To test the hypothesis that the strain relatedness of coagulase-negative staphylococci (CoNS) recovered from blood cultures can be inferred from automated antibiotic susceptibility testing (AST) results generated by Vitek 2, concordant or discordant AST results were compared with pulsed-field gel electrophoresis (PFGE) typing results for 119 CoNS blood culture isolate pairs. Concordant AST results were highly predictive of the strain relatedness of CoNS isolates.Clinicians and microbiologists are frequently facing the difficulty of determining the clinical significance of blood cultures positive for coagulase-negative staphylococci (CoNS). Various approaches and algorithms, including clinical and microbiological criteria, biomarkers, and mathematical models, have been developed to assist in the interpretation of CoNS-positive blood cultures (3, 8-10, 12-14, 18). A frequently used approach is to assess the identity or distinctness of CoNS isolates recovered from sequential blood cultures based on microbiologic routine testing results such as species identification, biotyping, and antimicrobial susceptibility testing (AST) (2,5,11,16). However, no precise criteria to predict the strain relatedness of CoNS based on AST results to help distinguish between true bacteremia and contamination have been developed to date.We propose a simple approach based on quantitative AST results to predict the strain relatedness of CoNS isolates recovered from two or more blood cultures from a patient with bacteremia. The aim of this study was to evaluate the sensitivity, specificity, and predictive values of the proposed approach by comparing the results obtained by application of the AST criteria with the results of pulsed-field gel electrophoresis (PFGE) of CoNS isolate pairs as the gold standard.(This work was presented in part at the 15th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2 April to 5 April 2005 [3a].)Blood culture isolates of all patients were prospectively collected at the Institute for Medical Microbiology, Immunology and Hygiene of the University of Cologne from 1 January 1999 to 31 December 2000 and stored at Ϫ70°C. Isolates were selected for this study if two or more blood cultures obtained from a patient within a 7-day interval had yielded CoNS. Stored isolates were subcultured and then identified to the species level and tested for antimicrobial susceptibility with Gram-positive identification (ID-GPC) and susceptibility testing (AST-P526) cards of the Vitek 2 system (bioMérieux, Marcy l'Etoile, France).Pairs of isolates were classified as concordant or discordant based on MIC data and the interpretative category (susceptible versus resistant) for each of the antimicrobial agents penicillin, oxacillin, ciprofloxacin, erythromycin, trimethoprim-sulfmethoxazole, tetracycline, gentamicin, fosfomycin, fusidic acid, and rifampin. Isolates were considered concordant when they had not more than two 2-fold dilution differences in MIC values and no differences in the resistan...