JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

Folia Microbiol DOI 10.1007/s12223-014-0369-4 1 3 2

4 Evaluation of the MALDI-TOF MS profiling for identification 5 of newly described spp.

79 Andrea Vávrová & Tereza Balážová & Ivo Sedláček & 8 Ludmila Tvrzová & Ondrej Šedo 10

11 Received: 8 April 2014 /Accepted: 2 December 2014 12 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014 OF 13 Abstract The genus Aeromonas comprises primarily aquatic Introduction 32 14 and also serious human and animal pathogens with 15 the occurrence in clinical material, drinking water, and food. The genus Aeromonas, isolated primarily from aquatic envi- 33 16 Aeromonads are typical for their complex and ronments (CarnahanPRO and Joseph 2005), involves enterotoxi- 34 17 nomenclature and for limited possibilities of identification to genic and enteroinvasive opportunistic human pathogens 35 18 the species level. According to studies describing the use of (AbbottD et al. 1992; Janda and Abbott 1998, 2010)andis 36 19 MALDI-TOF MS in diagnostics of aeromonads, this modern representedE by phenotypically identical species with high 37 20 chemotaxonomical approach reveals quite high percentage of percentage of 16S ribosomal RNA (rRNA) sequence similar- 38 21 correctly identified isolates. We analyzed 64 Aeromonas ref- ity of most species (Martínez-Murcia et al. 1992). 39 22 erence strains from the set of 27 species. After extending the Polymorphism of 16S rRNA has been described also, for 40 23 range of analyzed Aeromonas species by newly described example, among Aeromonas media and Aeromonas veronii 41 24 ones, we proved that MALDI-TOF MS procedure accompa- strains (Morandi et al. 2005). For example, Aeromonas 42 25 nied by Biotyper tool is not a reliable diagnostic technique for hydrophila ATCC 7966T and A. media ATCC 33907T differ 43 26 aeromonads. We obtained quite high percentage of false-pos- only in 3 bp of 16S rDNA (Martínez-Murcia et al. 1992; 44 27 itive, incorrect, and uncertain results. The identification of Yáñez et al. 2003). Multi-locus sequence typing of another 45 28 newly described species is accompaniedORRECT with misidentifica- house-keeping genes (gyrA, gyrB, rpoD, recA,anddnaJ gene) 46 29 tions that were observed also in theC case of pathogenic has been therefore evaluated as more promising in the field of 47 3031 aeromonads. phylogenetic analysis of aeromonads (Urwin and Maiden 48 Q1 2003). Biochemical identification systems and genotypic di- 49 UN agnostics methods have limited discriminatory power for 50 identification to the species and subspecies level. Routine 51 * : ž : A. Vávrová ( ) T. Balá ová L. Tvrzová biochemical identification of aeromonads differentiates iso- 52 Division of Microbiology, Department of Experimental Biology, “ ” 53 Faculty of Science, Masaryk University, Kamenice 735/5, 625 lates only to the level of complexes, i.e., three biochemically 00 Brno, Czech Republic differentiated groups—Aeromonas caviae, A. hydrophila,and 54 e-mail: [email protected] Aeromonas sobria complex (Carnahan and Altwegg 1996). 55 Taxonomy and nomenclature of aeromonads is being con- 56 T. Balážová : O. Šedo 57 Research Group Proteomics, CEITEC, Central European Institute of tinuously changed and extended by describing new taxa and Technology, Masaryk University, Kamenice 735/5, 625 00 Brno, rearrangements of the existing ones. It suffers from the use of 58 Czech Republic synonyms, such as “Aeromonas punctata” for A. caviae, 59 “Aeromonas trota” for Aeromonas enteropelogenes,and 60 I. Sedláček “ ” 61 Czech Collection of Microorganisms, Department of Experimental Aeromonas ichthiosmia for A. veronii (Collins et al. 1993; Biology, Faculty of Science, Masaryk University, Kamenice 735/5, Janda and Abbott 1998;Huysetal.2002a, 2002b; Saavedra 62 62500Brno,CzechRepublic et al. 2006). 63 The number of published works pointing out the occur- 64 O. Šedo 65 National Centre for Biomolecular Research, Faculty of Science, rence of Aeromonas spp. in clinical material, drinking water, Masaryk University, Kamenice 735/5, 625 00 Brno, Czech Republic and food has been significantly increasing during the last 66 JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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67 decades (Burke et al. 1984; Trust and Chipman 1979;Merino The aim of our study was to examine the suitability of 120 68 et al. 1995;Alavandietal.1998;Tsaietal.2006; Janda and Biotyper system for identification of the recently described 121 69 Abbott 2010;Chenetal.2012). These facts support the need Aeromonas species and species that have recently been syn- 122 70 for some accurate identification tests, enabling differentiation onymized. Up to now, newly described species 123 71 and classification of potentially pathogenic strains at the spe- A. cavernicola, A. diversa, A. fluvialis, A. rivuli, 124 72 cies and subspecies level. A. sanarellii,andA. taiwanensis have not been analyzed by 125 73 From 33 validly described taxa (http://www.bacterio.net/a/ MALDI-TOF MS yet. The mass spectral outputs were evalu- 126 74 aeromonas.html), 11 species were found in human clinical ated on the basis of the scoring algorithm involved in the 127 75 material. A. hydrophila, A. caviae, Aeromonas jandaei, A. Biotyper identification system and also by cluster analysis 128 76 veronii biovar sobria, A. veronii biovar veronii, Aeromonas based on mass spectra of type strains of 27 species with three 129 77 schubertii, A. media, Aeromonas encheleia,andAeromonas subspecies of A. hydrophila and five subspecies of Aeromonas 130 78 bestiarum (Miyake et al. 2000; Miñana-Galbis et al. 2009; salmonicida, including strain of A. cavernicola CCM 7641T 131 79 Janda and Abbott 2010) have been described as human (Martínez-Murcia et al. 2013), which has not been so far 132 80 pathogens, and also newly described species Aeromonas validly published in the International Journal of Systematic 133 81 aquariorum, Aeromonas taiwanensis,andAeromonas and Evolutionary Microbiology. 134 82 sanarellii have been isolated from wounds and clinical 83 material (Wu et al. 2012; Beaz-Hidalgo et al. 2012; Shin OF 84 et al. 2013). To date, there has been described a range of Material and methods 135 85 phenospecies and genospecies (Abbott et al. 1992; Carnahan 86 and Joseph 2005). New taxa from human clinical material Bacterial strains 136 87 and/or environment including A. aquariorum, Aeromonas PRO 88 australiensis, Aeromonas cavernicola, Aeromonas diversa, Well phenotypically and genetically characterized Aeromonas 137 89 Aeromonas fluvialis, Aeromonas piscicola, Aeromonas rivuli, strainsD (Table 1) were obtained from the Czech Collection of 138 90 A. sanarellii, A. taiwanensis,andAeromonas tecta have been MicroorganismsE (CCM, Brno, Czech Republic). In total, 64 139 91 described recently (Demarta et al. 2008; Martínez-Murcia strains have been analyzed including 26 type strains of validly 140 92 et al. 2008; Beaz-Hidalgo et al. 2009;Alperietal.2010a, described species and subspecies corresponding to current 141 93 2010b; Miñana-Galbis et al. 2010;Figuerasetal.2011; taxonomy, one type strain of A. cavernicola CCM 7641T 142 94 Aravena-Román et al. 2013; Martínez-Murcia et al. 2013; and reference strains. 143 95 Martino et al. 2014). The status of species A. aquariorum 96 and A. hydrophila subsp. dhakensis is problematic and ac- Growth conditions 144 97 cording to some publications, they should have been 98 reclassified (Martínez-Murcia et al. 2009; Beaz-Hidalgo All Aeromonas strains were cultivated aerobically on 145 99 et al. 2013), but reclassification has not beenORRECT validly published Tryptone soya agar (OXOID). Cell masses were collect- 146 100 in IJSEM so far. C ed after 24 h, if sufficient growth was monitored. If not, 147 101 Matrix-assisted laser desorption/ionization time of flight the period of cultivation was prolonged to 48 h. Strains 148 102 mass spectrometry (MALDI-TOF MS) is a modern approach were cultured at 22 °C or at 30 °C according to 149 103 widely used for identificationUN and typing of microorganisms, Catalogue of Cultures of Czech Collection of 150 104 rapid screening of pathogenic strains and species, and detec- Microorganisms. 151 105 tion of unique proteins and biomarkers. Its main advantage 106 consists in a short-time direct analysis without previous sam- Sample preparation prior to MALDI-TOF MS 152 107 ple separation or other extensive pretreatment methods 108 (Fenselau and Demirev 2001; Stackebrandt et al. 2002; Šedo Samples were prepared according to the standard extraction 153 109 et al. 2011). The rapid screening in medical diagnostics is protocol (Freiwald and Sauer 2009). Approximately 5–10 mg 154Q3 110 based on comparison with an enlarging database of reference of the bacterial culture was taken from Tryptone soya agar 155 111 spectra of clinically relevant microorganisms. Another advan- with the sterile inoculating loop (1 μL), washed by vortexing 156 112 tage of MALDI-TOF MS consists in its discriminatory power, with 1 mL of water in an Eppendorf tube, and centrifuged 157 113 as several studies reported distinguishing among bacterial (1365 g, 2 min). The supernatant was removed and 1.2 mL of 158 114 strains on the basis of their MALDI-TOF mass spectra (Seng 75 % ethanol was added to the pellet. The sample was centri- 159Q4 115 et al. 2010; Sandrin et al. 2013). In previous studies, MALDI- fuged again (12,100g, 2 min) and supernatant was discarded. 160 116 TOF MS had been already tested for detection and identifica- Sterilized cells were extracted by vortexing for 1 min with 10 161 117 tion of Aeromonas species (Donohue et al. 2006;Donohue to 50 μL of 70 % formic acid (depending on the size of the 162 118 et al. 2007; Beaz-Hidalgo et al. 2009; Lamy et al. 2011; pellet) and equal volume of acetonitrile. After centrifugation, 163 119 Benagli et al. 2012). the supernatant was deposited on three wells of the sample 164 JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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Q2 t1:1 Table 1 Aeromonas spp. cultures—newly described and validated species are in bold

t1:2 Aeromonas species CCM strain Equivalent code of the strain (if accessible) Source of isolation number

t1:3 A. allosaccharophila 4363T =ATCC 51208=CECT 4199=LMG 14059=C. Esteve E289 Diseased elver (Anguilla anguilla), Valencia, East Coast; Spain t1:4 4243 =H. Přepechalová 206 River water, Dalečín; Czech Republic t1:5 4531 =E. Aldová 29374 Urine, České Budějovice; Czech Republic t1:6 A. aquariorum 7695T =CECT 7289=DSM 18362=LMG 24688=strain MDC47 Aquaria of ornamental fish, Maia, Porto; Portugal t1:7 A. australiensis 8484T =CECT 8023=CIP 110626=LMG 2670=strain 266 Irrigation water system; Australia t1:8 A. bestiarum 4707T =ATCC 51108=CDC 9533–76=CIP 74.30=LMG Diseased fish 13444=M.Y. Popoff 218 t1:9 A. bivalvium 7467T =CECT 7113=CIP 109539=LMG 23376=strain 868E Ex cockles (Cardium sp.) obtained from a retail market, Barcelona; Spain t1:10 A. cavernicola 7641T – Water, Moravian Karst; Czech Republic t1:11 A. caviae 4491T =ATCC 15468=CCUG 25939=CIP 76.16=DSM 7323=JCM Epizootic of young guinea pigs 1060=LMG 3775=NCIMB 13016=NRRL B-968=WDCM 00062=B. Austin BA-31=M.Y. Popoff 545 t1:12 7231 =ATCC 15467=CCUG 25938=CIP 76.15=DSM UsedOF oil emulsions 30188=LMG 3755=M.Y. Popoff 544=R.H.W. Schubert 256 t1:13 A. diversa 7325T =ATCC 43946=CCUG 17321=CDC 2478–85=CECT Human leg wound, Louisiana, New Orleans; 4254=LMG 17321 USA t1:14 A. encheleia 4582T =CECT 4342=C. Esteve S181=LMG 16330 Healthy European eel (freshwater farm), PRO Valencia; Spain t1:15 7335 =ATCC 35941=CCUG 30364=CDC 1306–83=CECT 4253=CIP Ankle fracture; New Zealand 105413=LMG 13075=M. Altwegg A902 t1:16 7407 =strain P 1767=D 14/7D Fresh water from cave, Moravian karst; Czech E Republic t1:17 7408 =strain P 1688=C 9/4 Fresh water from cave, Moravian karst; Czech Republic t1:18 A. enteropelogenes 7243T =ATCC 49803=CECT 4487=CIP 104434=DSM Human stool; India 6394=JCM 8355=LMG 12646=M. Sanyal J11 t1:19 4358 =ATCC 49660=CCUG 30368=LMG 13081=strain AS 66 Human stool; Thailand t1:20 4368 =ATCC 49657=CCUG 29544=DSM 7312=JCM Human stool, Varasani; India 8315=LMG 12223=A.M. Carnahan AH2 t1:21 A. eucrenophila 4354T =ATCC 23309=CCTM La 885=CCUG 25942=CDC-RH63=CIP Fresh water fish 76.17=JCM 8238=LMG 3774=NCIMB 74=M. Popoff 546 t1:22 A. fluvialis 8437T =CECTORRECT 7401=LMG 24681=strain 717 Water from Muga river, Girona; Spain t1:23 A. hydrophila ssp. dhakensis 7146T =CCUG 45377=CIP 107500=LMG 19562=G. Huys Stool, child with diarrhea, Dhaka; Bangladesh CR-1999=I. Kühn P21 t1:24 7329 =E. Durnová 597/04=strain P 1097 Stool, acute profuse diarrhea (imported infection from Egypt), Ostrava; Czech Republic t1:25 A. hydrophila ssp. hydrophila 7232TUN =ATCC 7966=CCUG 14551=CDC 359–60=CIP Tin of milk with a fishy odor 76.14=DSM 30187=IAM 12460=JCM 1027=LMG 2844=NCIMB 9240=NCTC 8049=WDCM 00063 t1:26 2278 =ATCC 43409=JCM 3976=L.A. Page LAP-182 Red-legged frog (Rana auroa draytoni); California t1:27 2280 =JCM 3968=L.A. Page LAP-186 Patchnose snake (Salvadora hexalepsis virgultea) t1:28 4528 =E. Aldová 29352 Human stool, České Budějovice; Czech Republic t1:29 A. hydrophila ssp. ranae 7147T =CCUG 46211=LMG 19707=G. Huys R-5216=M. Pearson AU- Frog (Rana rugulosa) with liver septicemia, 1D12 Kamphaeng Phet; Thailand t1:30 7954 =P2335=strain 5 Leaf in water, Tad Fane waterfall; South-East Laos t1:31 A. jandaei 4355T =ATCC 49568=CCTM La 3019=CCUG 29543=CDC Human stool, diarrhea, Oregon; USA 0787–80=CIP 10471=DSM 7311=JCM 8316=LMG 12221 t1:32 4367 =ATCC 49570=CCUG 30352=LMG 13077=A. M. Carnahan Eye wound, Maryland; USA and S. W. Joseph WRII/4658 t1:33 A. media 3653T =ATCC 33907=CCTM La 3015=CCUG 14453=CIP River water, effluent from fish farm; UK 103208=CNCTC Aer 25/88=DSM 4881=JCM 2385=LMG 9073=NCIMB 2237=strain RM t1:34 3654 =CCUG 14454=JCM 2386=strain P43 River water, effluent from fish farm; UK JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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t1:36 Table 1 (continued)

Aeromonas species CCM strain Equivalent code of the strain (if accessible) Source of isolation number

t1:35 4242 =H. Přepechalová 739 River water, Dyje-Podhradí; Czech Republic t1:36 7321 =LMG 13461=M. Altwegg A75 Human feces; Switzerland t1:37 A. molluscorum 7245T =CECT 5864=LMG 22214=D. Minana-Galbis 848 Wedge-shells (Donax trunculus) from a retail market, Barcelona; Spain t1:38 A. piscicola 7715T =CECT 7443=LMG 24783=strain S1.2 Wild diseased salmon, Carballedo Galicia; Spain t1:39 A. popoffii 4708T =I. Kersters O-a-10-3 Drinking water production plant, Oelegem; Belgium t1:40 4961 =D. Baudišová A55 Vltava river, Praha, Troja; Czech Republic t1:41 7330 =B. Otipka Bol 20=strain P 661 Water, Frýdek-Místek; Czech Republic t1:42 A. rivuli 8438T =CECT 7518=DSM 22539=MDC 2511=strain WB4.1–19 Water, karst region, Westerhöfer Bach; Germany t1:43 A. salmonicida 1150 =V. Pleva 17 Carp t1:44 A. salmonicida 1275 =S.F. Snieszko 84 Brook trout (Salvelinus fontinalis) t1:45 A. salmonicida ssp. 7233T =ATCC 33659=CIP 104001=CCUG 42781=JCM 7875=LMG Brown trout (Salmo trutta), River Dee, achromogenes 14900=NCIMB 1110=I.W. Smith 6263/4/5 OFAberdeen; UK t1:46 A. salmonicida ssp. 4124T =ATCC 27013=CIP 103210=DSM 21760=IFO 13784=JCM Heart blood of “Sakuramasu” (Oncorhynchus masoucida 8191=LMG 3782=NCIMB 2020=T. Kimura 1-a-1 masou) t1:47 A. salmonicida ssp. 7020T =DSM 12609=NCIMB 13742=strain 34mel Water, Matanza River, Buenos Aires; Argentina pectinolytica t1:48 A. salmonicida ssp. 7246T =ATCC 33658=CIP 103209=JCM 7874=LMG 3780=NCIMBPROAtlantic salmon (Salmo salar) kill, River Cletter; salmonicida 1102=NCTC 12959=I.W. Smith 20 UK t1:49 1307 =JCM 8198=NCAIM B.01069=NCIMB 838=W.H.D Ewing 2998– Brown trout (Salmo trutta) 60 t1:50 1318 =CIP 64.50=DSM 46293=W.H. EwingE 3007–60 Unknown t1:51 A. salmonicida ssp. smithia 4103T =ATCC 49393=DSM 21293=NCIMB 13210=B. Austin Ulcer (Rutilus rutilus); UK 138=T. Owen AS20/1/1 t1:52 4105 =ATCC 49395=B. Austin 144=T. Owen AS20/9/3 Ulcer (Rutilus rutilus); UK t1:53 A. sanarellii 7886T =CECT 7402=CIP 110203=LMG 24682=strain A2-67 Wound of 70-year-old female with diabetes mellitus; Taiwan t1:54 A. schubertii 4356T =ATCC 43700=CCTM La 3016=CCUG 27820=CDC Forehead abscess, Texas; USA 2446-81=DSM 4882=JCM 7373=LMG 9074=NCIMB 13161 t1:55 4357 =AMC 1108=CCUG 25582=LMG 12655 Human leg wound, Maryland; USA t1:56 A. simiae 7234T =CCUG 47378=CIP 10778 Monkey feces, Strasbourg; France t1:57 A. sobria 2807T =ATCCORRECT 43979=CCTM La 3018=CIP 74.33=JCM Carp (Cyprinus carpio) 2139=LMG 3783=NCIMB 12065=M. Popoff 208 t1:58 2808C =L. Le Minor 17–72=M. Popoff 217 Pike (Esox lucius) t1:59 A. taiwanensis 7885T =CECT 7403=CIP 110204=LMG 24683=strain A2-50 Burn wound of 40-year-old male; Taiwan t1:60 A. tecta 7605T =CECT 7082=DSM 17300=MDC 91=strain F 518 Fecal sample of a 5-year-old child, UN Canton Ticino; Switzerland t1:61 A. veronii 4359T =ATCC 35624=CCUG 27821=CDC1169-83=CNCTC Sputum, drowning victim, Michigan; USA Aer 26/88=DSM 7386=JCM 7375=LMG 9075=NCIMB 13015 t1:62 1242 =ATCC 9071=CDC 360–60=CDC-RH 36=CIP 57.49=LMG Frog 3785=NCIMB 37=NCTC 7810=Canad-212 494=W.L. Kulp P2 t1:63 1254 =ATCC 11163=CDC 361–60=IAM 1646=NCIMB 73=S.F. Unknown Snieszko A t1:64 4529 =E. Aldová 28327 Stool, Praha; Czech Republic t1:65 7244 =ATCC 49904=CECT 4486=CIP 104613=DSM 6393=JCM Surface water 8354=LMG 12645 t1:66 7323 =CECT 5761=CIP 107763=LMG 21852=MTCC Midgut, mosquito 3249=NCIM 5147=G. Huys R-20199 (Culex quinquefasciatus), Pune; India

165 plate at a volume of 0.3 μL and, after drying at room temper- Instrumentation and data analysis 169 166 ature, overlaid with 0.3 μL of the saturated alpha-cyano-4- 167 hydroxycinnamic acid solution in acetonitrile to water to MALDI-TOF mass spectra of Aeromonas strains were obtain- 170 168 trifluoroacetic acid (50:47.5:2.5, v/v)mixture. ed with an Ultraflex III instrument (Bruker Daltonics, 171 JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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172 Bremen, Germany) operated in linear positive mode (25-kV From 14 remaining strains of species not included in the 220 173 acceleration voltage) under FlexControl 3.3 software. database, two strains (14 %) were identified correctly, one 221 Q5 174 External calibration of the mass spectra was performed using strain (A. piscicola 7715T) was assigned only to the genus 222 175 Escherichia coli DH5 alpha standard peaks (4346.3, 5095.8, level (2.0>Biotyper log(score)>1.7), and 11 strains (79 %) 223 176 5380.4, 6254.4, 7273.5, and 10,299.1 Da). Five independent yielded false-positive results including nine newly described 224 177 spectra accumulations comprising 1000 laser shots each were species. 225 178 acquired from each of the wells. Within an individual well, the As false-positive, we call results for strains that do not have 226 179 laser was directed according to a predefined lattice raster, reference spectra of their species included in database; never- 227 180 where a minimum of 200 and a maximum of 400 shots were theless, they are matched with other species with Biotyper 228 181 obtained from one raster position. Mass spectra were proc- log(score) >2000. The correct results for these cases should be 229 182 essed using Flex Analysis (version 3.4; Bruker Daltonics) and 1.999>Biotyper log(score)>1.700, which means probable 230 183 Biotyper software (version 3.0; MBT-BDAL-5 627 MSP li- identification of the genus. All strains were assigned to the 231 184 brary, Bruker Daltonics). Biotyper log(score) values bellow genus Aeromonas without yielding log(scores) exceeding 232 185 1.699 indicated no significant similarity of the spectra with 1.700 for any of Biotyper database entries representing differ- 233 186 any of the database entries; values 1.700-1.999 indicated ent genera. 234 187 probable identification of genus; log(score) 2.000–2.299 indi- These outputs reveal that the routinely used Biotyper iden- 235 188 cated genus identification and probable species identification; tification system may not beOF a suitable tool for reliable iden- 236 189 and values 2.300–3.000 indicated highly probable species tification of some of the Aeromonas spp. The existence of 237 190 identification. false-positive or uncertain results highlight the deficiency of 238 191 Signals present in at least 11 out of 15 spectra accumula- the commercial system, which may yield misidentifications. 239 192 tions obtained from each sample were taken into account for The strains yieldingPRO results concerning false-positive, incor- 240 193 cluster analysis. The MALDI-TOF mass spectra-based den- rect, and uncertain identification results are listed in Tables 2 241 194 drogram was generated, using the correlation distance mea- and 3.D Most of the false-positive identification results were 242 195 sure with the average linkage algorithm (unweighted average relatedE to members of species absent from the Biotyper data- 243 196 distance—UPGMA) settings. base (namely A. aquariorum, A. australiensis, Aeromonas 244 bivalvium, A. cavernicola, A. diversa, A. fluvialis, 245 197 Chemicals A. piscicola, A. rivuli, A. sanarellii, A. taiwanensis,and 246 A. tecta). These strains were always assigned to different 247 198 Alpha-cyano-4-hydroxycinnamic acid was obtained from species; in eight cases, the obtained scores even exceeded 248 199 Bruker Daltonics (Germany); ethanol, acetonitrile, the threshold of 2.3 (specified by the system provider as 249 200 trifluoroacetic acid, and formic acid from Sigma-Aldrich “highly probable species identification”). In case of five 250 201 (Germany); and water was prepared on a Milli-Q plus 185 strains, the Biotyper log(score) difference for two highest 251 202 apparatus (Millipore, Bilerica, MA, USA).ORRECTdatabase entries was lower than 0.1, which proves high degree 252 203 C of similarity between the spectra. Such a close similarity was 253 also found in the case of A. media and A. veronii, where two 254 strains yielded such ambiguous results. The remaining uncer- 255Q6 204 Results and discussion UN tain identification results were observed also for subspecies 256 that do not have their representative in the database, and their 257 205 MALDI-TOF MS profiles were compared with the commer- identification thus depends on their similarity to different 258 206 cially available Biotyper MBT-BDAL-5 627 MSP library. The subspecies of the same species (see Table 2). This was found 259 207 database does not include type strains of newly described for A. salmonicida subsp. smithia, A. salmonicida subsp. 260 208 species (bold in Table 1). All of the 64 analyzed strains yielded achromogenes,andA. hydrophila subsp. dhakensis. 261 209 MALDI-TOF MS profiles containing signals within the mass Three out of the four wrongly identified strains (e.g., strains 262 210 range 2–12 kDa. Fifty of the total of 64 analyzed strains belonging to species included in the database but assigned to 263 211 belong to the species included in the Biotyper database. different ones) concerned Aeromonas popoffii strains being 264 212 From this set, 41 strains (82 %) were correctly assigned to classified as A. bestiarum.Thisfollowsthefactthatwithin 265 213 the species level (Biotyper log(score) >2.0), including “A. hydrophila,” complex Aeromonas species are differentiat- 266 214 A. salmonicida subsp. smithia 4103T, that was identified cor- ed problematically, especially by phenotypical methods 267 215 rectly on the species but incorrectly on the subspecies level (Abbott et al. 2003;Saavedraetal.2006). Also, according 268 216 because of missing representatives of the subspecies in the to Miñana-Galbis et al. (2004), A. popoffii and A. bestiarum 269 217 database. Incorrect results were obtained for four strains (8 %) strains are in close genetic relationship. In agreement with our 270 218 and uncertain results (the score difference between the first findings, low degree of identification success rate for 271 219 and the second hit was lower than 0.1) for five strains (10 %). A. popoffii has also been reported by Benagli et al. (2012). 272 JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

Folia Microbiol t2:1 Table 2 Biotyper log(score) of incorrect and uncertain identifications of species present in MBT-BDAL-5627 MSP library, (Bruker Daltonics) t2:2 Aeromonas species CCM strain Identification Biotyper number log(score)a t2:3 Incorrect identification of species A. hydrophila subsp. dhakensis 7146T A. jandaei 2.271 t2:4 present in Biotyper database A. popoffii 4708T A. bestiarum 2.324 4961 A. bestiarum 2.316 t2:5 A. salmonicida subsp. salmonicida 2.245 t2:6 7330 A. bestiarum 2.244 t2:7 t2:8 Uncertain identification results A. hydrophila subsp. dhakensis 7329 A. hydrophila 2.365 A. jandaei 2.275 t2:9 t2:10 A. media 7321 A. media 2.351 A. veronii 2.350 t2:11 t2:12 A. veronii 1242 A. veronii 2.497 A. hydrophila 2.426 t2:13 t2:14 A. salmonicida subsp. 7233T A. bestiarum 2.391 achromogenes A. salmonicida subsp.OFsalmonicida 2.391 t2:15 t2:16 A. salmonicida subsp. smithia 4105 A. salmonicida subsp. salmonicida 2.385 A. bestiarum 2.296 t2:17 a Biotyper log(score) values bellow 1.699 indicated no significant similarity of the spectra withPRO any of the database entries; values 1.700–1.999 indicated probable identification of genus; and log(score) 2.000–2.299 indicated genus identification and probable species identification and values 2.300–3.000 indicated highly probable species identification D E t3:1 Table 3 False-positive identification outputs of newly described species including A. cavernicola (Martínez-Murcia et al. 2013) not present in MBT- BDAL-5627 MSP library (Bruker Daltonics) t3:2 Aeromonas species Taxonomical relationships Biotyper log(score)a LevelofsimilarityofMALDIMS profiles (Fig. 1) t3:3 A. aquariorum 7695T Heterotypic synonym of Aeromonas hydrophila A. jandaei 2.369 High for A. hydrophila and A. jandaei subsp. dhakensis ORRECTA. hydrophila 2.337 t3:4 t3:5 A. australiensis 8484T Assigned to A. fluvialis with 99.6 % similarity A. jandaei 2.376 High for A. australiensis CCM 8484T C T A. hydrophila 2.357 and A. fluvialis CCM 8437 t3:6 t3:7 A. cavernicola 7641T Low relatedness to other Aeromonas species A. media 2.144 Relatively distant position from the UN A. hydrophila 2.123 other aeromonads t3:8 t3:9 A. diversa 7325T Misidentified as A. schuberti A. schuberti 2.388 Subcluster with A. schubertii CCM 4356T t3:10 A. fluvialis 8437T 99.5 % similarity with A. veronii A. veronii 2.389 Subcluster with A. australiensis CCM 8484 T t3:11 Similarity with A. sobria and A. allosaccharophila A. hydrophila 2.381 t3:12 99.6 % similarity to A. australiensis t3:13 A. piscicola 7715T Related to species of A. salmonicida and A. bestiarum A. bestiarum 1.954 Most distant member of the genus A. salmonicida 1.870 t3:14 t3:15 A. rivuli 8438T Independent phylogenetic line with A. molluscorum A. molluscorum 2.362 Subcluster with A. molluscorum CCM 7245T and A. bivalvium t3:16 A. sanarellii 7886T 99.6-99.8 % similarity to A. caviae, A. caviae 2.441 Subcluster with A. caviae CCM 4491T A. enteropelogenes,andA. aquariorum A. hydrophila 2.406 t3:17 t3:18 A. taiwanensis 7885T 99.6-99.8 % similarity to A. caviae, A. caviae 2.381 Subcluster with A. caviae CCM 4491T A. enteropelogenes,andA. aquariorum A. hydrophila 2.378 t3:19 t3:20 A. tecta 7605T A. eucrenophila-like organism A. eucrenophila 2.425 Subcluster with A. eucrenophila CCM 4354T and A. media CCM 3653T

a Biotyper log(score) values bellow 1.699 indicated no significant similarity of the spectra with any of the database entries; values 1.700–1.999 indicated probable identification of genus; and log(score) 2.000–2.299 indicated genus identification and probable species identification and values 2.300–3.000 indicated highly probable species identification JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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273 The remaining wrongly identified strain was A. hydrophila CCM 7695, A. australiensis CCM 8484, A. cavernicola CCM 305 274 subsp. dhakensis CCM 7146T, identified as A. jandaei with 7641, A. diversa CCM 7325, A. fluvialis CCM 8437, 306 275 the log(score) of 2.271. A. piscicola CCM 7715, A. rivuli CCM 7438, A. sanarellii 307 276 Strains of newly described species were identified with the CCM 7886, A. taiwanensis CCM 7885, and A. tecta CCM 308 277 use of MALDI-TOF MS and Biotyper database as an incorrect 7605T), the number of incorrectly identified specimens in- 309 278 taxon. This could lead to misidentification of Aeromonas creased to 28 % because nine of previously correctly identi- 310 279 isolates in routine use of the technique. Opportunistic patho- fied species were matched incorrectly with one of the newly 311 280 gens absent from the database, namely A. aquariorum, added strains. This clearly proves that enlargement of the 312 281 A. taiwanensis,andA. sanarellii could be thereby identified database would, in the case of aeromonads, definitely deteri- 313 282 as common autochthonous aeromonads. The high values of orate the overall performance of the method. 314 283 Biotyper log(scores) indicate that the mass spectra similarity The first indication of the identification of aeromonads by 315 284 reaches almost the maximum degree within the possible var- using MALDI-TOF MS profiling not being a straightforward 316 285 iability of the method itself. This fact is in contrast with task was given by Donohue et al. (2007), whose identification 317 286 conclusions of the pilot study on the use of MALDI-TOF results obtained by using MALDI-TOF MS matched the 318 287 MS for Aeromonas identification, where practically non- phenotypic identification in all cases concerning strains with 319 288 problematic distinguishing among the species was indicated, a recognizable phenotypic profile. However, in a group of 320 289 while using sinapinic acid as a matrix and Phylogenetic strains with atypical phenotypicOF profile, the authors obtained 321 290 AnalysisUsingParsimony(PAUP*4.0bsoftware) 66 % identification success rate. The fact that the newly 322 291 (Donohue et al. 2006). Our results reveal that routine described aeromonads are assigned to other Aeromonas spp. 323 292 MALDI-TOF MS users encountered with novel Aeromonas has been noted by Beaz-Hidalgo et al. (2009), who obtained 324 293 species will not have doubts about their correct assignment, Biotyper log(scores)PRO indicating probable species identification 325 294 and the true identity of such strains will remain unrevealed. for A. popoffii, A. salmonicida,andA. bestiarum when ana- 326 295 Due to high values of the false positive scores, which indicate lyzingDA. piscicola, and by Lamy et al. (2011) who assigned 327 296 perfect similarity between tested and database entries, A.E aquariorum to Aeromonas eucrenophila, A. bivalvium to 328 297 MALDI-TOF MS connected to the scoring algorithm cannot A. hydrophila,andA. tecta to A. eucrenophila when identifi- 329 298 be used for reliable identification of aeromonads. A usual step cation against commercially available Biotyper database 330 299 which enhances the output of MALDI MS profiling consists (identification software version 2.0) was used. In the latter 331 300 in the addition of new entries to the current database. While work, small difference (<0.1) between the first and the second 332 301 10 % of strains (five of the total of 50 type and reference hit Biotyper log(scores) was described in the case of 35 % (42 333 302 strains of species included in the database) were identified of 118) analyzed strains; however, the top score in 82 % (32 334 303 incorrectly using Biotyper database, after enlarging the data- strains) of cases corresponded to the correct species. Also, in a 335 304 base with ten newly described type strainsORRECT (A. aquariorum study that identified 689 from 741 Aeromonas strains by 336 Fig. 1 Dendrogram based on C cluster analysis of MALDI-TOF MS profiles of 33 Aeromonas type strains UN JrnlID 12223_ArtID 369_Proof# 1 - 09/12/2014

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337 comparing the profiles against a home-made database of Alavandi S, Ananthan S, Kang G (1998) Prevalence, in-vitro secretory 388 389 338 SuperSpectra™ including 11 Aeromonas species (by using activity, and cytotoxicity of Aeromonas species associated with childhood gastroenteritis in Chennai (Madras), India. Jpn J Med 390 339 Shimadzu/BioMerieux system), 7 % of Aeromonas strains Sci Biol 51(1):1–12 391 340 were identified incorrectly because of missing reference Alperi A, Martínez-Murcia AJ, Ko WC, Monera A, Saavedra MJ, 392 341 SuperSpectra™ (23 strains) or because of insufficient coverage Figueras MJ (2010a) Aeromonas taiwanensis sp. nov. and 393 394 342 of the strain variability by the composition of the database (29 Aeromonas sanarellii sp. nov., clinical species from Taiwan. Int J Syst Evol Microbiol 60:2048–2055. doi:10.1099/ijs. 0.014621-0 395 343 strains) (Benagli et al. 2012). Alperi A, Martínez-Murcia AJ, Monera A, Saavedra MJ, Figueras MJ 396 344 Some problematic groups of species have already been (2010b) Aeromonas fluvialis sp. nov., isolated from a Spanish river. 397 345 indicated by the Biotyper supplier, who accompanied several Int J Syst Evol Microbiol 60:72–77. doi:10.1099/ijs. 0.011643-0 398 399 346 database entries with “matching hints” alerting the users to Aravena-Román M, Beaz-Hidalgo R, Inglis TJJ, Riley TV, Martínez- Murcia AJ, Chang BJ, Figueras MJ (2013) Aeromonas australiensis 400 347 species distinguishable with difficulties. This has been ob- sp. nov., isolated from irrigation water. Int J Syst Evol Microbiol 63: 401 348 served in case of particular species belonging, for example, 2270–2276. doi:10.1099/ijs. 0.040162-0 402 349 to genus Acinetobacter, Citrobacter, Clostridium, Beaz-Hidalgo R, Alperi A, Figueras MJ, Romalde JL (2009) Aeromonas 403 404 350 Corynebacterium, Salmonella, etc. Unfortunately, due to a piscicola sp. nov., isolated from diseased fish. Syst Appl Microbiol 32:471–479. doi:10.1016/j.syapm.2009.06.004 405 351 dynamic development of the bacterial taxonomy, the number Beaz-Hidalgo R, Shakèd T, Laviad S, Halpern M, Figueras MJ (2012) 406 352 of problematic species will probably increase quite rapidly as Chironomid egg masses harbour the clinical species Aeromonas 407 353 well. Early indication of such cases by the scientific commu- taiwanensis and AeromonasOF sanarellii. FEMS Microbiol Lett 408 – 409 354 nity is thus necessary to avoid further misclassifications. 337(1):48 54. doi:10.1111/1574-6968.12003 Beaz-Hidalgo R, Martínez-Murcia A, Figueras MJ (2013) 410 355 To express the mutual similarity of MALDI MS profiles of Reclassification of Aeromonas hydrophila subsp. dhakensis Huys 411 356 all analyzed strains, the MALDI-TOF mass spectra were et al. 2002 and Aeromonas aquariorum Martínez-Murcia et al. 2008 412 357 subjected to cluster analysis. The MALDI-TOF MS misiden- as AeromonasPRO dhakensis sp. nov. comb nov. and emendation of the 413 – 414 358 tification outputs for newly described species correspond with species Aeromonas hydrophila. Syst Appl Microbiol 36(3):171 6. doi:10.1016/j.syapm.2012.12.007 415 359 mutual positions in dendrogram (Fig. 1). Biotyper log(score) BenagliD C, Demarta A, Caminada A, Ziegler D, Petrini O, Tonolla M 416 360 of “false-positive” results and similarity of MALDI MS pro- E(2012) A rapid MALDI-TOF MS identification database at 417 361 files are summarized in Table 3. genospecies level for clinical and environmental Aeromonas strains. 418 362 PLoS ONE 7(10):e48441. doi:10.1371/journal.pone.0048441 419 In conclusion, MALDI-TOF MS in its current state is not a 420 363 Burke V,Robinson J, Gracey M, Peterson D, Partridge K (1984) Isolation reliable method for identification of Aeromonas isolates. of Aeromonas hydrophila from a metropolitan water supply: sea- 421 364 Several Aeromonas species, especially the newly described sonal correlation with clinical isolates. Appl Environ Microbiol 422 365 ones, are yielding unsatisfactory identification outputs with 48(2):361–366 423 366 Carnahan A, Altwegg M (1996) Taxonomy. In: Austin B, Altwegg M, 424 the use of Biotyper identification system and identification, 425 367 Gosling PJ, Joseph S (eds) The genus Aeromonas. Wiley, which may lead to misleading conclusions. On the other hand, Chichester, pp 1–38 426 368 the outputs of MALDI-TOF mass spectrometry were found to Carnahan MC, Joseph SW (2005) Order XII. ord. nov. 427 369 be in a very good agreement with alreadyORRECT known genetic and In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s 428 370 manual of systematic bacteriology, 2nd edn. vol. 2 (the 429 phenotypic similarities within AeromonasC spp., which proves 430 371 ), part B (the ). Springer, New that MALDI-TOF MS can be used as a rapid tool providing York, p 556 431 372 valuable taxonomic information. Chen PL, Ko WC, Wu CJ (2012) Complexity of β-lactamases among 432 UN clinical Aeromonas isolates and its clinical implications. J Microbiol 433 Immunol Infect 45(6):398–403. doi:10.1016/j.jmii.2012.08.008 434 373 Acknowledgments This work was supported by project CEITEC— Collins MD, Martínez-Murcia AJ, Cai J (1993) Aeromonas 435 374 Central European Institute of Technology (CZ.1.05/1.1.00/02.0068)— enteropelogenes and Aeromonas ichthiosmia are identical to 436 375 and CEB—Center of Experimental Biomedicine (CZ.1.07/2.3.00/ Aeromonas trota and Aeromonas veronii, respectively, as revealed 437 376 20.0183)—and funded by the European Regional Development Fund, by small-subunit rRNA sequence analysis. Int J Syst Bacteriol 43: 438 377 by the Ministry of Agriculture of the Czech Republic (QI101A094 and 855–856 439 378 MZE0002716202), and by Student Project Grant at Masaryk University Demarta A, Küpfer M, Riegel P, Harf-Monteil C, Tonolla M, Peduzzi R, 440 379 (specific research, rector’s program) MUNI/A/0884/2013. 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