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Antimicrobial Agents and Chemotherapy, December 2009, p. 5088-5094, Vol. 53, No. 12
0066-4804/09/$08.00+0     doi:10.1128/AAC.00518-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Methicillin-Resistant Staphylococcus aureus Screening by Online Immunometric Monitoring of Bacterial Growth under Selective Pressure{triangledown}

Teppo Stenholm,1* Antti J. Hakanen,2 Jonne Vaarno,3 Sari Pihlasalo,3 Perttu Terho,4 Pekka E. Hänninen,3 Jaana Vuopio-Varkila,2 Pentti Huovinen,2 and Pirkko Kotilainen1

Department of Medicine, Turku University Hospital, and University of Turku, Turku, Finland,1 National Institute for Health and Welfare (THL), Turku and Helsinki, Finland,2 Laboratory of Biophysics and Medicity, Institute of Biomedicine,3 Centre for Biotechnology, Cell Imaging Core, University of Turku, Turku, Finland4

Received 20 April 2009/ Returned for modification 29 May 2009/ Accepted 8 September 2009

Rapid, high-throughput screening tools are needed to contain the spread of hospital-acquired methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains. Most techniques used in current clinical practice still require time-consuming culture for primary isolation of the microbe. We present a new phenotypic assay for MRSA screening. The technique employs a two-photon excited fluorescence (TPX) detection technology with S. aureus-specific antibodies that allows the online monitoring of bacterial growth in a single separation-free process. Different progressions of fluorescence signals are recorded for methicillin-susceptible and -resistant strains when the growth of S. aureus is monitored in the presence of cefoxitin. The performance of the new technique was evaluated with 20 MRSA strains, 6 methicillin-susceptible S. aureus strains, and 7 coagulase-negative staphylococcal strains and two different monoclonal S. aureus-specific antibodies. When either of these antibodies was used, the sensitivity and the specificity of the TPX assay were 100%. All strains were correctly classified within 8 to 12 h, and up to 70 samples were simultaneously analyzed on a single 96-well microtiter plate. As a phenotypic method, the TPX assay is suited for screening purposes. The final definition of methicillin resistance in any S. aureus strain should be based on the presence of the mecA gene. The main benefit afforded by the initial use of the TPX methodology lies in its low cost and applicability to high-throughput analysis.

* Corresponding author. Mailing address: Antimicrobial Resistance Unit, National Institute for Health and Welfare (THL), P.O. Box 51, Turku 20521, Finland. Phone: 358 505427406. Fax: 358 20610 6699. E-mail: teppo.stenholm{at}utu.fi

{triangledown} Published ahead of print on 14 September 2009.

Antimicrobial Agents and Chemotherapy, December 2009, p. 5088-5094, Vol. 53, No. 12
0066-4804/09/$08.00+0     doi:10.1128/AAC.00518-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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