519) 763-9544. E-mail: larroyo{at}uoguelph.ca. Back


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REFERENCES
 
    1
  1. Barbut, F., G. Corthier, Y. Charpak, M. Cerf, H. Monteil, T. Fosse, A. Trevoux, B. De Barbeyrac, Y. Boussougant, S. Tigaud, F. Tytgat, A. Sedallian, S. Duborgel, A. Collignon, M. E. Le Guern, P. Bernasconi, and J. C. Petit. 1996. Prevalence and pathogenicity of Clostridium difficile in hospitalized patients. A French multicenter study. Arch. Intern. Med. 156:1449-1454.[Abstract/Free Full Text]
    2. 2
  2. Barbut, F., M. Delmee, J. S. Brazier, J. C. Petit, I. R. Poxton, M. Rupnik, V. Lalande, C. Schneider, P. Mastrantonio, R. Alonso, E. Kuipjer, M. Tvede, and the ESCMID Study Group on Clostridium difficile (ESGCD). 2003. A European survey of diagnostic methods and testing protocols for Clostridium difficile. Clin. Microbiol. Infect. 9:989-996.[CrossRef][Medline]
    3. 3
  3. Bliss, D. Z., S. Johnson, C. R. Clabots, K. Savik, and D. N. Gerding. 1997. Comparison of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance of hospitalized patients. Diagn. Microbiol. Infect. Dis. 29:1-4.[CrossRef][Medline]
    4. 4
  4. Borriello, S. P., and H. Honour. 1981. Simplified procedure for the routine isolation of Clostridium difficile from faeces. J. Clin. Pathol. 34:1124-1127.[Abstract/Free Full Text]
    5. 5
  5. Buchanan, A. G. 1984. Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin. J. Clin. Microbiol. 20:74-76.[Abstract/Free Full Text]
    6. 6
  6. Buggy, B. P., C. C. Hawkins, and R. Fekety. 1985. Effect of adding sodium taurocholate to selective media on the recovery of Clostridium difficile from environmental surfaces. J. Clin. Microbiol. 21:636-637.[Abstract/Free Full Text]
    7. 7
  7. Clabots, C. R., S. J. Gerding, M. M. Olson, L. R. Peterson, and D. N. Gerding. 1989. Detection of asymptomatic Clostridium difficile carriage by an alcohol shock procedure. J. Clin. Microbiol. 27:2386-2387.[Abstract/Free Full Text]
    8. 8
  8. Conover W. J. 1980. Practical nonparametric statistics, 2nd ed. John Wiley & Sons, Inc., New York, N.Y.
    9. 9
  9. Eggertson, L. 2004. C. difficile hits Sherbrooke, Que., hospital: 100 deaths. Can. Med. Assoc. J. 31:436.
    10. 10
  10. Fedorko, D. P., and E. C. Williams. 1997. Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile. J. Clin. Microbiol. 35:1258-1259.[Abstract]
    11. 11
  11. Freeman, J., and M. H. Wilcox. 2003. The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces. J. Clin. Pathol. 56:126-128.[Abstract/Free Full Text]
    12. 12
  12. Kuijper, E. J., J. de Weerdt, H. Kato, N. Kato, A. P. van Dam, E. R. van der Vorm, J. Weel, C. van Rheenen, and J. Dankert. 2001. Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain. Eur. J. Clin. Microbiol. Infect. Dis. 20:528-534.[Medline]
    13. 13
  13. Levett, P. N., and G. Margaritis-Bassoulis. 1985. Enrichment media for isolation of Clostridium difficile from faeces. Eur. J. Clin. Microbiol. 4:135-136.[CrossRef][Medline]
    14. 14
  14. Marler, L. M., J. A. Siders, L. C. Wolters, Y. Pettigrew, B. L. Skitt, and S. D. Allen. 1992. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J. Clin. Microbiol. 30:514-516.[Abstract/Free Full Text]
    15. 15
  15. McFarland, L. V. 1998. Epidemiology, risk factors and treatments for antibiotic-associated diarrhea. Dig. Dis. 16:292-307.[CrossRef][Medline]
    16. 16
  16. O'Farrell, S., M. Wilks, J. Q. Nash, and S. Tabaqchali. 1984. A selective enrichment broth for the isolation of Clostridium difficile. J. Clin. Pathol. 37:98-99.[Free Full Text]
    17. 17
  17. Pepin, J., L. Valiquette, M. E. Alary, P. Villemure, A. Pelletier, K. Forget, K. Pepin, and D. Chouinard. 2004. Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. Can. Med. Assoc. J. 31:466-472.
    18. 18
  18. Pituch, H., A. Van Belkum, N. Van Den Braak, P. Obuch-Woszczatynski, H. Verbrugh, F. Meisel-Mikolajczyk, and M. Luczak. 2003. Recent emergence of an epidemic clindamycin-resistant clone of Clostridium difficile among Polish patients with C. difficile-associated diarrhea. J. Clin. Microbiol. 41:4184-4187.[Abstract/Free Full Text]
    19. 19
  19. Riley, T. V., J. S. Brazier, H. Hassan, K. Williams, and K. D. Phillips. 1987. Comparison of alcohol shock enrichment and selective enrichment for the isolation of Clostridium difficile. Epidemiol. Infect. 99:355-359.[Medline]
    20. 20
  20. Wilson, K. H. 1983. Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination. J. Clin. Microbiol. 18:1017-1019.[Abstract/Free Full Text]
    21. 21
  21. Wilson, K. H., M. J. Kennedy, and F. R. Fekety. 1982. Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J. Clin. Microbiol. 15:443-446.[Abstract/Free Full Text]

Journal of Clinical Microbiology, October 2005, p. 5341-5343, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5341-5343.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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  • Martirosian, G. (2006). Recovery of Clostridium difficile from Hospital Environments.. J. Clin. Microbiol. 44: 1202-1203 [Full Text]  

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