G Model SYAPM-25346; No. of Pages 5 ARTICLE IN PRESS Systematic and Applied Microbiology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting Valeria Gaia a,b,∗, Simona Casati a,b, Mauro Tonolla a,c a Cantonal Institute of Microbiology, Bellinzona, Switzerland b National Reference Centre for Legionella, Bellinzona, Switzerland c Microbial Ecology, Microbiology Unit, Plant Biology Department, University of Geneva, Switzerland article info abstract Keywords: A set of reference strains representing 38 different Legionella species were submitted to Whole Cell Mass Legionella spp. Spectrometry (WCMS) with MALDI-TOF. MALDI-TOF MS The dendrogram computed from strain mass spectral patterns obtained by WCMS was compared to the mip phylogenetic tree obtained from macrophage infectivity potentiator (mip) sequences. The trees inferred Identification from these two methods revealed significant homologies. Sequencing Taxonomy Using 453 Legionella isolates previously characterized by genotyping, it was possible to create species- specific SuperSpectra, using appropriate sets of spectral masses, allowing unambiguous differentiation and identification of the most frequently isolated Legionella species. These SuperSpectra were tested for their suitability to identify Legionella strains isolated from water samples, cooling towers, potting soils and patient specimens deposited at the Swiss National Reference Centre for Legionella and previously identified by molecular methods such as mip gene sequencing. 99.1% of the tested strains isolated from the environment could be correctly identified by comparison with the new SuperSpectra. The identification of Legionella spp. by MALDI-TOF MS is rapid, easy to perform and has the advantage of being time- and cost-saving, in comparison to sequence-based identification. © 2010 Elsevier GmbH. All rights reserved. Introduction infectivity potentiator), rpoB (␤-subunit of DNA-dependent RNA polymerase) or gyrA (gyrase A) [7,11,22], as well as immunolog- Species of Legionella cause severe forms of pneumonia called ical tests with species-specific antibodies (serotyping) [13–16]. Legionnaires’ disease. The first outbreak occurred in 1976 in a hotel L. pneumophila and L. anisa can be rapidly identified using com- in Philadelphia, where the state convention of the American Legion mercially available agglutination tests that show a very high was taking place: of 182 legionnaires who contracted the disease, specificity (100% for L. pneumophila and 99.5% for L. anisa) 29 died. Legionella pneumophila was reported as the causal agent [17,29]. For all other species, identification is usually per- for the first time a few months later [10,26]. Legionella spp. are formed by mip sequencing [28]. An internet-available database naturally present in water and soil, in association with amoebae (www.ewgli.org) allows fast retrieval of known, deposited DNA and other protozoa and in biofilms [8,30]. The family Legionel- sequences; sequencing, however, not only requires a considerable laceae includes 53 recognized species in one genus, 20 of which amount of time and work but is also coupled with relatively high are considered to be human pathogens [2,9]. The majority of cases costs. of Legionnaires’ diseases in Europe are caused by L. pneumophila It is almost impossible to distinguish different Legionella species serogroup 1 [1]. based on the appearance of the colonies on agar media. A rapid The reference methods used for the identification of Legionella and inexpensive technique for direct screening of colonies would spp. include sequencing of specific genes, such as mip (macrophage be very useful, in particular when studying environmental sam- ples from cooling towers, potting soils, and composts, for example, where many non-L. pneumophila species, such as L. bozemanii, L. longbeachae, L. cincinnatiensis, L. jamestowniensis, L. micdadei and L. Abbreviations: MALDI-TOF MS, Matrix Assisted Laser Desorption Ionization – oakridgensis, can be simultaneously present [4,5,20,23,32]. Time Of Flight Mass Spectrometry; SARAMISTM, Spectral Archive And Microbial Whole Cell MALDI-TOF MS (WCMS) is increasingly used for the Identification System. ∗ identification of bacterial strains [3,6,18,19]. Only small amounts Corresponding author at: Cantonal Institute of Microbiology, Bellinzona, of cells are required; the procedure can be performed using the Switzerland. Tel.: +41 91 814 60 18; fax: +41 91 814 60 19. E-mail address: [email protected] (V. Gaia). biomass from single colonies. A recent paper showed the usefulness 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.11.007 Please cite this article in press as: V. Gaia, et al., Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting, Syst. Appl. Microbiol. (2011), doi:10.1016/j.syapm.2010.11.007 G Model SYAPM-25346; No. of Pages 5 ARTICLE IN PRESS 2 V. Gaia et al. / Systematic and Applied Microbiology xxx (2010) xxx–xxx Table 1 isolated during the last 10 years by the National Reference Centre Reference strains obtained from culture collections and used in this study, and (NRC) for Legionella in Bellinzona. accession numbers for mip sequences. To avoid variations in protein spectra that could result from the Species Strain No. NCBI sequence different ecological provenances of the isolates, all strains were accession number purified and cultivated on buffered charcoal yeast extract agar (mip) (BCYE) (bioMérieux, Geneva, Switzerland) at 36 ◦C for 48–72 h. 1 Legionella adelaidensis ATCC 49625 U91606 2 Legionella anisa ATCC 35292 U91607 Identification of the Legionella spp. studied 3 Legionella beliardensis ATCC 700512 AF047756 4 Legionella birminghamensis ATCC 43702 U91608 5 Legionella bozemanii ATCC 33217 U91609 Sequences of the mip gene of the 38 reference strains were 6 Legionella brunensis ATCC 43878 U92227 obtained from NCBI GenBank (Table 1). Isolates of L. pneumophila 7 Legionella cherrii ATCC 35252 U91635 and L. anisa (243 strains) were identified by the agglutination test 8 Legionella cincinnatiensis ATCC 43753 U91636 9 Legionella drozanskii ATCC 700990 AF148983 (Slidex Legionella, bioMérieux, Switzerland). Isolates for which 10 Legionella dumoffii ATCC 33279 U91637 no agglutination was observed (210 strains) were analyzed by 11 Legionella erythra ATCC 35303 U92203 mip-sequencing as previously described [28] and the sequences 12 Legionella fallonii ATCC 700992 AF148987 compared to those included in the mip identification database 13 Legionella feelei ATCC 35072 U92205 (www.ewgli.org). 14 Legionella geestiana ATCC 49504 FJ534536a 15 Legionella gormanii ATCC 33297 U91638 16 Legionella gresilensis ATCC 700509 AF047755 Sequence data analysis of reference strains 17 Legionella hackeliae ATCC 35250 U92207 18 Legionella impletisoli DSM 18493 AB233217 The nucleotide (nt) sequences of the mip gene of the reference 19 Legionella israelensis ATCC 43119 U92208 20 Legionella jamestowniensis ATCC 35298 U92228 strains were downloaded from NCBI (Table 1). For most species 21 Legionella jordanis ATCC 33623 U92209 sequences of 557 nt were obtained, whereas for L. yabuuchiae and 22 Legionella lansingensis ATCC 49751 U92210 L. impletisoli, only 417 nt were available. L. geestiana, which failed to 23 Legionella londiniensis ATCC 49505 U92229 produce an amplicon of the correct size (only 194 nt were available) 24 Legionella longbeachae ATCC 33462 X83036 [28] was not included in the alignment. 25 Legionella maceachernii ATCC 35300 U92211 26 Legionella micdadei ATCC 33218 FJ534537 The sequence similarities of strains were analyzed and phyloge- 27 Legionella moravica ATCC 43877 U92212 netic trees were constructed, using the Neighbor joining algorithm, 28 Legionella oakridgensis ATCC 33761 U92214 included in the MEGA 4 package. Confidence levels of inferred rela- 29 Legionella parisiensis ATCC 35299 U92215 tionships were estimated following 1000 bootstrap iterations [24]. 30 Legionella pneumophila ATCC 33152 AJ878849 31 Legionella quinlivanii ATCC 43830 U92217 32 Legionella rubrilucens ATCC 35304 U92218 MALDI-TOF MS 33 Legionella sainthelensi ATCC 35248 U92219 34 Legionella steigerwaltii ATCC 35302 U92223 A small amount (approx. 0.5 ␮l) of material was taken from 35 Legionella taurinensis ATCC 700508 AF022342 freshly grown colonies and transferred with a plastic loop into 36 Legionella tucsonensis ATCC 49180 U92224 TM 37 Legionella wadsworthii ATCC 33877 U92225 a well of a 48-well stainless steel FLEXImass target plate 38 Legionella yabuuchiae DSM 18492 AB233215 (Shimadzu Biotech, Kyoto, Japan). Analyses were run in dupli- a Partial sequence of 194 nt. cate by spotting a colony into two different wells. The bacteria were overlaid with 0.5 ␮l of matrix solution containing 75 mg/ml 2,5dihydroxybenzoic acid (DHB) in acetonitrile/ethanol/water of WCMS for the rapid and inexpensive identification of twenty-one (1:1:1) supplemented with 3% trifluoroacetic acid and allowed to human pathogenic Legionella species [27]. dry at room temperature until the DHB crystals became visible. The SARAMIS database (Spectral ARchiving And Microbial Iden- MS analyses were performed in positive linear mode in the tification System, AnagnosTec GmbH, Potsdam, Germany) was range of 2000–20,000 masses-to-charge ratio (m/z), with an AXIMA developed for the rapid identification of bacterial strains [21]. ConfidenceTM