title: Association of Fidaxomicin with C. difficile Spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production.
abstract: BACKGROUND
  We have previously shown that fidaxomicin instillation prevents spore recovery in an in-vitro gut model, whereas vancomycin does not. The reasons for this are unclear. Here, we have investigated persistence of fidaxomicin and vancomycin on C. difficile spores, and examined post-antibiotic exposure spore recovery, outgrowth and toxin production.
  
  
  METHODS
  Prevalent UK C. difficile ribotypes (n = 10) were incubated with 200mg/L fidaxomicin, vancomycin or a non-antimicrobial containing control for 1 h in faecal filtrate or Phosphate Buffered Saline. Spores were washed three times with faecal filtrate or phosphate buffered saline, and residual spore-associated antimicrobial activity was determined by bioassay. For three ribotypes (027, 078, 015), antimicrobial-exposed, faecal filtrate-washed spores and controls were inoculated into broth. Viable vegetative and spore counts were enumerated on CCEYL agar. Percentage phase bright spores, phase dark spores and vegetative cells were enumerated by phase contrast microscopy at 0, 3, 6, 24 and 48 h post-inoculation. Toxin levels (24 and 48h) were determined by cell cytotoxicity assay.
  
  
  RESULTS
  Fidaxomicin, but not vancomycin persisted on spores of all ribotypes following washing in saline (mean = 10.1mg/L; range = 4.0-14mg/L) and faecal filtrate (mean = 17.4mg/L; 8.4-22.1mg/L). Outgrowth and proliferation rates of vancomycin-exposed spores were similar to controls, whereas fidaxomicin-exposed spores showed no vegetative cell growth after 24 and 48 h. At 48h, toxin levels averaged 3.7 and 3.3 relative units (RU) in control and vancomycin-exposed samples, respectively, but were undetectable in fidaxomicin-exposed samples.
  
  
  CONCLUSIONS
  Fidaxomicin persists on C. difficile spores, whereas vancomycin does not. This persistence prevents subsequent growth and toxin production in vitro. This may have implications on spore viability, thereby impacting CDI recurrence and transmission rates.
authors.affiliation: Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Caroline H
authors.identifier: http://orcid.org/0000-0002-8076-1699
authors.initials: CH
authors.lastname: Chilton
authors.affiliation: Faculty of Science and Engineering, Manchester Metropolitan University, Manchester, United Kingdom.
authors.forename: Grace S
authors.initials: GS
authors.lastname: Crowther
authors.affiliation: Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Ashwin
authors.affiliation: Astellas Pharma Europe Ltd, Chertsey, Surrey, United Kingdom.
authors.forename: Chris M
authors.initials: CM
authors.lastname: Longshaw
authors.affiliation: Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom.|Department of Microbiology, Leeds Teaching Hospitals NHS Trust, The General Infirmary, Old Medical School, Leeds, United Kingdom.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Wilcox
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000096:biosynthesis*; D016360:Clostridioides difficile / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology*; D003015:Clostridium Infections / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D000077732:Fidaxomicin; D006801:Humans; D008826:Microbial Sensitivity Tests; D013171:Spores, Bacterial / Q000187:drug effects*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001427:Bacterial Toxins; D000077732:Fidaxomicin