Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars

D S Pickering; J J Vernon; J Freeman; M H Wilcox; C H Chilton

doi:10.1016/j.anaerobe.2018.01.010

Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars

Anaerobe. 2018 Apr:50:38-43. doi: 10.1016/j.anaerobe.2018.01.010. Epub 2018 Feb 1.

Authors

D S Pickering¹, J J Vernon², J Freeman³, M H Wilcox⁴, C H Chilton²

Affiliations

¹ Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK. Electronic address: umdsp@leeds.ac.uk.
² Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK.
³ Microbiology, Leeds Teaching Hospitals Trust, Leeds, UK.
⁴ Healthcare Associated Infections Research Group, Molecular Gastroenterology, Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, West Yorkshire, UK; Microbiology, Leeds Teaching Hospitals Trust, Leeds, UK.

PMID: 29408598
DOI: 10.1016/j.anaerobe.2018.01.010

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.

Keywords: Culture media; Germination; Lysozyme; MIC; Spores; Taurocholate.

MeSH terms

Agar
Amino Acids / chemistry
Amino Acids / pharmacology
Clostridioides difficile / drug effects
Clostridioides difficile / physiology*
Culture Media* / chemistry
Culture Media* / pharmacology
Microbial Sensitivity Tests
Spores, Bacterial* / drug effects

Substances

Amino Acids
Culture Media
Agar