Systematic and Applied Microbiology 36 (2013) 75–81 Contents lists available at SciVerse ScienceDirect Systematic and Applied Microbiology jo u rn al homepage: www.elsevier.de/syapm Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting Cristina Andrés-Barrao a , Cinzia Benagli b , Malou Chappuis a , Ruben Ortega Pérez a , Mauro Tonolla b,c , Franc ¸ ois Barja a,∗ a Microbiology Unit, Botany and Plant Biology Dept., University of Geneva, Ch. Des Embrouchis 10, CH-1254 Jussy-Geneva, Switzerland b Cantonal Institute of Microbiology, Via Mirasole 22A, CH-6500 Bellinzona, Switzerland c Microbial Ecology, Microbiology Unit, Botany and Plant Biology Dept., University of Geneva, Quai Ernest-Anserment 30, CH-1211 Geneva, Switzerland a r t i c l e i n f o Article history: Received 15 March 2012 Received in revised form 5 September 2012 Accepted 7 September 2012 Keywords: MALDI-TOF MS Whole-cell identification Acetic acid bacteria Vinegar a b s t r a c t Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. © 2012 Elsevier GmbH. All rights reserved. Introduction Acetic acid fermentation is a process whereby ethanol is transformed into acetic acid by a particular group of microor- ganisms known as acetic acid bacteria (AAB). These widespread microorganisms play an important role in multiple natural pro- cesses that result in high-value food and beverage products, such as vinegar, chocolate and kombucha, as well as chemicals of industrial interest, including cellulose, ascorbic acid (vitamin C) or dihydroxyacetone (DHA). Some symbiotic N 2 -fixing AAB strains are currently studied in order to improve non-legume coffee, rice and sugarcane cultures. However, a subset of these bacteria has recently been described as emerging opportunistic human pathogens that are resistant to several microbicidal agents [24,25,1,8]. Over the last few years, the taxonomy of AAB has been sub- jected to continuous remodeling because of the availability of new molecular techniques for bacterial identification and clas- sification [29,23,6]. Currently, AAB are classified in the family Acetobacteraceae and grouped within twelve genera including Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, Asaia, ∗ Corresponding author. Tel.: +41 22 379 3750; fax: +41 22 379 3756. E-mail address: Francois.Barja@unige.ch (F. Barja). Neoasaia, Kozakia, Frauteria, Granulibacter, Saccharibacter, Swami- nathania and Tanticharoenia [38,36]. Of these, particularly those classified in the genera Acetobacter (A. aceti, A. pasteurianus, A. cerevisiae, A. oeni, A. malorum, A. estunensis and A. pomorum), Gluconobacter (G. oxydans) and Gluconacetobacter (Ga. europaeus, Ga. hansenii, Ga. entanii, Ga. intermedius, Ga. oboediens, Ga. liq- uefaciens, and Ga. xylinus) are involved in industrial vinegar production because of their capacity to oxidize ethanol to acetic acid and their extreme resistance to high acetic acid concentrations [9,37,18,10,33]. For the identification and typing of AAB, several DNA-based techniques have been developed, such as restriction fragment length polymorphism (RFLP), sequence analysis of several genomic targets, and fingerprinting analysis of genomic repetitive elements [19,11,7,17]. An alternative to DNA-based molecular typing meth- ods is provided by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), a recently developed protein fingerprinting method that allows organisms to be distin- guished at the species or even subspecies level [28,3]. The aim of this study was to test the suitability of MALDI-TOF MS for the rapid identification of the most important culturable acetic acid bacteria belonging to the genera Acetobacter, Gluconobac- ter and Gluconacetobacter using the SARAMIS TM software package (Spectral Archive and Microbial Identification System; Anagnostec Gmbh, Germany). 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.09.002