View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital Commons@Becker Washington University School of Medicine Digital Commons@Becker Open Access Publications 2013 Optimizing identification of clinically relevant gram-positive organisms by use of the bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system Erin McElvania TeKippe Washington University School of Medicine in St. Louis Sunni Shuey St. Louis Children's Hospital David W. Winkler St. Louis Children's Hospital Meghan A. Butler St. Louis Children's Hospital Carey-Ann Burnham Washington University School of Medicine in St. Louis Follow this and additional works at: http://digitalcommons.wustl.edu/open_access_pubs Recommended Citation McElvania TeKippe, Erin; Shuey, Sunni; Winkler, David W.; Butler, Meghan A.; and Burnham, Carey-Ann, ,"Optimizing identification of clinically relevant gram-positive organisms by use of the bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system." Journal of Clinical Microbiology.51,5. 1421-1427. (2013). http://digitalcommons.wustl.edu/open_access_pubs/2333 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry System Downloaded from Erin McElvania TeKippe, Sunni Shuey, David W. Winkler, Meghan A. Butler and Carey-Ann D. Burnham J. Clin. Microbiol. 2013, 51(5):1421. DOI: 10.1128/JCM.02680-12. Published Ahead of Print 20 February 2013. http://jcm.asm.org/ Updated information and services can be found at: http://jcm.asm.org/content/51/5/1421 These include: REFERENCES This article cites 30 articles, 21 of which can be accessed free at: http://jcm.asm.org/content/51/5/1421#ref-list-1 on March 8, 2014 by Washington University in St. Louis CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System Erin McElvania TeKippe,a Sunni Shuey,b David W. Winkler,b Meghan A. Butler,b Carey-Ann D. Burnhama Downloaded from Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USAa; St. Louis Children’s Hospital, St. Louis, Missouri, USAb Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identifica- tion of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical speci- mens. We evaluated 4 direct-smear methods, including “heavy” (H) and “light” (L) smears, with and without a 1-␮l direct for- mic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or “score.” We ؉ found that a heavy smear with a formic acid overlay (H FA) produced optimal MALDI-TOF MS identification scores and the http://jcm.asm.org/ highest percentage of correctly identified organisms. Using a score of >2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of >1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identi- on March 8, 2014 by Washington University in St. Louis fication of Gram-positive bacteria by use of MALDI-TOF MS. henotypic methods for the identification of bacteria in the cess, from both accuracy and workflow standpoints. We evaluated a Pclinical laboratory vary by laboratory and often include sub- method of bacterial preparation that is an intermediate between these jective test interpretation. An alternative method for bacterial two well-described methods, a 1-␮l formic acid overlay applied di- identification that is emerging in clinical microbiology is matrix- rectly over the dried organism on the stainless steel target (19). assisted laser desorption ionization–time of flight mass spectrom- The objective of this study was to evaluate the ability of etry (MALDI-TOF MS). MALDI-TOF MS has been described as a MALDI-TOF MS to accurately identify clinically relevant aerobic rapid, cost-effective, and reliable method for the identification of Gram-positive bacteria using a formic acid overlay method, and bacteria in the clinical laboratory (1, 2, 3, 4, 5, 6, 7, 8, 9). During we focused on the optimization of methods to achieve the highest MALDI-TOF MS identification, a spectral profile representing a rate of identification without introducing misidentifications. In “fingerprint” of bacterial proteins is generated. The spectrum gen- addition, this study evaluates the impact of different incubation erated from a particular isolate can be compared to that of a ref- temperatures, media types, and subculture frequency to deter- erence database for organism identification. Ribosomal proteins mine if these conditions impact MALDI-TOF MS identification. are primarily used for identification due to their relative abun- An accompanying paper by Ford and Burnham (20) aims to val- dance in the bacterial cell (10). idate the Bruker Biotyper system for clinical use for identification Many studies have been published that demonstrate the utility of Gram-negative bacteria. of MALDI-TOF MS for the identification of specific groups of (This work was presented in part at the 112th General Meeting of Gram-positive bacteria, including Staphylococcus aureus, coagu- the American Society for Microbiology, San Francisco, CA, June lase-negative staphylococci, Streptococcus agalactiae, viridans 2012.) group streptococci, atypical catalase-negative Gram-positive cocci, Listeria spp., and Corynebacterium spp. (11, 12, 13, 14, 15, MATERIALS AND METHODS 16, 17, 18). These publications have focused on two methods by Bacterial isolates. Two hundred thirty-nine aerobic Gram-positive or- which bacterial isolates can be prepared for MALDI-TOF MS ganisms isolated from clinical specimens were included in this study. This analysis. The first is the direct application of a thin film or smear of bacteria onto the stainless steel target from an isolated colony, which is a rapid-identification method. Due to the thick pepti- Received 5 October 2012 Returned for modification 30 October 2012 doglycan cell walls of Gram-positive bacteria, this method of Accepted 27 December 2012 preparation can sometimes result in poor MALDI-TOF MS spec- Published ahead of print 20 February 2013 tra (6). To achieve better spectra, a protein extraction may be Address correspondence to Carey-Ann D. Burnham, [email protected]. performed (1, 6). This process can be relatively time- and labor- Copyright © 2013, American Society for Microbiology. All Rights Reserved. intensive and can be disruptive to the workflow of the clinical doi:10.1128/JCM.02680-12 laboratory. We sought to optimize the sample preparation pro- May 2013 Volume 51 Number 5 Journal of Clinical Microbiology p. 1421–1427 jcm.asm.org 1421 McElvania TeKippe et al. TABLE 1 Organisms tested in this study TABLE 2 Summary of isolates, extent of analysis, and primary media from which isolates were tested Organism No. of isolates Abiotrophia defectiva 1 No. of isolates (of a total % of isolates Actinomyces spp. 2 Category of 239) in category in category Aerococcus urinae 1 Source of isolates Aerococcus viridans 1 Fresh 222 92.9 Arthrobacter spp. 2 Frozen 17 7.1 Bacillus cereus/thuringiensis 2 Bacillus megaterium 1 Extent of analysis Bacillus spp. 9 Direct smear 219 91.6 Downloaded from Cellulosimicrobium spp. 3 Repeat direct smear 20 8.4 Coagulase-negative Staphylococcus spp. 18 Full extraction 7 2.9 Corynebacterium pseudodiphtheriticum 1 16S rRNA gene sequencing 17 7.1 Corynebacterium spp. 13 Corynebacterium striatum 5 Medium for isolate testing Corynebacterium urealyticum 1 BAP 229 95.8 Dolosigranulum pigrum 1 Chocolate 5 2.1 Enterococcus avium 1 CNA 5 2.1 Enterococcus casseliflavus 1 http://jcm.asm.org/ Enterococcus faecalis 2 Enterococcus faecium 8 performed to the same level of resolution used for the clinical reporting of Enterococcus spp. 12 these isolates. Granulicatella spp. 1 Validation study design. To validate MALDI-TOF MS for use with Group A alpha-hemolytic Streptococcus spp. 30 Gram-positive bacteria in our laboratory, we planned to test at least 200 Group B alpha-hemolytic Streptococcus spp. 20 isolates, representing a minimum of 15 species, but using no more than 35 Group C alpha-hemolytic Streptococcus spp. 1 isolates of a single species. The isolates were to represent the regular flow Group G alpha-hemolytic Streptococcus spp. 4 of organisms in the clinical lab, with the supplementation of organisms on March 8, 2014 by Washington University in St. Louis Kocuria spp. 1 rarely seen in the laboratory. All isolates were tested in quadruplicate as Lactobacillus spp.