Detection of intrinsic oxacillin resistance in non-multiresistant, oxacillin-resistant Staphylococcus aureus (NORSA)

Iain B. Gosbell1,2,*, Stephen A. Neville1, Joanne L. Mercer1, Lorna A. Fernandes3 and Clarence J. Fernandes3

1 Department of Microbiology and Infectious Diseases, South Western Area Pathology Service, Liverpool; 2 School of Pathology, Faculty of Medicine, University of New South Wales, Kensington; 3 Department of Microbiology, Royal North Shore Hospital, St Leonards, Sydney, Australia

Keywords: intrinsic methicillin resistance, Staphylococcus aureus

Sir,

Community-acquired, non-multiresistant, oxacillin-resistant Staphylococcus aureus (NORSA) strains, unrelated to nosocomial strains, have emerged in many parts of the world.1 These strains, if truly community-acquired, are usually susceptible to most, if not all, of the non-ß-lactam antibiotics.2 MICs of methicillin for NORSA strains are significantly lower than those for multiresistant ORSA (MORSA) strains.3 Consequently, we studied a selection of oxacillin-susceptible S. aureus (OSSA), NORSA and MORSA strains with respect to the ease of detection of oxacillin resistance.

The organisms selected for this study were collected in our previous studies of community-acquired ORSA infection.4 Thirty-four isolates of hospital-acquired MORSA (10 resistant to three, three resistant to four, 10 resistant to five and one resistant to seven non-ß-lactams); 27 isolates of OSSA (10 resistant to none, 10 resistant to one, five resistant to two and two resistant to five non-ß-lactams); and 34 NORSA strains (20 resistant to none, six resistant to one and eight resistant to two non-ß-lactams) were included.

PCR for the mecA and nuc genes was performed on all isolates. MICs were determined by agar dilution. Methicillin and oxacillin were tested with and without NaCl (2% w/v) incorporated into the agar. Oxacillin plates were incubated at 35 or 30°C for 24 h, and methicillin plates at 35°C for 24 h, before reading. Antimicrobial susceptibility testing was performed with the VITEK GPS-IX card and ATB Staph strips (bioMérieux-Vitek, Australia; Pty Ltd), following the manufacturer’s instructions. Disc diffusion testing for oxacillin resistance was performed using the NCCLS methodology. The presence of heterogeneous or homogeneous resistance to methicillin, in ORSA isolates only, was detected using disc testing, as previously described.5

Agar dilution MICs, using various substrates and various conditions, are shown in Table 1. The performance of the various phenotypical methods at detecting methicillin resistance, using PCR as the gold standard, is shown in Table 2. All NORSA strains fully susceptible to non-ß-lactams demonstrated heterogeneous oxacillin resistance. Three of six NORSA strains resistant to one non-ß-lactam, and one of eight NORSA strains resistant to two non-ß-lactams demonstrated heterogeneous resistance. No MORSA isolates demonstrated heterogeneous oxacillin resistance.


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Table 1.  Agar dilution MICs (mg/L) to methicillin and oxacillin
 

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Table 2.  Detection of intrinsic oxacillin resistance using PCR as the gold standard
 
The detection of intrinsic oxacillin resistance is known to be difficult.6 The gold standard method is detection of the mecA determinant or its product, PBP 2a.6 Phenotypical methods to detect intrinsic oxacillin resistance can have problems with sensitivity and specificity.3 Recommendations about incubation times and adding NaCl to the media are critical to the optimum performance of the tests.3

In our study, we found that agar dilution using oxacillin provided the best agreement with PCR, with a sensitivity of 100% and a specificity of 96%. Using methicillin, agar dilution gave a sensitivity of 98% and a specificity of 96%. Without NaCl in the media, the sensitivity fell to 84% with oxacillin, because nine NORSA strains were labelled as susceptible; the sensitivity fell to 97% with methicillin, and 10 NORSA isolates had MICs on the breakpoint of 16 mg/L. The multiresistance of MORSA strains indicates that the strain may be methicillin-resistant Staphylococcus aureus (MRSA), even if the testing for methicillin resistance was negative; this might prompt the laboratory to perform other tests for methicillin resistance. This identifying factor is missing with NORSA isolates, and total reliance is placed on the methicillin result.

Specificity in the detection of methicillin resistance was suboptimal with the VITEK and ATB systems, and with NCCLS disc testing, as found previously.5 Two strains that tested negative by PCR gave positive results in these three tests: one was a multiresistant strain of phage type 85/88, the most common MRSA phage type currently in our hospitals (this was negative with the MRSA Screen Test for PBP 2a; data not shown), and the other was a non-multiresistant strain. Another multiresistant strain (also negative for PBP 2a) tested resistant to oxacillin with ATB, and with the disc method.

Nimmo et al.2 determined that community-acquired gentamicin-susceptible Western Pacific ORSA strains (now known as Oceanian ORSA/MRSA) also demonstrated heterogeneous resistance, and hospital-acquired MORSA strains homogeneous oxacillin resistance, a finding mirrored in our study.

NORSA strains are usually heteroresistant, with MICs that can approach the breakpoints.2,3 Detection of intrinsic oxacillin resistance in NORSA strains is thus a potential difficulty, and it is vital to follow the NCCLS methodology exactly. The VITEK GPS-IX card, ATB Staph and NCCLS disc methods demonstrated good sensitivity, but at the expense of specificity. NORSA strains are increasingly seen, and laboratories need to be aware that the detection of their intrinsic oxacillin resistance can be a problem.

Acknowledgements

The authors would like to acknowledge Kelly Glass and Helen Ziochos for maintaining the organism cultures and performing the testing with VITEK GPS-IX cards, ATB Staph strips and discs, and Rosemary Munro and Philip Jones for supervising the project.

Footnotes

* Corresponding author. Tel: +61-2-9828-5128; Fax: +61-2-9828-5129; E-mail: i.gosbell{at}unsw.edu.au Back

References

1 . Chambers, H. F. (2001). The changing epidemiology of Staphylococcus aureus? Emerging Infectious Diseases 7, 178–82.[ISI][Medline]

2 . Nimmo, G. R., Schooneveldt, J., O’Kane, G., McCall, B. & Vickery A. (2000). Community acquisition of gentamicin-sensitive methicillin-resistant Staphylococcus aureus in southeastern Queensland, Australia. Journal of Clinical Microbiology 38, 3926–31.[Abstract/Free Full Text]

3 . Merlino, J., Watson, J., Rose, B., Beard-Pegler, M., Gottlieb, T., Bradbury, R. et al. (2002). Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney, Australia. Journal of Antimicrobial Chemotherapy 49, 793–801.[Abstract/Free Full Text]

4 . Gosbell, I. B., Mercer, J. L., Neville, S. A., Crone, S. A., Chant, K. G., Jaluludin, B. B. et al. (2001). Non-multiresistant and multiresistant methicillin-resistant Staphylococcus aureus in community-acquired infections. Medical Journal of Australia 174, 627–30.[ISI][Medline]

5 . Frebourg, N. B., Nouet, D., Lemee, L., Martin, E. & Lemeland, J. F. (1998). Comparison of ATB staph, rapid ATB staph, Vitek, and E-test methods for detection of oxacillin heteroresistance in staphylococci possessing mecA. Journal of Clinical Microbiology 36, 52–7.[Abstract/Free Full Text]

6 . Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews 10, 781–91.[Abstract]