Differences in potential for selection of clindamycin-resistant mutants between inducible erm(A) and erm(C) Staphylococcus aureus genes

Abstract

In staphylococci, inducible macrolide-lincosamide-streptogramin B (MLS(B)) resistance is conferred by the erm(C) or erm(A) gene. This phenotype is characterized by the erythromycin-clindamycin "D-zone" test. Although clindamycin appears active in vitro, exposure of MLS(B)-inducible Staphylococcus aureus to this antibiotic may result in the selection of clindamycin-resistant mutants, either in vitro or in vivo. We have compared the frequencies of mutation to clindamycin resistance for 28 isolates of S. aureus inducibly resistant to erythromycin and bearing the erm(C) (n = 18) or erm(A) (n = 10) gene. Seven isolates susceptible to erythromycin or bearing the msr(A) gene (efflux) were used as controls. The frequencies of mutation to clindamycin resistance for the erm(A) isolates (mean +/- standard deviation, 3.4 x 10(-8) +/- 2.4 x 10(-8)) were only slightly higher than those for the controls (1.1 x 10(-8) +/- 6.4 x 10(-9)). By contrast, erm(C) isolates displayed a mean frequency of mutation to clindamycin resistance (4.7 x 10(-7) +/- 5.5 x 10(-7)) 14-fold higher than that of the S. aureus isolates with erm(A). The difference was also observed, although to a lower extent, when erm(C) and erm(A) were cloned into S. aureus RN4220. We conclude that erm(C) and erm(A) have different genetic potentials for selection of clindamycin-resistant mutants. By the disk diffusion method, erm(C) and erm(A) isolates could be distinguished on the basis of high- and low-level resistance to oleandomycin, respectively.

FIG. 1.

Frequencies of mutation to clindamycin resistance of S. aureus strains, including 18 with erm(C) (solid circles), 10 with erm(A) (solid triangles), and 7 controls (solid diamonds). Open circle and triangle represent RN4220erm(C) and RN4220erm(A), respectively. Approximately 10⁸ to 5 × 10⁹ CFU of cells was spread onto agar plates containing 20 μg/ml of clindamycin. After 48 h of incubation at 35°C, colonies were counted, and the mutation frequencies were determined relative to the total count of viable organisms plated. Each data point represents the mean mutation frequency calculated from three experiments for one strain.

FIG. 2.

Schematic presentation of the regulatory regions of the inducibly expressed erm(C) gene from S. aureus 9 (A) and the constitutively expressed erm(C) genes selected in this study (B, C, and D). RBS1 and RBS2, ribosome binding sites of the leader peptide and the erm(C) gene, respectively. Arrows indicate the inverted repeated sequences IR1 to IR4. (A) Wild-type erm(C) gene; (B) derivatives with 15- and 16-bp deletions in IR3; (C) derivative with a 50-bp deletion (deletion of the stop codon of the leader peptide generates a translational fusion with the methylase); (D) derivative with tandem duplication of a 16-bp sequence (insertion of a new IR2).