A Rapid MALDI-TOF MS Identification Database at Genospecies Level for Clinical and Environmental Aeromonas Strains Cinzia Benagli1*, Antonella Demarta1, AnnaPaola Caminada1, Dominik Ziegler2, Orlando Petrini1, Mauro Tonolla1,3 1 Institute of Microbiology, Bellinzona, Switzerland, 2 Mabritec AG, Riehen, Switzerland, 3 Microbial Ecology, Microbiology Unit, Plant Biology Department University of Geneva, Gene`ve, Switzerland Abstract The genus Aeromonas has undergone a number of taxonomic and nomenclature revisions over the past 20 years, and new (sub)species and biogroups are continuously described. Standard identification methods such as biochemical characterization have deficiencies and do not allow clarification of the taxonomic position. This report describes the development of a matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF MS) identification database for a rapid identification of clinical and environmental Aeromonas isolates. Citation: Benagli C, Demarta A, Caminada A, Ziegler D, Petrini O, et al. (2012) A Rapid MALDI-TOF MS Identification Database at Genospecies Level for Clinical and Environmental Aeromonas Strains. PLoS ONE 7(10): e48441. doi:10.1371/journal.pone.0048441 Editor: Mikael Skurnik, University of Helsinki, Finland Received May 10, 2012; Accepted September 25, 2012; Published October 31, 2012 Copyright: ß 2012 Benagli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No current external funding sources for this study. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Dominik Ziegler is employed by Mabritec AG. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction [15,16,17,18,19,20]. One major problem in Aeromonas identifica- tion relies on the fact that some species are phenotypically very Bacteria belonging to the genus Aeromonas are widely distributed similar (e.g. A. caviae and A. media, A. veronii and A. sobria). Several in freshwater and brackish environments, and have long been molecular methods have been therefore applied as an alternative recognized as etiologic agents for fish diseases [1]. They are to the laborious DNA-DNA hybridization technique for resolving included into the class Gammaproteobacteria, comprising Gram- the Aeromonas taxonomy and even though the sequence analysis of negative, non-spore-forming rod-shaped bacteria, are facultative ribosomal RNA genes allowed for the discrimination of the anaerobic oxidase- and catalase-positive, glucose-fermenting, genospecies [6,21,22], other more discriminating housekeeping resistant to the vibriostatic agent O/129, and generally motile [2]. genes such as gyrB and rpoD are now increasingly used Aeromonas play also a significant role as opportunistic pathogens [8,23,24,25,26]. Nevertheless, sequencing and phylogenetic meth- for humans causing gastroenteritis, septicemia, pneumonia, ods are costly, time consuming and therefore not appropriate for a meningitis, and wound infections in immunocompetent as well rapid species identification in the diagnostic laboratory. A valid as in compromised patients. A. hydrophila, A. caviae and A. veronii alternative to conventional methods of bacterial identification and (biovar sobria and biovar veronii), are clinically the most significant classification, based on the characterization of biomarker mole- species [3]. cules, but definitely more rapid and reliable is the mass So far, the genus Aeromonas comprises 21 validly proposed spectrometry technique [27]; MALDI-TOF MS (matrix assisted species: A. allosaccharophila, A. aquariorum, A. bestiarum, A. bivalvium, laser desorption ionization mass spectrometry – time of flight) A. caviae (synonym: A. punctata) A. culicicola, A. encheleia (corresponds combined with a reliable database is a powerful method for the to HG 11), A. eucrenophila, A. hydrophila, A. jandaei, A. media, A. identification and comparison of microbial isolates based on molluscorum, A. popoffii, A. salmonicida, A. schubertii, A. sharmana, A. protein fingerprints analysis of whole cells [28]. MALDI-TOF MS simiae, A. sobria, A. tecta, A. trota (synonym: A. enteropelogenes), A. applications in microbiology are important for proteomic and veronii (synonym: A. ichthiosmia). It has to be noted that within natural product analyses [29]. This technique can be used to these proposed species the position of A. allosaccharophila, A. detect non-volatile and thermally unstable molecules from a few to culicicola and A. sharmana has to be clarified since the first two several hundred kDa, the most applicable range used for the might belong to A. veronii and the last one seems not belong to analysis is 2–20 kDa. The identification of microorganisms by the genus Aeromonas at all [4,5]. MALDI-TOF MS is based on the detection of mass signals from Several phylogenetic studies on Aeromonas allowed the elevation biomarkers that are specific at genus, species or sub-group level. of the genus name to the rank of family [2,6,7,8]. Nevertheless the All mass spectra were generated in positive linear mode by taxonomy of this genus is rather complex and has been submitted scanning the sample spot with the laser beam, and after signal to ongoing changes due to newly described species acquisition, the raw mass spectra are processed automatically by [9,10,11,12,13,14] and rearrangements of existing taxa smoothing, baseline correction and peak recognition [30]. The PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48441 Identification of Aeromonas essential information used for microbial identification is contained Phylogenetic tree was constructed using the Neighbour-Joining in a peak list containing m/z values and intensities. This list is method with genetic distances computed by employing Kimura’s analysed by comparison to the database SARAMISTM (Spectral 2-parameter method [41]. Archive And Microbial Identification System), in which the identification at the species level is based on a percentage of MALDI-TOF MS TM confidence referred to reference spectra (SuperSpectra ) that Strains were transferred from the colony directly on a 48- contain family, genus and species specific m/z biomarkers, as position stainless steel FlexiMassTM target plate (Shimadzu described in the SARAMISTM user manual. For the generation of Biotech, Kyoto, Japan) using a plastic loop. The transferred TM one SuperSpectra some representatives isolates of one species colony material was then overlaid with 0.5 ml of Matrix (DHB from different locations (hospitals, reference centers and strain 75%) solution containing 75 mg/ml 2, 5-dihydroxybenzoic acid in culture collections) are needed. Beside the FingerprintSpectra acetonitrile/ethanol/water (1:1:1) supplemented with 3% trifluor- every isolate will be determined by accredited and published oacetic acid. All mass spectra were acquired using an AXIMA microorganism identification procedures. The SuperSpectraTM ConfidenceTM (Shimadzu Biotech, Kyoto, Japan) mass spectrom- are generated based on measurements of well known microor- eter, equipped with a nitrogen laser (pulse width: 3 ns) operated in ganisms and contain sets of genus, species and strain biomarkers positive linear mode. The measured mass range of spectra was which are characteristic for the respective group of microorgan- 2000–20,000 Da. A minimum of 20 laser shots per sample was isms. SuperspectaTM are computed from typical strains covering used to generate each ion spectrum. For each bacterial sample, 50 more than 90% of the intraspecific diversity in most species. protein mass fingerprints were averaged and processed. Accuracy of the identification strongly relies upon the robust- All spectra were processed by the MALDI-TOF MS Launchpad ness of the database and the choice of reference isolates. This is 2.8 software (Shimadzu Biotech, Kyoto, Japan). especially important when considering genera comprising species of clinical and environmental origin presenting a high genetic Data Analysis diversity. A database identification system was established analyzing 92 There are excellent precedents for the application of MALDI- morphologically and genetically well characterized Aeromonas TOF MS for taxonomic studies [31,32,33,34], as well as for strains belonging to all known species of the genus. The resulting routine diagnostic [35]. peak lists of these samples were exported to the SARAMISTM Previous studies proved the applicability of this technique for software package (bioMe´rieux, France) and submitted to single- the identification of the Aeromonas species [36,37,38]. The major linkage cluster analysis to produce taxonomic trees. These trees aim of this study was to establish a rapid and reliable species were compared to a gyrB phylogenetic tree (Neighbour-Joining). identification tool for the genus Aeromonas using the SARAMISTM Specific biomarkers containing sets of genus, species and strain identification system based on a relatively high number of characteristic masses were used for the creation of species-specific phylogenetically well characterized isolates of clinical and envi- SuperSpectraTM recognizing the most frequently encountered ronmental origin. species. 11 different SuperSpectraTM were created that allow identifications of: A. hydrophila,