RESEARCH LETTER Combination of capillary electrophoresis, PCR and physiological assays in di¡erentiation of clinical strains of Staphylococcus aureus Katarzyna Hrynkiewicz 1 , Ewa Klodzi ´ nska 2 , Hanna Dahm 1 , Jacek Szeliga 3 , Marek Jackowski 3 & Boguslaw Buszewski 2 1 Department of Microbiology, Institute of General and Molecular Biology, Nicolaus Copernicus University, Toru´ n, Poland; 2 Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Toru´ n, Poland; and 3 Department of Surgery, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Toru´ n, Poland Correspondence: Katarzyna Hrynkiewicz, Department of Microbiology, Institute of General and Molecular Biology, Nicolaus Copernicus University, Gagarina 9, PL-87-100 Toru ´ n, Poland. Tel.: 148 56 61 14 447; fax: 148 56 61 14 772; e-mail: hrynk@biol.uni.torun.pl Received 10 March 2008; accepted 22 May 2008. First published online 9 July 2008. DOI:10.1111/j.1574-6968.2008.01245.x Editor: William Wade Keywords Staphylococcus aureus ; capillary zone electrophoresis (CZE); PCR; RFLP. Abstract Fast, sensitive and cheap determination of pathogenic bacteria is extremely important in many branches, for example biotechnology, quality control, analysis of samples and antimicrobial therapy. The development and application of analytical techniques in practice could provide new possibilities in this regard. The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality. Rapid and sensitive determination is therefore very important. In the present study, novel methods, based on capillary zone electrophoresis and (as confirmation of these results) molecular analysis of a part of the coag gene, were developed for identification and differentiation of three S. aureus strains. The electrophoretic measurements rely on the differential mobility of bacteria in the fused silica capillary under the direct current electric field. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the S. aureus gene were amplified using the PCR technique followed by restriction enzyme digestion and analysis of restriction fragment length poly- morphism patterns as well as sequencing. Finally, the results of electrophoretic measurements with molecular analysis were compared. Introduction Staphylococcus aureus is perhaps the greatest nosocomial pathogen of our time. This Gram-positive bacterium causes a wide range of human diseases ranging from minor skin infections, such as folliculitis and impetigo, to more invasive diseases including endocarditis, sepsis, and toxic shock syndrome (Weems, 2001). Staphylococcus aureus is the leading cause of infectious complications within the hospital environment and the incidence of multidrug resistant isolates is increasing (Reniere et al., 2007). An increasing number of infections are related to medical developments, including the use of joint prostheses, immunosuppressants and catheters (Casey et al., 2007). These facts warrant the rapid identification of infected and colonized patients as well as the interruption of strain transmission. Control of S. aureus infection can be accompanied by epidemiological typing of S. aureus. This may clarify whether environmental strains from staff members are related to those that cause infection and whether the isolates from one patient belong to one genotype. Most screening is still carried out using plate-based methods, but because a growing number of strains are resistant to multiple antibacterial chemotherapies, their resistance phenotypes are only of little discriminatory value. Therefore, alternative methods such as broth culture, chro- mogenic media, rapid screening kits, molecular assays and automated systems are increasingly being used (Casey et al., 2007). Molecular methods are valuable not only in the identification of bacterial strains but also in demonstrating the evolutionary and clonal relationships between the iso- lates. In the present study we evaluated the capacity of coagulase gene typing to discriminate between unrelated strains of S. aureus. Coagulase production is the principal criterion used in clinical microbiology laboratories for the identification of S. aureus isolates from human infections. FEMS Microbiol Lett 286 (2008) 1–8 c  2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved