Inability of L22 ribosomal protein alteration to increase macrolide MICs in the absence of efflux mechanism in Haemophilus influenzae HMC-S.
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.
Peric
Mihaela
M
Department of Pathology, Hershey Medical Center, Mail code H083, 500 University Dr., Hershey, PA 17033, USA.
Bozdogan
Bülent
B
Galderisi
Chad
C
Krissinger
Dan
D
Rager
Terry
T
Appelbaum
Peter C
PC
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
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D018942:Macrolides; D012329:RNA, Bacterial; D012269:Ribosomal Proteins; D004917:Erythromycin; D017291:Clarithromycin