Optimization of Routine Identification of Clinically Relevant Gram-Negative Bacteria by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and the Bruker Biotyper

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) might complement and one day replace phenotypic identification of bacteria in the clinical microbiology laboratory, but there is no consensus standard regarding the requirements for its validation prior to clinical use in the United States. The objective of this study was to assess the preanalytical variables influencing Gram-negative identification by use of the Bruker Biotyper MALDI-TOF MS system, including density of organism spotting on a stainless steel target plate and the direct overlay of organisms with formic acid. A heavy smear with formic acid overlay was either superior or equivalent to alternative smear conditions. Microbiological preanalytical variables were also assayed, such as culture medium, growth temperature, and use of serial subculture. Postanalytical analysis included the application of modified species-level identification acceptance criteria. Biotyper identifications were compared with those using traditional phenotypic methods, and discrepancies were resolved with 16S rRNA gene sequencing. Compared to the recommended score cutoffs of the manufacturer, the application of optimized Biotyper score cutoffs for species-level identification increased the rate of identification by 6.75% for the enteric Gram-negative bacteria and 4.25% for the nonfermenting Gramnegative bacteria. Various incubation temperatures, growth medium types, and repeat subcultures did not result in misidentification. We conclude that the Bruker MALDI Biotyper is a robust system for the identification of Gram-negative organisms in the clinical laboratory and that meaningful performance improvements can be made by implementing simple pre-and postanalytical techniques.M atrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) employs soft ionization to detect individual intact biomolecules within complex solutions. Practical use of MALDI-TOF has been facilitated by the development of matrices, such as ␣-cyano-4-hydroxycinnamic acid (1). While the potential for the identification of bacteria by their individual mass spectrometric "fingerprints" has long been appreciated (2), the adoption of MALDI-TOF MS in clinical microbiology laboratories in the United States has been hindered until recently by a lack of available platforms with databases of bacterial whole-cell MALDI-TOF reference spectra.Recent studies using the Bruker Biotyper MALDI-TOF MS platform have revealed that this system might correctly identify bacteria to the species level 95% of the time, with the remaining 5% comprising unidentified or erroneously identified isolates (3, 4). These studies invariably used Bruker's recommended scoring cutoffs (a Biotyper score of Ն2.0 for species-level identification and Ն1.7 for genus-level identification) to define the confidence with which a correct identification had been made. Alatoom and colleagues (5) noted that the preparatory extraction of the protein fraction of Gram-positive organisms was ne...

Two Cases of Kerstersia gyiorum Isolated from Sites of Chronic Infection

“…The isolate was subsequently analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and identified as Kerstersia gyiorum on the BioTyper system (software version 3.0; Bruker-Daltonics, Billerica, MA). BioTyper scores of 2.3 and 2.4 (excellent identification) were obtained with and without a formic acid overlay, respectively (1). The isolate was unidentified on the Vitek MS (database version 2.0; bioMérieux).…”

High-throughput discrimination of bacteria isolated fromAstacus astacusandA. leptodactylus

“…MALDI-TOF MS can examine the pattern of proteins detected directly from intact bacteria, giving a reproducible spectra consisting of a series of peaks corresponding to mass-to-charge ratios of ions released from bacterial proteins during laser desorption (Dupont et al, 2010). MALDI-TOF MS is considered a tool with potential to replace phenotypic identification of bacteria in clinical microbiology laboratories (Bizzini et al, 2010;Ford and Burnham, 2013;Jamal et al, 2013;Kok et al, 2013), especially due to its time-saving benefit where the extensive time needed with culture-based methods is reduced to a few minutes. In this study, we isolated bacteria from various tissues of apparently healthy Astacus astacus and A. leptodactylus and compared the performances of the API 20E panels to the Bruker Biotyper MALDI-TOF MS (Bruker Daltonics, Billerica, MA) for the identification of bacterial isolates.…”

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…MALDI-TOF MS is a rapid, inexpensive, and accurate approach for species-level identification of Gram-negative and Gram-positive bacteria and yeasts (1). It has been recently optimized for routine use in clinical microbiology laboratories (2,3). This method has shown to be sufficiently robust to be applicable with fastidious bacteria, such as anaerobic bacteria, Legionella, Bartonella, or mycobacteria (4).…”