title: In vitro emergence of rifampicin resistance in Propionibacterium acnes and molecular characterization of mutations in the rpoB gene.
abstract: OBJECTIVE
  Activity of rifampicin against Propionibacterium acnes biofilms was recently demonstrated, but rifampicin resistance has not yet been described in this organism. We investigated the in vitro emergence of rifampicin resistance in P. acnes and characterized its molecular background.
  
  
  METHODS
  P. acnes ATCC 11827 was used (MIC 0.007 mg/L). The mutation rate was determined by inoculation of 10(9) cfu of P. acnes on rifampicin-containing agar plates incubated anaerobically for 7 days. Progressive emergence of resistance was studied by serial exposure to increasing concentrations of rifampicin in 72 h cycles using a low (10(6) cfu/mL) and high (10(8) cfu/mL) inoculum. The stability of resistance was determined after three subcultures of rifampicin-resistant isolates on rifampicin-free agar. For resistant mutants, the whole rpoB gene was amplified, sequenced and compared with a P. acnes reference sequence (NC006085).
  
  
  RESULTS
  P. acnes growth was observed on rifampicin-containing plates with mutation rates of 2 ± 1 cfu  ×  10(-9) (4096× MIC) and 12 ± 5 cfu  ×  10(-9) (4 × MIC). High-level rifampicin resistance emerged progressively after 4 (high inoculum) and 13 (low inoculum) cycles. In rifampicin-resistant isolates, the MIC remained >32 mg/L after three subcultures. Mutations were detected in clusters I (amino acids 418-444) and II (amino acids 471-486) of the rpoB gene after sequence alignment with a Staphylococcus aureus reference sequence (CAA45512). The five following substitutions were found: His-437 → Tyr, Ser-442 → Leu, Leu-444 → Ser, Ile-483 → Val and Ser-485 → Leu.
  
  
  CONCLUSIONS
  The rifampicin MIC increased from highly susceptible to highly resistant values. The resistance remained stable and was associated with mutations in the rpoB gene. To our knowledge, this is the first report of the emergence of rifampicin resistance in P. acnes.
authors.affiliation: Infectious Diseases Service, Department of Medicine,  Lausanne University Hospital, Lausanne,  Switzerland.
authors.forename: Ulrika
authors.initials: U
authors.lastname: Furustrand Tafin
authors.forename: Andrej
authors.initials: A
authors.lastname: Trampuz
authors.forename: Stéphane
authors.initials: S
authors.lastname: Corvec
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011425:Propionibacterium acnes / Q000187:drug effects* / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D012692:Serial Passage
chemical_list: D000900:Anti-Bacterial Agents; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin