Erythromycin-heteroresistant methicillin-resistant Staphylococcus aureus isolates from Turkey

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We read with interest the Editorial in CMI [1] concerning the heteroresistance of organisms such as Staphylococcus aureus, coagulase-negative staphylococci, Acinetobacter baumannii, Mycobacterium tuberculosis, Streptococcus pneumoniae, Enterococcus faecium and Cryptococcus neoformans. The Editorial focused especially on heteroresistant vancomycin-intermediate S. aureus and heteroresistant glycopeptide-intermediate S. aureus strains. We would like to draw attention to another heteroresistance phenomenon that we observed among methicillin-resistant S. aureus (MRSA) isolates in Turkiye Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey [2].

MRSA isolates from various clinical samples obtained from hospitalised patients at Turkiye Yuksek Ihtisas Teaching and Research Hospital were identified by Gram’s stain, tube coagulase tests and oxacillin agar screening tests. Erythromycin heteroresistance was determined using a standard disk method [3]. After realising that tiny colonies grew in the erythromycin inhibition zone, we decided to test the effect of this heteroresistance on the expression of inducible clindamycin resistance using D-tests [4]. In brief, a 15-μg erythromycin disk was placed 15 mm from the centre of a 2-μg clindamycin disk. Inhibition of the circular zone around the clindamycin disk (i.e., flattening of the zone towards the erythromycin disk) was considered to be a positive indication of inducible clindamycin resistance.

The results of detailed studies with these erythromycin-heteroresistant isolates were as follows: (i) colonies growing within the erythromycin inhibition zones were so tiny and so few in number that they were hardly visible after incubation for 24 h at 37°C, and the isolate could easily have been evaluated as erythromycin-sensitive; (ii) after the same plates were incubated at 37°C for a further 24 h, the intra-zone colonies became more visible; (iii) using the double-disk-approximation method, these isolates showed an inducible clindamycin resistance phenotype, which was more visible after incubation at 37°C for 48 h; (iv) when the erythromycin disk-diffusion test was performed with a heavier inoculum (e.g., a McFarland turbidity of 1 or 2), confluent growth was observed in the erythromycin inhibition zone; and (v) the colonies growing within the erythromycin inhibition zone were of varying sizes, and there was no zone around the disk when the erythromycin disk-diffusion susceptibility test was repeated with pure cultures of these inter-zone colonies (i.e., the cultures were fully resistant to erythromycin). The size of the colonies did not influence the results, and all the colonies also showed a D-zone around a clindamycin disk. Repeated studies using the same strains yielded identical results.

In conclusion, in order to identify such erythromycin-heteroresistant MRSA strains, all MRSA isolates should be tested by the disk-approximation method, using erythromycin and clindamycin disks. The plates should be examined carefully for the existence of tiny colonies, with re-incubation for a further 24 h if necessary.

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