Identification of Anaerobic Bacteria by Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry with On-Plate Formic Acid Preparation

Abstract

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria. Identification of anaerobic bacteria using phenotypic methods is time-consuming and may produce inconclusive results, leading to application of costly methods, such as 16S rRNA gene sequencing. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used predominantly for aerobic bacterial identification and is now in use in many clinical laboratories (1-5). A small number of studies have preliminarily evaluated this technology for identification of anaerobic bacteria (4, 6-12), but this application is not in widespread clinical use.We showed that off-plate protein extraction yields increased scores and, consequently, a higher percentage of isolates identified compared to direct on-plate testing of colonies for Corynebacterium species and Gram-positive cocci using the Bruker Biotyper system (Bruker Daltonics, Billerica, MA) (1, 13). Others have applied this strategy to anaerobic bacteria (6,14,15). In our experience, off-plate extraction is cumbersome for laboratory technologists and results in waste production and high cost. We recently showed that an easier-to-use onplate testing method using 70% formic acid demonstrated results comparable to those of off-plate extraction for Corynebacterium species and yeast (16). Using the Bruker Biotyper system, Justesen and coworkers performed a similar on-plate preparation technique using 1 l of 70% formic acid for anaerobic bacteria which were not identified using direct on-plate testing without an extraction step (7). Unpublished studies conducted by our group have shown that on-plate formic acid testing yields results equivalent to those of off-plate extraction for staphylococci, streptococci, and aerobic and facultatively anaerobic Gram-negative bacilli. Universal application of this method for identification of bacterial colonies is potentially fast and cost-effective and allows application of MALDI-TOF MS without antecedent Gram staining.We evaluated the Bruker MALDI Biotyper for identification of clinically isolated anaerobic bacteria using on-plate formic acid preparation. MATERIALS AND METHODS Bacterial isolates.A total of 253 clinical isolates of anaerobic bacteria were obtaine...

Comparison of two matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry methods and API 20AN for identification of clinically relevant anaerobic bacteria

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