Pickering, DS, Vernon, JJ, Freeman, J et al. (2 more authors) (2018) Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars. Anaerobe, 50. pp. 38-43. ISSN 1075-9964
Abstract
Background: A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, L-histidine & L-phenylalanine) on C. difficile spore outgrowth were investigated. Methods: Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,L-histidine,L-phenyalanine). Results: CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ± 0.02 vs 0.12 ± 0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, L-histidine and L-phenylalanine. Conclusions: We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,L-histidine,L-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 Elsevier Ltd. This is an author produced version of a paper published in Anaerobe. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Lysozyme; Germination; Spores; MIC; Culture media; Taurocholate |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > Institute of Molecular Medicine (LIMM) (Leeds) > Section of Molecular Gastroenterology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 26 Apr 2018 13:33 |
Last Modified: | 01 Feb 2019 01:50 |
Status: | Published |
Publisher: | Elsevier Masson |
Identification Number: | 10.1016/j.anaerobe.2018.01.010 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:130099 |