Aeromonas Australiensis Sp. Nov., Isolated from Irrigation Water

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2270–2276 DOI 10.1099/ijs.0.040162-0

Aeromonas australiensis sp. nov., isolated from irrigation water

Max Aravena-Roma´n,1,2 Roxana Beaz-Hidalgo,3 Timothy J. J. Inglis,1,2 Thomas V. Riley,1,2 Antonio J. Martı´nez-Murcia,4 Barbara J. Chang1 and Maria Jose Figueras3

Correspondence 1School of Pathology and Laboratory Medicine, the University of Western Australia, Crawley, WA, Max Aravena-Romań Australia [email protected] 2Division of Microbiology and Infectious Diseases, PathWest Laboratory Medicine, Nedlands, WA, Maria Jose Figueras Australia [email protected] 3Unitat de Microbiologia, Department de Cie`nces Me`diques Ba´siques, Facultat de Medicina i Cie`nces de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain 4Departamento de Produccioń Vegetal y Microbiologıá, EPSO, Universidad Miguel Hernańdez, Orihuela 03312 (Alicante), Spain

A Gram-negative, facultatively anaerobic bacillus, designated strain 266T, was isolated from an irrigation water system in the south-west of Western Australia. Analysis of the 16S rRNA gene sequence confirmed that strain 266T belonged to the genus Aeromonas, with the nearest species being Aeromonas fluvialis (99.6 % similarity to the type strain, with 6 nucleotide differences) followed by Aeromonas veronii and Aeromonas allosaccharophila (both 99.5 %). Analysis of gyrB and rpoD sequences suggested that strain 266T formed a phylogenetic line independent of other species in the genus. This was confirmed using the concatenated sequences of six housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA and dnaX) that also indicated that A. veronii and A. allosaccharophila were the nearest relatives. DNA–DNA reassociation experiments and phenotypic analysis further supported the conclusion that strain 266T represents a novel species, for which the name Aeromonas australiensis sp. nov. is proposed, with type strain 266T (5CECT 8023T 5LMG 2670T).

The genus Aeromonas consists of Gram-negative, non- A. trota, A. allosaccharophila, A. encheleia, A. popoffii (Martin- spore-forming bacilli or coccobacilli, motile by polar Carnahan & Joseph, 2005), A. molluscorum (Minãna-Galbis flagella, that are oxidase- and catalase-positive, reduce et al., 2004), A. simiae (Harf-Monteil et al., 2004), A. bivalvium nitrate to nitrite and are usually resistant to the vibriostatic (Minãna-Galbis et al., 2007), A. aquariorum (Martıńez-Murcia agent O/129. The genus Aeromonas resides within the et al., 2008), A. tecta (Demarta et al., 2008), A. piscicola (Beaz- family Aeromonadaceae that belongs to the class Gamma- Hidalgo et al., 2009), A. fluvialis (Alperi et al., 2010a), A. proteobacteria (Abbott et al., 2003; Martin-Carnahan & taiwanensis and A. sanarellii (Alperi et al., 2010b), A. diversa Joseph, 2005). Aeromonas species are found globally in (formerly Aeromonas group 501) (Minãna-Galbis et al., 2010) water and soil environments and are often associated with and A. rivuli, isolated from a karst water rivulet in Germany fish and human disease (Janda & Abbott, 1998, 2010; (Figueras et al., 2011a). Figueras, 2005). At the time of writing, the genus includes 25 species with validly published names: Aeromonas The complex taxonomy of this genus has been attributed to hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, a lack of definitive biochemical markers and poor A. eucrenophila, A. sobria, A. veronii, A. jandaei, A. schubertii, congruence between genotypic and phenotypic methods (Soler et al., 2003; Figueras, 2005; Ørmen et al., 2005; Beaz- Abbreviation: MLPA, multilocus phylogenetic analysis. Hidalgo et al., 2009; Figueras et al., 2011b). In addition, the 16S rRNA gene sequences, which are classically considered The GenBank/EMBL/DDBJ accession numbers for the rpoD, gyrB, dnaX, gyrA, recA, dnaJ and 16S rRNA gene sequences of strain 266T are an important taxonomic tool, do not have the resolving respectively FN773335, FN691773, HE611951, HE611952, power to discriminate between closely related Aeromonas HE611953, HE611954 and HE611955. species. This is due to the high interspecies sequence Two supplementary tables and eight supplementary figures are available similarity of this gene, ranging from 96.7 to 100 % with the online version of this paper. (Martıńez-Murcia et al., 2007; Figueras et al., 2011b), and

Downloaded from www.microbiologyresearch.org by 2270 040162 G 2013 IUMS Printed in Great Britain IP: 54.70.40.11 On: Tue, 30 Oct 2018 06:55:21 Aeromonas australiensis sp. nov. the existence of sequence variation in copies of the gene tested in parallel under identical conditions in two (microheterogeneities) in some species (Morandi et al., laboratories (in Spain and in Australia). A total of 36 2005; Alperi et al., 2008; Roger et al., 2012a). Dependence phenotypic tests for the characterization of strain 266T on the 16S rRNA gene sequence to differentiate between were selected from those performed by Abbott et al. (2003) Aeromonas species has been superseded by the use of following the procedure described in Alperi et al. (2010a) single-copy genes that perform essential functions in with some exceptions, i.e. utilization of citrate, which was bacteria, e.g. rpoD, gyrB, dnaJ, recA and cpn60, which are determined by the method of Ha¨nninen (1994) and using known as housekeeping genes (Yań˜ez et al., 2003; Soler Simmon’s method (Cowan & Steel, 1993), oxidation of et al., 2004; Ku¨pfer et al., 2006; Nhung et al., 2007; Sepe potassium gluconate, production of lipase, urease, Jordan’s et al., 2008; Minãna-Galbis et al., 2009). In this sense, the tartrate, malonate, phenylalanine deaminase, nitrate reduc- rpoD and gyrB genes were employed for the first time in the tion (MacFaddin, 1976) and bacteriolytic activity (expres- definition of the species A. tecta and A. aquariorum sion of a stapholysin) (Satta et al., 1977). Acid production (Demarta et al., 2008; Martıńez-Murcia et al., 2008), while from carbohydrate was performed in broth (Excel) at a four concatenated housekeeping gene sequences were used final concentration of 1 % (w/v) of the desired sugar and in the description of A. piscicola and A. diversa (Beaz- 1 % (v/v) Andrade’s indicator as well as by the method Hidalgo et al., 2009; Minãna-Galbis et al., 2010). In the case described by Alperi et al. (2010a). The following carbohy- of A. piscicola, the four genes employed were rpoD, gyrB, drates were used: adonitol, amygdalin, L-arabinose, cellobiose, dnaJ and recA (Beaz-Hidalgo et al., 2009), while, in the case dulcitol, glucose, glucose 1-phosphate, glucose 6-phosphate, of A. diversa, recA was replaced by cpn60 (Minãna-Galbis glycerol, myo-inositol,lactose,lactulose,maltose,mannose, et al., 2010). According to the ad hoc committee for the re- D-mannitol, melibiose, methyl a-D-glucoside, raffinose, L- evaluation of the species definition in bacteriology, there is rhamnose, ribose, salicin, D-sorbitol, sucrose and trehalose. a current consensus that ‘an informative level of phylo- Additional carbohydrate fermentation was investigated with genetic data would be obtained from the determination of the API 20E and API CH50 systems (bioMe´rieux). a minimum of five genes under stabilizing selection for The ability to grow at different temperatures was assayed encoded metabolic functions (housekeeping genes) on TSA supplemented with sheep blood agar at 4, 25, 30, (Stackebrandt et al., 2002). A. fluvialis, A. taiwanensis, A. 35 and 44 uC. Acid production from carbohydrates, sanarellii and A. rivuli are the first species of Aeromonas hydrolysis of aesculin, urea and DNA and production of that included in their description the concatenated hydrogen sulfide from cysteine were evaluated for 7 days. sequences of five genes (rpoD, gyrB, dnaJ, recA and gyrA) Other tests were read as described by Abbott et al. (2003). (Alperi et al., 2010a, b; Figueras et al., 2011a). Recently, a Appropriate positive and negative controls were included. multilocus phylogenetic analysis (MLPA) of the complete T The inability of strain 266 to produce acid from D- genus with information derived from seven concatenated mannitol was a significant phenotypic marker, as the genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD) majority of Aeromonas species can produce acid from this demonstrated concordance with the species delineation carbohydrate, with the exception of A. schubertii, A. simiae based on DNA–DNA results (Martıńez-Murcia et al., and A. diversa and some strains of A. trota (Table 1 and 2011). Almost the same phylogenetic conclusions were Table S1, available in IJSEM Online). Key differentiating recently inferred by Roger et al. (2012b) using an MLPA, phenotypic features between these D-mannitol-negative also based on seven housekeeping genes (dnaK, gltA, gyrB, species and strain 266T are indicated in Table S1. Strain radA, rpoB, tsf and zipA), six of which were different from T 266 can be differentiated from A. schubertii by producing those employed by Martıńez-Murcia et al. (2011). indole from tryptophan and acid from sucrose; from A. The purpose of the present study was to use a polyphasic simiae by being haemolytic (strain 266T exhibited b- approach to characterize the taxonomic position of strain haemolysis, while A. simiae did not) and positive for 266T. The results revealed that this strain represents an Voges–Proskauer and indole reactions; from A. diversa by independent phylogenetic line within the genus Aeromonas. its ability to decarboxylate lysine and produce acid from sucrose and from A. trota by being positive for the Voges– Strain 266T was isolated by membrane filtration on an Proskauer reaction but negative for the utilization of citrate enriched lauryl sulfate agar (50 mm) plate while deter- (Tables 1 and S1). A small zone of inhibition (~10.8 mm in mining a total coliform count from a treated effluent used diameter) with a 150 mg disc containing the vibriostatic for irrigation at a sports ground in the south-west of agent O/129 was observed after overnight incubation on Western Australia. blood agar. This susceptibility to the vibriostatic agent is Cell size, morphology and the presence of flagella were uncommon in the genus and was previously reported for determined by electron microscopy following procedures two strains of A. eucrenophila and one of A. veronii by described previously (Collado et al., 2009). Abbott et al. (2003), and recently for the novel species ‘Aeromonas cavernicola’ (Martıńez-Murcia et al., 2013). Biochemical and physiological tests for the characterization of strain 266T were performed at 30 and 35 uC. All type The antimicrobial susceptibility of strain 266T was strains of species belonging to the genus Aeromonas were determined by the agar dilution method according to

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 2271 IP: 54.70.40.11 On: Tue, 30 Oct 2018 06:55:21 M. Aravena-Roma´n and others A. C. ) ) ) ) ) ) ) ) ) )

u standards of the CLSI (2011). Antimicrobial agents tested et al. 2 2 2 2 2 + 2 2 2 ( ( + ( ( ( ( ( ( ( ( , 2004; C); 26, included amikacin, amoxicillin, amoxicillin/clavulanate, u V , 2010b; 2 2 2 2 2 2 ND ND + ) ) ) ) ) ) ) ) ) ) aztreonam, cephalothin, cefazolin, cefepime, cefoxitin, et al. 2 2 2 2 2 + + + 2 + ( ( ( ( ( ( ( ( ( et al. ( ceftazidime, ceftriaxone, ciprofloxacin, colistin, gentami- V 2 2 2 2 2 2 + + + ) ) ) ) ) ) ) ) ) )

bv. veronii; 11, cin, meropenem, moxifloxacin, nalidixic acid, nitrofuran- + 2 2 + 2 2 + 2 + + , 2011a; 30 ( ( ( ( ( ( ( ( ( (

(Alperi toin, norfloxacin, pipercillin/tazobactam, tetracycline, 2 2 2 2 2 + + + + ND ana-Galbis et al. ˜ ) ) ) ) ) ) ticarcillin/clavulanate, tobramycin, trimethoprim and tri- ) ) ) ) + 2 2 + 2 2 + + + + ( ( ( ( ( ( ( ( ( ( A. veronii

(Min methoprim/sulfamethoxazole. Interpretative criteria were 2 2 2 2 + + + + + ND in accordance with the CLSI (2006). Strain 266T was A. fluvialis ) ) ) ) ) ) ) ) ) ) ; 10, 2 + + + + + + 2 + 2 ( (Figueras ( ( ( ( ( ( ( ( ( resistant to amoxicillin and cefazolin and was susceptible to , 2013). 2 2 ND + + + + + + + the other antimicrobial agents tested. ) ) ) ) ) ) ) ) ) ) C); 21, u 2 + + 2 + 2 2 2 2 + et al. ( ( ( ( ( ( ( ( ( ( A. rivuli A. jandaei 2 2 2 2 2 ND + + +

ND Cellular fatty acid methyl ester (FAME) analyses were A. molluscorum ) ) ) ) ) ) ) ) ) ) interpreted as described by Huys et al. (1994). In brief, + + + 2 2 + 2 + + + ( ( ( ( ( ( ( ( ( ( ; 17, V ] V V V 2 2 C); 25, 2 + + + isolates were inoculated onto trypticase soy agar (TSA) and , 2008; 30 u C ) ) ) ) ) ) ) ) u ) ) nez-Murcia u

´ incubated at 28 C for 48 h in air. Approximately 20 mg + + + + + + 2 + + 2 ( ( ( ( ( ( ( ( ( ( et al. bv. sobria; 9, 2 2 biomass of each strain was harvested and extracts were + + + + + + ND ND ) ) ) ) ) ) ) ) ) ) prepared using the standardized protocol of the Microbial + + + + + + + 2 2 2 ( ( ( ( ( ( ( ( ( ( , 2010b; 30 2 2 2 Identification System (MIDI; Microbial ID) and analysed + + + + + + + (2004) at 30 (Demarta ) A. veronii ) ) ) ) ) ) ) ) ) with a Hewlett Packard model HP6890 series GLC as et al. + + 2 + + + 2 2 2 2 ( ( ( ( ( ( ( ( ( ( ;8, , No data available. Results in parentheses were obtained in this study , 2010a, b; Martı V V 2 2 2 described by Osterhout et al. (1991). All cultures had + + + + ND et al. ND D ) ) ) ) ) ) ) ) ) A. tecta ) similar physiological age when they were harvested and 2 + 2 2 2 2 2 2 et al. (Alperi 2 + ( ( ( ( ( ( ( ( C. ( ( u

V cellular fatty acid analyses were performed in triplicate. 2 2 2 2 2 2 ND + A. sobria ND ) ) ) ) ) ) ) ) ) ) Identification of the fatty acids was performed by the C); 20, + 2 2 2 + + 2 + + + ;7, ( ( ( ( ( ( ( ( u ( ( V V 2 2 2 2 + + + + Sherlock MIDI software package (version 6). Table S2 , 16–84 % of strains positive. Data in columns 1–15 were obtained from Abbott ) ) ) ) ) ) V

) ) T ) ) A. sanarellii + 2 2 + 2 + shows cellular fatty acid profiles of strain 266 and the type 2 2 + + ( ( ( ( ( ( ( ( ( ( V V V V 2 2 2 + + + strains of species included in the genus Aeromonas. The ) ) ) ) ) ) , 2008; 25 ) ) ) )* T 2 2 + 2 2 2 2 + + + ( ( ( ( ( ( cellular fatty acid profile of strain 266 contained summed C); 24, ( ( ( ( u V V V 2 2 2 2 2 + 2 A. eucrenophila et al. feature 3 (28.7 %; C16 : 1v7c and/or C16 : 1v6c), summed ) ) ) ) ) ) ) ) ) ) 2 + 2 2 2 + + + + + ;6, ( ( ( ( ( ( ( ( ( ( feature 8 (11.4 %; C18 : 1v7c and/or C18 : 1v6c), C16 : 0 , which were tested at 25 bv. sobria ATCC 9071) performed in two laboratories (in Spain and in Australia) at 30 and 35 V V V data obtained by Harf-Monteil 2 2 2 2 + + + [ (11.3 %), C16 : 1v7c alcohol (7.20 %), C12 : 0 (6.0 %), D ) ) ) ) ) ) ) ) ) ) 2 2 2 2 2 species 2 + + + , 2010b; 30 ( ( ( ( ( + ( ( ( ( summed feature 2 (5.6 %; one of more of C aldehyde?, ( 12 : 0 V A. media V V V nez-Murcia 2 2 2 2 2 V ´ A. sobria iso-C I and C 3-OH), summed feature 9 (3.7 %; 10- ) ) ) ) ) ) ) 16 : 1 14 : 0 ) ) et al. ) A. veronii ;5, A. simiae + + + + + 2 2 + + 2 ( ( ( ( ( ( ( ( ( , 0–15 % of strains positive; ( methyl C or iso-C v9c), iso-C (3.5 %), C v8c

and 16 : 0 17 : 1 15 : 0 17 : 1 V (Martı 2 2 2 + + + + + + + ) ) ) ) ) ) ; 16, ) ) ) ) (3.3 %), iso-C17 : 0 (3.2 %), C14 : 0 (2.5 %) and C16 : 0 N + + + + + + 2 2 2 2 (Alperi ( ( ( ( ( ( ( ( ( ( Aeromonas

A. caviae alcohol (1.7 %). The similarity index (SI) obtained for 2 2 2 2 + + + + + + T ) ) ) ) ) ) ) ) ) ) strain 266 varied between 0.200 and ,0.300 and, in most ;4, + + + + + 2 2 2 A. popoffii + 2 ( ( ( ( ( ( ( ( is negative, as opposed to previous results (Alperi ( ( A. popoffii V 2 2 2 instances, named A. schubertii as a possible match. 2 + + + + + ) ) ) ) ) ) ) ) ) ) A. aquariorum According to the MIDI system documentation, SI values + + 2 + 2 2 2 + 2 + ( ( ( ( ( ( ( ( ( ; 15, from other A. taiwanensis V 2 2 2 2 2 T + + + of ,0.300 may represent an atypical strain of the species ) ) ) ) ) ) )v( ) ) ) + 2 2 + 2 2 C); 19, 2

+ + + named first in the chromatogram. This identification was ( ( ( ( ( ( A. encheleia ( ( ( ( u A. salmonicida V V V V 2 2 2 2 + +

C); 23, consistent with the fact that A. schubertii showed a similar u ) ) ) ) ) ) ) ) ) ;3, ) T + + 2 2 2 2 + + + + ( ( ( ( (

( biochemical profile to strain 266 . The FAME composi- ( ( ( ( , 85–100 % of strains positive; V V V V V 2 2 2 + + ) ) ) tions of the Aeromonas strains analysed in the present study ) + ) ) ) ) ) ) , 2007; 30 2 + + + 2 2 2 2 C with the exceptions of + + ( ( ( ( ( ( ( ( ( ( u differed significantly from previous reports (Lambert et al., A. allosaccharophila C). , 2009; 25 V V V 2 2 2 + + + + u et al. ) ) ) ) ) A. bestiarum ) ) ) 1983; Huys et al., 1994; Ka¨mpfer et al., 1994). Consistent ) ) 2 + + + 2 2 2 + + + ; 14, ( ( ( ( ( et al. ( ( ( ( ( with previous observations, variations in FAME data can be ;2, V V V V V 2 + + + + C. ) ) ) ) ) ) ) ) ) )

u attributed to differences in culture conditions, sets of + + 2 + 2 + + + + + ( ( ( ( ( ( ( ( ( ( , 2010; 30 V V V V V 2 2 ana-Galbis strains used and the type of media and equipment + + + ˜ ) ) ) ) ) ) ) ) ) ) et al. A. encheleia and the type strains of all species (and reference strain employed to analyse results (Huys et al. 1994). Our results + + + + + + + 2 + + ( ( ( ( ( ( ( ( ( ( (Min T T V V V + + + + A. hydrophila indicate that subtle differences exist between strain 266 (Beaz-Hidalgo ; 13, D 2 2 2 2 2 2 D ;1. ++ ++ ++ and other -mannitol-negative Aeromonas strains (Table + T

C but not at 35 S2) and that Aeromonas species belong to one of two u ana-Galbis ˜ A. trota clusters, those that produce alcohols (C16 : 1v7c alcohol and A. bivalvium A. piscicola Key tests for the differentiation of strain 266 (Min C N alcohol), and those that do not. However,

; 12, 16 : 0 identification of bacteria by analysis of their FAMEs is -Lactate -Mannitol -Arabinose C); 22, C); 18, Salicin Citrate L D DL u u -Haemolysis reaction more suitable for slowly growing bacteria such as non- Hydrolysis of aesculin Glucose (gas) Voges–Proskauer Acid from: Utilization of: Character A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 b Lysine decarboxylase Positive at 30 30 schubertii 25 A. diversa from tests for strain 266 (2003); these authors performed tests at 35 D Table 1. Taxa: A, strain 266 *Retested in this study, showing that the type strain of fermenters (Osterhout et al., 1991) and, in agreement with

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T the comments by Ka¨mpfer et al. (1994), fatty acid patterns 89 A. bestiarum CIP 7430 (X60406) 72 A. piscicola CECT 7443 T (HQ832417) show limited resolution to split Aeromonas species. 27 A. salmonicida NCIMB 1102T(X60405) 0.002 T T A. sobria NCIMB 12065 (X60412) Protein analysis of strain 266 was performed using a 45 A. rivuli DSM 22539T (FJ976900) A. molluscorum CECT 5864T (AY532691) Bruker microflex LT MALDI-TOF mass spectrometer 38 61 A. popoffii CECT 5176T (HQ832415) (Bruker Daltonic). Protein extraction was performed as A. bivalvium CECT 7113T (DQ504429) T described previously (Beaz-Hidalgo et al., 2009). Peaks in 50 A. encheleia CECT 4342 (HQ832414) T T 37 A. eucrenophila NCIMB 74 (X60411) the mass spectrum of strain 266 ranged from 2000 to 57 A. tecta CECT 7082T (HQ832416) 20 11 300 Da and differed from those of the closest related A. media ATCC 33907T (X60410) species, A. allosaccharophila, A. fluvialis and A. veronii (Fig. 98 A. hydrophila ATCC 7966T (X60404) 40 A. allosaccharophila CECT 4199T (S39232) T S1). 67 A. taiwanensis CECT 7403 (FJ230077) A. sanarellii CECT 7402T (FJ230076) Methods of DNA extraction and the conditions and 21 99 A. aquariorum CECT 7289T (EU085557) T primers for amplification of the 16S rRNA, gyrB, rpoD, 67 A. trota ATCC 49657 (X60415) 64 A. caviae NCIMB 13016T (X60408) recA, dnaJ, gyrA and dnaX genes were as described A. jandaei ATCC 49568T (X60413) previously (Martıńez-Murcia et al., 1992, 2011). Purified A. veronii ATCC 35624T (X60414) T PCR products were prepared for sequencing by using the 31 A. fluvialis CECT 7401 (FJ230078) 84 A. australiensis 266T (HE611955) BigDye Terminator version 1.1 cycle sequencing kit A. simiae MDC 54T (GQ860945) T (Applied Biosystems) and sequencing was performed with 97 A. schubertii ATCC 43700 (X60416) 76 T an ABI PRISM 310 and ABI 3130XL Genetic Analyzer A. diversa CECT 4254 (GQ365710) (Applied Biosystems). Using the CLUSTAL_X program, version 1.8 (Thompson et al., 1997), the sequences Fig. 1. Unrooted neighbour-joining phylogenetic tree derived from obtained were aligned independently with sequences of 16S rRNA gene sequences showing the relationships of strain 266T type and reference strains of all members of the genus with all other Aeromonas species. The phylogenetic tree was Aeromonas taken from our in-house database (Martıńez- constructed from an alignment of 1503 nt. Numbers at nodes Murcia et al., 2011) and some 16S rRNA gene sequences indicate bootstrap values. Bar, 0.002 estimated substitutions per retrieved from GenBank. Genetic distances and clustering site. were determined using Kimura’s two-parameter model (Kimura, 1980) and evolutionary trees were constructed by the neighbour-joining method (Saitou & Nei 1987) using T 81 A. veronii CECT 5761 the MEGA 5 program (Tamura et al., 2007). The stability of 89 A. australiensis 266 T 98 T relationships was assessed by the bootstrap method (1000 0.01 A. allosaccharophila CECT 4199 replications). 99 A. fluvialis CECT 7401T 69 A. sobria CECT 5254T Strain 266T (1503 bp) showed the highest 16S rRNA gene A. jandaei CECT 4228T T 59 A. hydrophila CECT 839 sequence similarity to the type strain of A. fluvialis (99.6 %) 100 A. aquariorum CECT 7289T followed by those of A. allosaccharophila and A. veronii, A. trota CECT 4255T both with a similarity of 99.5 %, these also being the closest A. taiwanensis CECT 7402T T 100 A. caviae CECT 838 neighbours in the phylogenetic tree (Fig. 1). The latter two T T 71 A. sanarellii CECT 7403 T species were also the most related to strain 266 on the 95 A. eucrenophila CECT 4224 basis of the MLPA constructed with the concatenated 93 A. tecta CECT 7082T 85 T sequences of six genes (gyrB, rpoD, recA, dnaJ, gyrA and A. encheleia CECT 4342 A. media CECT 4232T T dnaX; 3606 bp) from all the type strains (Fig. 2). Strain 100 A. molluscorum CECT 5864 T 266 showed the minimum interspecies divergence from A. 99 A. rivuli DSM 22539T veronii (3.2 %), which was higher than those obtained A. bivalvium CECT 7113T A. salmonicida CECT 894T between A. piscicola and A. bestiarum (approx. 2.1 %) or A. T 100 A. popoffii CECT 5176 T allosaccharophila and A. veronii (approx. 2.9 %) as reported 100 A. piscicola CECT 7443 by Martıńez-Murcia et al. (2011). As pointed out by 99 A. bestiarum CECT 4227T A. simiae CIP 107798T Ka¨mpfer & Glaeser (2012) and Martıńez-Murcia et al. T 100 A. diversa CECT 4254 (2011), a critical comparison of the different tree 100 A. schubertii CECT 4240T topologies based on single genes is important to determine genes that may be affected by lateral gene transfer or subsequent recombination events. The trees reconstructed Fig. 2. Unrooted neighbour-joining phylogenetic tree derived from from the six individual genes showed that, in all them, MLPA of concatenated sequences of six housekeeping genes strain 266T formed a clearly distinctive branch, but always (gyrB, rpoD, recA, dnaJ, gyrA and dnaX) sequences showing the T clustered near the species A. veronii, A. fluvialis and A. relationships of strain 266 with the type strains of all other allosaccharophila (Figs S2–S7). Aeromonas species. The phylogenetic tree was constructed from an alignment of 3606 nt. Numbers at nodes indicate bootstrap values. DNA–DNA hybridization studies were performed between Accession numbers for individual sequences are provided in Figs strain 266T and the type strains of A. veronii (CECT 4257T), S2–S7. Bar, 0.02 estimated substitutions per site.

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Table 2. DNA–DNA relatedness between strain 266T and related type strains

Results are expressed as means (SD) of three determinations. 2, Not determined.

Source of labelled DNA DNA–DNA hybridization (%) with strain:

1234

1. Strain 266T 100 56.8 (6.5) 64.8 (1.5) 65.0 (0.5) 2. A. fluvialis CECT 7401T 47.6 (6.5) 100 22 3. A. veronii CECT 4257T 62.6 (1.5) 2 100 2 4. A. allosaccharophila CECT 4199T 65.5 (0.5) 22100

A. allosaccharophila (CECT 4199T) and A. fluvialis (CECT reaction is positive when tested by disc (Rosco) but not in 7401T), as these were the phylogenetically closest species the API 20E strip. Does not utilize citrate (Simmon’s and both in the 16S rRNA gene sequence analysis and the Hanninen’s methods) or malonate or produce gas from MLPA. DNA extraction was performed using an Easy DNA glucose, but a positive citrate reaction is observed with the kit (Invitrogen) and DNA–DNA hybridization experiments API 20E strip. DL-Lactate is utilized at 30 uC but not at were conducted using the methods described by Ziemke 35 uC. Hydrogen sulfide, urease and elastase are not et al. (1998) and Urdiain et al. (2008). Reassociation was produced and it does not hydrolyse aesculin. No clearing performed under optimal conditions at 70 uC; single- and of tyrosine-containing medium, but starch hydrolysis is double-stranded DNA molecules were separated by the use positive after 5 days. Produces DNase and lipase and of hydroxyapatite and colour development was measured oxidizes potassium gluconate. Dehydrolyses arginine and at 405 nm using a Biotec Power safe XS2 microplate lysine is decarboxylated, but not ornithine. Utilizes acetate and urocanic acid and it is positive for the Voges– reader. Reported mean DNA–DNA relatedness values and Proskauer reaction and hydrolysis of gelatin. No bacteri- standard deviations were based on a minimum of three olytic activity (stapholysin) is detected. Negative for hybridizations for both direct and reciprocal reactions T phenylalanine deaminase, alkylsulfatase, pyrazinamidase (Table 2). DNA–DNA hybridization between strain 266 and Jordan’s tartrate. Acid is produced from the following and the type strains of A. allosaccharophila, A. veronii and carbohydrates: fructose, galactose, glucose, glycerol, gly- A. fluvialis was 65.3, 63.7 and 52.2 %, respectively, all below cogen, glucose 1-phosphate, glucose 6-phosphate, maltose, the 70 % limit for species delineation (Wayne et al., 1987; mannose, N-acetylglucosamine, ribose, sucrose and treha- Stackebrandt & Goebel, 1994). The MLPA once more lose, but not from adonitol, amygdalin, L-arabinose, showed perfect agreement with the DNA–DNA hybridiza- cellobiose, dulcitol, myo-inositol, lactose, lactulose, D- T tion results; both showed that strain 266 represents a mannitol, melibiose, methyl a-D-glucoside, raffinose, L- novel species within the genus Aeromonas (Fig. 2). These rhamnose, salicin or D-sorbitol. Acid production is results were further supported by phenotypic and chemo- observed for the following carbohydrates with the API T taxonomic analysis and confirmed that strain 266 50CH strip: glycerol, D-ribose, D-galactose, D-glucose, D- represents a novel species, for which the name Aeromonas fructose, D-mannose, N-acetylglucosamine, maltose, suc- australiensis sp. nov. is proposed. rose, trehalose, starch, glycogen and potassium gluconate. The type strain is 266T (5CECT 8023T 5LMG 26707T), Description of Aeromonas australiensis sp. nov. isolated from treated effluent in the south-west region of Aeromonas australiensis (aus.tra.li.en9sis. N.L. fem. adj. Western Australia. australiensis of or belonging to Australia). Motile rods with polar flagella (Fig. S8). Cells are Gram- Acknowledgements negative, straight, non-spore-forming and non-encapsu- We thank Dr J. P. Euze´by (E´ cole Nationale Ve´te´rinaire, France) for lated rods, 0.6–0.9 mm wide and 1.8–2.7 mm long, oxidase- etymological advice and Catalina Nu´ n˜ez for technical support. This and catalase-positive, reduce nitrate to nitrite and are work was supported in part by the Ministerio de Ciencia e Innovacio´n susceptible to the vibriostatic agent O/129 (150 mg). (Spain) (project reference AGL2011-30461-C02-02). Colonies on TSA plus sheep blood are 1.5–2.0 mm in diameter, glossy, circular and beige in colour after 24 h at 35 uC. No brown diffusible pigment is produced on TSA at References 35 uC. Growth occurs at 25, 30 and 35 uC, but not at 4 or Abbott, S. L., Cheung, W. K. W. & Janda, J. M. (2003). The genus 44 uC after 24 h on TSA plus sheep blood. b-Haemolysis is Aeromonas: biochemical characteristics, atypical reactions, and observed on sheep (5 %) blood agar. Grows on MacConkey phenotypic identification schemes. J Clin Microbiol 41, 2348–2357. (Difco) and thiosulfate-citrate-bile-sucrose agar (Difco) Alperi, A., Figueras, M. J., Inza, I. & Martı´nez-Murcia, A. J. (2008). and in nutrient broth in 0 and 3 % NaCl, but not at 6 % Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains NaCl. Indole is produced from tryptophan. The ONPG and their impact in species delineation. Int Microbiol 11, 185–194.

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