title: Inability of L22 ribosomal protein alteration to increase macrolide MICs in the absence of efflux mechanism in Haemophilus influenzae HMC-S.
abstract: BACKGROUND
  Haemophilus influenzae HMC-C with high-level macrolide resistance after multi-step selection by clarithromycin reverted spontaneously and became hypersusceptible to macrolides.
  
  
  OBJECTIVE
  Determination of macrolide resistance mechanism(s) in hypersusceptible and hyperresistant strains.
  
  
  METHODS
  The presence of macrolide efflux in the strains was studied by radioactive erythromycin accumulation. Ribosomal mutations were investigated by sequencing. The possible role of acrAB clusters in macrolide resistance was studied by sequencing and expression analysis.
  
  
  RESULTS
  The parent strain had no ribosomal alteration, but both high-level resistant and hypersusceptible strains had R88P mutations in ribosomal protein L22. Radioactive macrolide accumulation studies pointed to the presence of macrolide efflux in the high-level resistant and parent strains, but not in the hypersusceptible derivative. Transformation of hypersusceptible strains using total DNA from the parent strain restored the macrolide efflux system in the hypersusceptible strain, which was confirmed by MIC levels and radioactive erythromycin accumulation similar to that of the mutant resistant strain. Analysis of sequence and transcription of acrAB gene clusters showed no significant differences between resistant and hypersusceptible derivatives.
  
  
  CONCLUSIONS
  Mutation in ribosomal protein L22 alone does not confer high-level macrolide resistance unless efflux is present.
authors.affiliation: Department of Pathology, Hershey Medical Center, Mail code H083, 500 University Dr., Hershey, PA 17033, USA.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Peric
authors.forename: Bülent
authors.initials: B
authors.lastname: Bozdogan
authors.forename: Chad
authors.initials: C
authors.lastname: Galderisi
authors.forename: Dan
authors.initials: D
authors.lastname: Krissinger
authors.forename: Terry
authors.initials: T
authors.lastname: Rager
authors.forename: Peter C
authors.initials: PC
authors.lastname: Appelbaum
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015139:Blotting, Southern; D017291:Clarithromycin / Q000494:pharmacology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D005810:Multigene Family / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012269:Ribosomal Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D018942:Macrolides; D012329:RNA, Bacterial; D012269:Ribosomal Proteins; D004917:Erythromycin; D017291:Clarithromycin