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A panel of 24 methicillin-resistant Staphylococcus aureus strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.

M atrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been routinely used for the identification of bacteria and fungi from agar culture in many centers in Europe and is increasingly used in North America and elsewhere for primary identification of microorganisms (1, 2, 25). MALDI-TOF instruments use an ionizing laser to vaporize the abundant structural elements (primarily ribosomal proteins) of bacteria and yeasts and analyze the weight and relative abundance of each particle to generate a spectrum. Spectra are compared to a computer database of reference or user-defined organism spectra, and identification is obtained by matching the most similar spectrum in the database to the unknown organism. Performance of the Bruker MALDI BioTyper has been extensively studied in multiple centers and confirms that reliable identification can be obtained for Ͼ95% of the isolates grown on solid media routinely encountered in the clinical laboratory (1,4,5,8,19).More recently, protocols for the direct identification of pathogens from positive blood culture broths have been developed (3,12,16,21,22). Although blood culture broths are usually monobacterial (or monofungal) cultures, the presence of proteins from red cells, white blood cells, and serum interferes with the analysis by adding spectral peaks not found in the organism database. Furthermore, interfering substances such as charcoal (when present) and low organism numbers (as might be encountered with slow-growing or contaminating bacteria) present additional challenges in the use and interpretation of MALDI-TOF spectra for identification pathogens directly from positive blood cultures (24). As a result, many centers have developed in-house methods for preprocessing of blood cultures to optimize recovery of the bacterial proteome. More recently, a commercial kit (Bruker Sepsityper) has been released to simplify the processing steps required for the purification and extraction of the bacterial proteome from positive blood cultures. The system serves to facilitate preprocessing and minimize the impact of the interfering human proteome on the MALDI-TOF analysis. Here we report the performance of the Sepsityper system on the Bruker MALDI BioTyper for the direct identification of pathogens from blood cultures and a cost and turnaround time analysis of the results. MATERIALS AND METHODS Blood cultures.Blood was collected at the bedside and directly inoculated into BacT/Alert SA (aerobic culture) and/or SN (anaerobic) (bioMérieux, Marcy l'Etoile, France). Both bottle types are charcoal free. Bottles were loaded onto the a BacT/Alert instrument (bioMérieux, Marcy l'Etoile, France) and incubated. Bottles were incubated for up to 5 days, and when the operator was notified of a positive blood culture, a Gram stain was performed. All positive bottles were subjected to subculture and routine

The 16S rRNA sequencing data and metadata generated in this study have been submitted to the NCBI Sequence Read Archive (SRA: http://www.ncbi.nlm.nih.gov/bioproject/381931).