Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci

Abstract

Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC). MLSB resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLSB resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA, ermC, and msrA. Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC, 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLSB resistance in this study.

FIG. 1.

Disk induction testing. (A) Positive disk induction test within a routine disk diffusion format (CNS; University Hospital). Erythromycin and clindamycin disks were placed in adjacent central positions in a standard disk dispenser. (B to D) Constitutive lincosamide resistance by disk induction testing. Growth extends to the edge of the clindamycin disk. (B) S. haemolyticus (ermA ermC msrA); (C) S. epidermidis (ermA), demonstrating a “double zone” of growth around the clindamycin disk; (D) S. aureus (ermA) showing a double-zone of growth and blunting of the outer clindamycin zone (discernible at 15 and 20 mm). (E and F) Positive disk induction test indicating inducible lincosamide resistance. Flattening of the clindamycin inhibition zone adjacent to the erythromycin disk is easily discernible at 15 and 20 mm (disks placed by hand), as well as at 26 mm. (E) S. hominis (ermC); (F) S. aureus (ermC). (G and H) Negative disk induction test indicating the absence of inducible lincosamide resistance. No flattening or distortion of the clindamycin zone adjacent to the erythromycin disk can be detected at a disk spacing of 15, 20, or 26 mm. (G) S. epidermidis (msrA); (H) S. aureus (msrA).