HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS REGISTER		Author Keyword(s) Vol Page [Advanced]

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this link to a friend Similar articles in this journal Similar articles in PubMed Add article to my folders Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Freeman, J Articles by Wilcox, M H Search for Related Content PubMed PubMed Citation Articles by Freeman, J Articles by Wilcox, M H

The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces

Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK

Correspondence to:
Dr M H Wilcox, Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK;
markwi{at}pathology.leeds.ac.uk Aims:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated the effects of storage conditions upon the viability of this organism and its toxin.

Methods: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic strain, and an environmental isolate) were grown anaerobically at 37°C for 72 hours in a pool of five faecal emulsions. Aliquots of each emulsion were stored at either -20°C (frozen) or 4°C (refrigerated). Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days 1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing.

Results: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing adversely affected the viability of C difficile vegetative cells or spores. Single and multiple exposures of samples to 4°C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three strains.

Conclusion: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal effects upon the viability of C difficile or its spores. Storage at 4°C has no discernible effect on C difficile cytotoxin. However, storage at -20°C has a detrimental effect upon C difficile cytotoxin, and multiple cycles of freezing and thawing may further adversely effect toxin titres.

Keywords: Clostridium difficile; storage temperature; viability; toxin degradation

Abbreviations: CCEYL, cycloserine-cefoxitin egg yolk agar plates with lysozyme; PBS, phosphate buffered saline; PCR, polymerase chain reaction

This article has been cited by other articles:

				J. Halsey Current and future treatment modalities for Clostridium difficile-associated disease Am. J. Health Syst. Pharm., April 15, 2008; 65(8): 705 - 715. [Abstract] [Full Text] [PDF]

				R. L. P. Jump, M. J. Pultz, and C. J. Donskey Vegetative Clostridium difficile Survives in Room Air on Moist Surfaces and in Gastric Contents with Reduced Acidity: a Potential Mechanism To Explain the Association between Proton Pump Inhibitors and C. difficile-Associated Diarrhea? Antimicrob. Agents Chemother., August 1, 2007; 51(8): 2883 - 2887. [Abstract] [Full Text] [PDF]

				R. J. van den Berg, N. Vaessen, H. P. Endtz, T. Schulin, E. R. van der Vorm, and E. J. Kuijper Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study J. Med. Microbiol., January 1, 2007; 56(1): 36 - 42. [Abstract] [Full Text] [PDF]

				N. J. Asha, W. N. Fawley, J. Freeman, and M. H. Wilcox Increased Rate of DNA Recovery from United Kingdom Epidemic Clostridium difficile PCR Ribotype 1 Strains Stored Cryogenically J. Clin. Microbiol., November 1, 2005; 43(11): 5794 - 5795. [Abstract] [Full Text] [PDF]

				L. G. Arroyo, J. Rousseau, B. M. Willey, D. E. Low, H. Staempfli, A. McGeer, and J. S. Weese Use of a Selective Enrichment Broth To Recover Clostridium difficile from Stool Swabs Stored under Different Conditions J. Clin. Microbiol., October 1, 2005; 43(10): 5341 - 5343. [Abstract] [Full Text] [PDF]

				S. Goh, B. J Chang, and T. V Riley Effect of phage infection on toxin production by Clostridium difficile J. Med. Microbiol., February 1, 2005; 54(2): 129 - 135. [Abstract] [Full Text] [PDF]

				G. Ackermann, S. Thomalla, F. Ackermann, R. Schaumann, A. C Rodloff, and B. R Ruf Prevalence and characteristics of bacteria and host factors in an outbreak situation of antibiotic-associated diarrhoea J. Med. Microbiol., February 1, 2005; 54(2): 149 - 153. [Abstract] [Full Text] [PDF]

				C. Thomas, M. Stevenson, and T. V. Riley Antibiotics and hospital-acquired Clostridium difficile-associated diarrhoea: a systematic review J. Antimicrob. Chemother., June 1, 2003; 51(6): 1339 - 1350. [Abstract] [Full Text] [PDF]