Emergence of two oxacillin-susceptible mecA-positive Staphylococcus aureus clones in a Greek hospital

E. Petinaki, F. Kontos and A. N. Maniatis*

Department of Microbiology, Medical School, University of Thessalia, Papakyriazi 22, Larissa, Greece

Sir,

Methicillin resistance in Staphylococcus aureus (MRSA) isolates is due to the acquisition of the mecA gene, which encodes the low-affinity penicillin-binding protein PBP2a.1 The NCCLS breakpoint for oxacillin resistance is >=4 mg/L for S. aureus.2 Although sometimes S. aureus isolates expressing borderline resistance (MIC 2 mg/L) carry the mecA gene, isolates possessing the mecA gene but susceptible to oxacillin (MIC < 2 mg/L) have not yet been reported.1

From January to December 2001, a subset of 25 oxacillin-susceptible isolates were selected from 225 consecutive S. aureus isolates, sporadically recovered from various clinical materials (blood, pus and other wounds) from hospitalized patients in 10 different departments of the University Hospital of Thessalia, Central Greece. Bacteria were identified as S. aureus by Gram’s stain, catalase and coagulase tests and the API Staph System (bioMerieux). The isolates were classified as oxacillin susceptible by both the agar dilution (performed according to NCCLS guidelines3) and disc diffusion methods and by the Vitek automated system (bioMerieux). MICs of oxacillin for the isolates ranged from 0.5 to 1 mg/L. Population analysis, performed as described previously4 revealed that the isolates were heterogeneously resistant to oxacillin and belonged to class 1 (1 x 10–6 cells that grew at 4 mg/L).

Colonies of the 25 oxacillin-susceptible S. aureus isolates were examined by MRSA latex agglutination test (Slidex MRSA Detection, bioMerieux), and despite their susceptibility to oxacillin, gave a positive latex reaction, so must express PBP2a. PCR, performed as described previously1,5 confirmed the presence of the mecA gene in all the isolates. The isolates were then subjected to an analysis of their levels of resistance to a variety of antibiotics, and their genotypes.

Resistance to 14 antimicrobial agents (penicillin, co-trimoxazole, ofloxacin, clindamycin, erythromycin, gentamicin, tobramycin, kanamycin, amikacin, rifampicin, tetracycline, fusidic acid, vancomycin and teicoplanin) was tested using an agar diffusion method according to NCCLS guidelines,2 and the MICs of tetracycline, kanamycin and fusidic acid were determined by an agar dilution method, also according to NCCLS guidelines.3 A large majority of the isolates (20/25) exhibited resistance to tetracycline, fusidic acid and kanamycin (TFK), while the remaining isolates (5/25) were susceptible to these antibiotics. MICs of tetracycline, fusidic acid and kanamycin for the TFK-resistant isolates were 32, 64 and 32 mg/L, respectively. The presence of the tetK and aph(3')-IIIa genes was detected by PCR in all TFK-resistant isolates, indicating that the mechanism of tetracycline resistance is probably mediated by the TetK tetracycline efflux protein, and the mechanism of kanamycin resistance is probably mediated by the Aph(3')-IIIa aminoglycoside modifying enzyme.1 The five isolates with a TFK-susceptible pattern gave negative PCR results for tetM, tetK, tetL, tetO, ant(4')-Ia and aph(3')-IIIa genes.

PFGE analysis5 revealed the presence of two different clones amongst the 25 isolates (Figure 1). All TFK-resistant isolates belong to the same clone (pattern I), which differs significantly from that including the TFK-susceptible isolates (pattern II) (Figure 1). This second clone includes three subtypes (IIa, IIb, IIc) that differ from each other by two bands.



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Figure 1  SmaI macrorestriction patterns of representative isolates. Lanes 1, 8, 15 and 20, standards; lanes 2–4, patterns IIa, IIb and IIc; lanes 5–7, 9–14 and 16–19, pattern I.

 
In conclusion, this is the first report of the emergence of oxacillin-susceptible MRSA clones in Greece. These clones have emerged in our clinical setting during 2001 and differ significantly from those that predominated in the area of Thessalia in recent years.5 There is a possibility that these isolates were already circulating but were not detected by conventional susceptibility tests. The difficulty in differentiating oxacillin-susceptible MRSA strains from fully susceptible ones (MSSA) may have implications for infection control practice.

Footnotes

* Corresponding author. Tel: +30-41-068-2514; Fax:+30-41-068-2508; E-mail: anm{at}otenet.gr Back

References

1 . Fluit, A. C., Visser, M. R. & Schmitz, F. J. (2001). Molecular detection of antimicrobial resistance. Clinical Microbiology Reviews 14, 836–71[Abstract/Free Full Text]

2 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing. M100-S10. NCCLS, Wayne, PA, USA.

3 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

4 . Tomasz, A., Nachman, S. & Leaf, H. (1991). Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci. Antimicrobial Agents and Chemotherapy 35, 124–9.[ISI][Medline]

5 . Petinaki, E., Miriagou, V., Tzouvelekis, L. S., Pournaras, S., Hatzi, F., Kontos, F. et al. (2001). Methicillin-resistant Staphylococcus aureus in the hospitals of central Greece. International Journal of Antimicrobial Agents 18, 61–5.[ISI][Medline]





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