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 Aeromonas 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 bacteria 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 taxonomy 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 Folia Microbiol 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 Folia Microbiol Q2 t1:1 Table 1 Aeromonas spp.