Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

Two novel protocols for inactivation and extraction were developed and used to identify 107 Mycobacterium clinical isolates, including Mycobacterium tuberculosis complex, from solid cultures using Vitek matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry. The protocol using heat inactivation with sonication and cell disruption with glass beads resulted in 82.2% and 88.8% species and genus level identifications, respectively. The Mycobacterium genus consists of over 100 species of rapidly growing and slow-growing acid-fast bacilli (AFB) (1-4). Rapid and accurate diagnosis of mycobacteria infections is important to patient care and public health (4). Inappropriate treatment may lead to unnecessary exposure to toxic drugs or drug resistance (5). Rapid identification (ID) of mycobacteria has proven difficult due in part to their fastidious growth requirements and low growth rate (3, 6). Molecular probes and DNA hybridization are relatively fast and simple but are available only for a limited number of clinically common species (2,3,5,7,8). High-performance liquid chromatography (HPLC) (2, 3, 9) and electrospray ionizationtandem mass spectrometry analysis (2) have recently been used to analyze mycolic acid but are labor-intensive and require technical expertise (1, 2).Recent studies have shown that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an accurate and rapid method for identifying bacteria and yeast from solid culture media (6, 10-13). MALDI-TOF MS has also recently been adapted for the identification of mycobacteria (1-5, 8, 9, 14), mostly using a Bruker Daltonics Flex system (1, 2, 4, 5, 8). We previously evaluated one inactivation procedure, described in a 2010 training manual from bioMérieux, that suspended mycobacteria in trifluoroacetic acid (TFA) for 30 min (15) and found that both the procedure and the database were ineffective (data not shown). There is no standard inactivation procedure currently available for identifying mycobacteria by using MALDI-TOF MS for either the Bruker or bioMérieux system. This study evaluates two novel inactivation and extraction protocols used to identify clinical mycobacterial isolates, including Mycobacterium tuberculosis complex (MTC), from solid culture media. In addition, this study evaluates an improved database for mycobacteria by using Vitek MALDI-TOF MS RUO (Vitek MS) (bioMérieux, Durham, NC) with a reference database developed in-house.(Parts of these results were presented at the 113th General Meeting of the American Society for Microbiology, 18 to 21 May 2013.)Clinical mycobacterial isolates from a culture positive for AFB were identified by either a DNA probe or HPLC. Organisms grown on BBL Middlebrook 7H11 solid agar media (Becton, Dickinson [BD], Sparks, MD) and incubated at 37°C in 4.0 to 8.0% CO 2 were used for identification by Vitek MS.Due to the lack of a mycobacterial superspectrum in the RUO database at the time, we developed a reference database consisting of 50 r...

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