title: Proteomic analysis of a NAP1 Clostridium difficile clinical isolate resistant to metronidazole.
abstract: BACKGROUND
  Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates.
  
  
  RESULTS
  NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R.
  
  
  CONCLUSIONS
  This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Chong
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Tarah
authors.initials: T
authors.lastname: Lynch
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Stuart
authors.initials: S
authors.lastname: McCorrister
authors.affiliation: Victoria General Hospital, Victoria, British Columbia, Canada.
authors.forename: Pamela
authors.initials: P
authors.lastname: Kibsey
authors.affiliation: Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Miller
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Denise
authors.initials: D
authors.lastname: Gravel
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Garrett R
authors.initials: GR
authors.lastname: Westmacott
authors.affiliation: Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mulvey
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D016360:Clostridioides difficile / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D016000:Cluster Analysis; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008795:Metronidazole / Q000494:pharmacology*; D020543:Proteome; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D020543:Proteome; D008795:Metronidazole