Low level resistance to oleandomycin as a marker of ermA in staphylococci

Vicenza Di Modugnoa, Massimo Guerrinia, Saroj Shahb and Jeremy Hamilton-Millerb,*

a Department of Microbiology, Research Laboratories, GlaxoSmithKline Medicines Research Centre, via Fleming 4, Verona 37135, Italy; b Department of Medical Microbiology, Royal Free and University College Medical School, London NW3 2PF, UK

Sir,

During a study of the activity of the ketolide telithromycin against staphylococci,1 we identified two subgroups within the inducible MLSB phenotype. Such strains are routinely recognized by being resistant to erythromycin and susceptible to clindamycin, but resistance to the latter is induced by erythromycin. There were 129 strains with this phenotype: in 114 (subgroup 1) both erythromycin and oleandomycin always induced resistance not only to clindamycin but also to telithromycin, quinupristin and rokitamycin, while for the remaining 15 strains (subgroup 2), erythromycin but not oleandomycin was an inducer of resistance.

We have now further investigated, by genotyping, the 15 subgroup 2 strains together with 15 strains from subgroup 1 selected to match numbers of Staphylococcus aureus (n = 11) and coagulase-negative staphylococci (n = 4). Coagulase-negative strains were identified by API Staph (API, Basingstoke, UK). MICs were determined by plate dilution following NCCLS guidelines. Genotyping was carried out by PCR using specific primers for the msr(A), erm(A) and erm(C) genes, which are associated in staphylococci with different mechanisms of resistance to macrolides. Primers were designed from published sequences for msr(A), erm(A) and erm(C) genes in S. aureus;2,3 the expected PCR products were obtained from each primer (340, 526 and 349 bp, respectively). Genomic DNA was prepared by the method of Eady et al.,4 and also (for two strains) using the PrepMan Ultra Sample Preparation Reagent (AB Applied Biosystems, Foster City, CA, USA).

Results are shown in the Table. Subgroup 1 strains were all of genotype erm(C) and highly resistant to the 14- and 15-membered macrolides erythromycin, oleandomycin, azithromycin and clarithromycin, but were susceptible to the 16-membered macrolide tylosin (mean MIC 3 mg/L). On the other hand, 14 of the 15 strains in subgroup 2 contained erm(A), and were much less resistant to clarithromycin and oleandomycin and somewhat less resistant to erythromycin and azithromycin; they were also significantly more susceptible to tylosin (mean MIC 1.7 mg/L) than were the group 1 strains. During MIC determinations with erythromycin it was clear that there were two end-points, growth changing from ‘confluent’ to ‘light’ at concentrations between 8 and 64 mg/L (depending on the individual strain), and from ‘light’ to none at the concentration shown in the Table.

For four strains in group 2 (Staphylococcus haemolyticus 29, Staphylococcus simulans 190 and 416, and S. aureus 304) MICs of the macrolides were greater than shown by the remaining 11; in our earlier study, we had found antagonism between oleandomycin and telithromycin for these four strains, but this was detectable only by chequerboard titration, not by the disc test, suggesting a low level of telithromycin resistance induction. Two of these strains showed macrolide resistance factors in addition to erm(A): S. haemolyticus 29 had the msr(A) gene (efflux pump) and S. simulans 190 the erm(C) gene. We cannot explain the higher resistance in the other two strains; this may be due to resistance genes other than erm(A), erm(C) and msr(A) (it is interesting to note that strain 416 is less susceptible to clindamycin than any other strain).

The staphylococcal ermC protein is known to bring about macrolide resistance by dimethylating a specific residue in 23S rRNA, whereas other genetic determinants cause monomethylation at the same site. These variations in mechanism may explain the subtle differences we have observed in degrees of resistance conferred to the macrolides tested.

The MIC of 14- and 15-membered macrolides in general, and oleandomycin in particular, against S. aureus could be useful as a marker of the genetic determinant of resistance, higher level resistance being conferred by the erm(C) gene.


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Table. Genotypes and macrolide susceptibility pattern of 30 staphylococci
 
Notes

* Corresponding author. Tel +44-20-7794-0500; Fax +44-20-7435-9694; E-mail: j.hamilton-miller{at}rfc.ucl.ac.uk Back

References

1 . Hamilton-Miller, J. M. T. & Shah, S. (2000). Patterns of phenotypic resistance to the macrolide-lincosamide-ketolide-streptogramin group of antibiotics in staphylococci. Journal of Antimicrobial Chemotherapy 46, 941–9.[Abstract/Free Full Text]

2 . Shortridge, V. D., Flamm, R. K., Ramer, N., Beyer, J. & Tanaka, S. K. (1996). Novel mechanisms of macrolide resistance in Streptococcus pneumoniae. Diagnostic Microbiology and Infectious Disease 26, 73–8.[ISI][Medline]

3 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

4 . Eady, E. A., Ross, J. I., Tipper, J. L., Walters, C. E., Cove, J. H. & Noble, W. C. (1993). Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pump in epidemiologically distinct groups of staphylococci. Journal of Antimicrobial Chemotherapy 31, 211–7.[Abstract]





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