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Clinical Therapeutics

Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

Hannah C. Harris, Emma L. Best, Charmaine Normington, Nathalie Saint-Lu, Frédérique Sablier-Gallis, Jean de Gunzburg, Antoine Andremont, Mark H. Wilcox, Caroline H. Chilton
Hannah C. Harris
aHeath Care Associated Infection Research Group, Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom
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Emma L. Best
bDepartment of Microbiology, Leeds Teaching Hospitals NHS Trust, The General Infirmary, Leeds, United Kingdom
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Charmaine Normington
aHeath Care Associated Infection Research Group, Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom
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Nathalie Saint-Lu
cDa Volterra, Paris, France
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Frédérique Sablier-Gallis
cDa Volterra, Paris, France
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Jean de Gunzburg
cDa Volterra, Paris, France
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Antoine Andremont
cDa Volterra, Paris, France
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Mark H. Wilcox
aHeath Care Associated Infection Research Group, Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom
bDepartment of Microbiology, Leeds Teaching Hospitals NHS Trust, The General Infirmary, Leeds, United Kingdom
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Caroline H. Chilton
aHeath Care Associated Infection Research Group, Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom
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DOI: 10.1128/AAC.01401-20
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ABSTRACT

A healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

FOOTNOTES

    • Received 7 July 2020.
    • Returned for modification 9 August 2020.
    • Accepted 21 October 2020.
    • Accepted manuscript posted online 2 November 2020.
  • Supplemental material is available online only.

  • Copyright © 2020 American Society for Microbiology.

All Rights Reserved.

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Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics
Hannah C. Harris, Emma L. Best, Charmaine Normington, Nathalie Saint-Lu, Frédérique Sablier-Gallis, Jean de Gunzburg, Antoine Andremont, Mark H. Wilcox, Caroline H. Chilton
Antimicrobial Agents and Chemotherapy Dec 2020, 65 (1) e01401-20; DOI: 10.1128/AAC.01401-20

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Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics
Hannah C. Harris, Emma L. Best, Charmaine Normington, Nathalie Saint-Lu, Frédérique Sablier-Gallis, Jean de Gunzburg, Antoine Andremont, Mark H. Wilcox, Caroline H. Chilton
Antimicrobial Agents and Chemotherapy Dec 2020, 65 (1) e01401-20; DOI: 10.1128/AAC.01401-20
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KEYWORDS

Clostridioides difficile
antibiotics
colonization resistance
dysbiosis

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