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1 Institute of Bacteriology UPRES-EA 3432, Louis Pasteur University, Strasbourg University Hospital, 3 rue Koeberlé, F-67000 Strasbourg, France
2 Unité de Biodiversité des Bactéries Pathogènes Emergentes, U389 INSERM, Institut Pasteur, 28 rue du Docteur Roux, F-75724 Paris, France
Correspondence
Colette Harf-Monteil
colette.harf-monteil{at}medecine.u-strasbg.fr
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Published online ahead of print on 3 October 2003 as DOI 10.1099/ijs.0.02786-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IBS S6874T is AJ536821.
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, 2002; Pidiyar et al., 2002; Esteve et al., 2003).
Members of the genus Aeromonas are widespread in environmental habitats, such as soil (Brandi et al., 1996), fresh and brackish water, sewage and wastewater (Holmes et al., 1996). They are implicated as pathogens of cold-blooded animals and various mammals, including humans, where they cause severe gastroenteritis, soft-tissue infections and bacteraemia (Janda & Abbott, 1998). Aeromonas isolates have been recovered from faeces in acute cases of gastroenteritis, as well as in asymptomatic cases (Hänninen & Siitonen, 1995). During a routine survey to determine the presence of possible pathogens in the intestinal tract of monkeys (Macaca fascicularis) from Mauritius that had been put in quarantine in the Centre for Primatology, Louis Pasteur University, Strasbourg, France, two phenotypically related strains, which were identified tentatively as Aeromonas sp., were isolated. These strains could not be identified as members of any previously described Aeromonas species. In the current investigation, phylogenetic analysis of 16S rRNA gene sequences, DNA–DNA hybridization and extensive phenotypic tests were undertaken to determine the taxonomic position of the two monkey isolates. Based on the reported data, we describe a novel species of the genus Aeromonas, for which the name Aeromonas simiae sp. nov. is proposed.
Two strains (Institute of Bacteriology of Strasbourg strain IBS S6874T=CIP 107798T=CCUG 47378T and IBS S6652=CIP 107797) were each isolated from a different sample of faeces from two healthy monkeys (M. fascicularis) from Mauritius that were kept in quarantine in the Centre for Primatology, Louis Pasteur University, Strasbourg, France, and were sampled routinely in September 1999. Reference strains of other Aeromonas species were obtained from the Collection de l'Institut Pasteur (CIP), Paris, France.
Strains were cultivated aerobically on sheep-blood agar [trypticase soy agar (bioMérieux) that was supplemented with 5 % defibrinated sheep blood] and on Mueller–Hinton agar (Bio-Rad) plates at 30 °C unless indicated otherwise.
Cell shape, Gram-stain and biochemical activities were determined with cultures grown overnight on Mueller–Hinton medium (Smibert & Krieg, 1981). Kovacs cytochrome oxidase was tested by using commercially available discs that were impregnated with dimethylparaphenylene diamine oxalate (Bio-Rad). D-Glucose fermentation by the method of Hugh & Leifson (1953), salt tolerance and resistance to vibriostatic agent O/129 (150 µg) were determined by methods described by Lee & Donovan (1985). Staining of flagella was performed according to the Rhodes method (Buttiaux et al., 1966).
Together with three reference strains of Aeromonas (Aeromonas veronii CIP 103438T, Aeromonas schubertii CIP 103437T and Aeromonas caviae CIP 76.16T), they were tested for physiological and biochemical properties according to the identification scheme described by Abbott et al. (1992). The following biochemical tests were performed by using previously described methods and incubation was carried out at 30 °C for 48 h: indole production by using peptone water [1 % (w/v) peptone supplemented with 0·5 % tryptophan] (Smibert & Krieg, 1981); gas production from D-glucose (Lee et al., 1981); lysine and ornithine decarboxylase and arginine dihydrolase activities were determined in Fay–Barry medium as described by Altwegg et al. (1987). Acetoin production (Voges–Proskauer test) and aesculin hydrolysis were performed as described by Lee & Donovan (1985). Fermentation of carbohydrates (acid production) was determined in nutrient broth (Difco) that was supplemented with 0·002 % phenol red and one of the following substrates: lactose, D-mannitol, sucrose, L-arabinose, salicin or D-cellobiose. Yellow colour of the liquid medium was considered to be a positive reaction. Haemolysis of sheep blood was determined on trypticase soy agar supplemented wih 5 % defibrinated sheep blood. Pyrazinamidase activity was determined by using pyrazinamidase diagnostic tablets (Eurobio Rosco) according to the manufacurer's instructions. For additional biochemical tests, API 50CH strips (bioMérieux) were used according to the manufacturer's instructions. Growth at 37 and 42 °C was tested in nutrient broth (Difco); cultures were incubated for 24 h. Susceptibility to cephalothin and ampicillin was tested by the Bauer–Kirby disc diffusion method (Bauer et al., 1966).
For small-subunit rDNA sequencing and phylogenetic study, strains were cultured on Columbia agar (Difco) at 37 °C. DNA was extracted and purified with an IsoQuick Nucleic Acid Extraction kit (ORCA Research). Using universal primers, 1·5 kb of the 16S rRNA gene was amplified by PCR: the primers used were A, corresponding to positions 8–28 of the Escherichia coli 16S rRNA gene (Edwards et al., 1989), and rJ, complementary to positions 1510–1492 (Janvier & Grimont, 1995).
The amplification reaction was carried out in a final volume of 100 µl, with 1 µl template, 20 pmol each primer ml-1, 200 µM each dNTP, 50 µM MgCl2, 2·5 U Taq polymerase and 10 µl 10x reaction buffer (Amersham Biosciences). Initial denaturation was carried out for 4 min at 94 °C. Amplification (35 cycles) was performed in a DNA Thermal Cycler 9700 (PE Applied Biosystems). Each cycle consisted of three steps: denaturation at 94 °C for 1 min, annealing at 49 °C for 1 min and extension at 72 °C for 2 min. A final extension was performed at 72 °C for 5 min.
PCR products were detected by electrophoresis on 0·8 % agarose gels in Tris/borate/EDTA buffer (0·089 M Tris/borate, 0·018 M EDTA) with 1 kb DNA ladder (Gibco-BRL) as the molecular size marker.
The amplified product was sequenced by Genome Express (Meylan, France) with three primers, E, rE and D, which are located in conserved regions of the E. coli 16S rRNA gene. Primer E corresponded to positions 787–806, primer rE was complementary to primer E and primer D corresponded to positions 519–536 [numbering according to Brosius et al. (1978)].
Sequences of strains IBS S6874T (GenBank accession no. AJ536821) and IBS S6652 (AJ536820) were compared and aligned with those of other strains, which were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) by using BLAST (ncbi) and the megalign module of the lasergene software (DNASTAR). A phylogenetic tree was generated by using the neighbour-joining algorithm (Saitou & Nei, 1987).
DNA–DNA hydridization was performed as described previously (Riegel et al., 1994). Briefly, DNA was extracted from SDS-treated cells of strains IBS S6874T, IBS S6652, A. schubertii CIP 103437T and Aeromonas trota CIP 103677T by using a phenol/chloroform-based procedure. DNA was quantified spectrophotometrically and the purity of each preparation was ascertained by measuring the A280/A260 and A230/A260 ratios. Hybridization between labelled DNA and the fragmented DNA preparation was carried out at 60 °C for 16 h in 0·42 M NaCl by the nuclease/trichloroacetic acid method (Grimont et al., 1980). Values of DNA–DNA relatedness that are given are means of the results of three independent experiments.
Isolates IBS S6874T and IBS S6652, which are studied herein, were recovered on blood agar plates from different faeces samples from two healthy monkeys. These two isolates were identified as members of the genus Aeromonas, as they were Gram-negative rods with polar flagella that were oxidase-positive, glucose-fermenting, facultatively anaerobic, unable to grow in nutrient broth with 6 % NaCl and resistant to vibriostatic agent O/129 (150 µg). The biochemical patterns of these two strains were nearly identical and did not permit their assignment to any recognized Aeromonas species. In addition, there was 89 % DNA–DNA hybridization between isolates IBS S6874T and IBS S6652, indicating that these two strains should be assigned to the same genomospecies.
In order to determine the taxonomic position of these two strains, which differ from recognized Aeromonas species, we studied them and reference strains by performing DNA–DNA hybridization analysis and small-subunit rDNA sequence analysis. The almost-complete sequence of strain IBS S6874T (1452 bp), and a large part of the sequence of strain IBS S6652 (1239 bp), were determined. Phylogenetic analyses confirmed that the two strains belonged to the robust monophyletic unit that groups all available Aeromonas sequences together (Fig. 1). Comparison of 16S rDNA sequences revealed that those from the two strains exhibited a high level of similarity to each other (0·1 % nucleotide substitution), but that they represented an unknown phylogenetic lineage within the genus Aeromonas. The lowest level of nucleotide substitution between strain IBS S6874T and a currently recognized Aeromonas species was 1·7 % (A. schubertii); nucleotide substitution with other representatives of the genus Aeromonas (listed in Fig. 1) ranged from 2·0 to 3·5 %. It is noteworthy that these values are above the nucleotide substitution values that are usually obtained between recognized species of the genus Aeromonas (Martínez-Murcia et al., 1992a, b; Pidiyar et al., 2002). According to the limitations of 16S rDNA analysis in the determination of novel taxa (Martínez-Murcia, 1999), DNA–DNA hybridization was performed between strain IBS S6874T and reference strains A. trota CIP 103677T and A. schubertii CIP 103437T, yielding values of 18 and 16 %, respectively. Thus, DNA–DNA hybridization clearly segregated strain IBS S6874T from A. trota and A. schubertii, which were closely related taxa on the basis of 16S rDNA sequence comparison analysis. Therefore, according to the level of phylogenetic diversity within the genus Aeromonas and following the criteria proposed by Wayne et al. (1987) and Stackebrandt & Goebel (1994), our 16S rDNA sequence and DNA–DNA hybridization data clearly show that the two monkey isolates form a novel genomic species within the genus Aeromonas.
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shows selected biochemical characters for phenotypic differentiation from other Aeromonas species. Thus, the two isolates differ from most previously described mesophilic Aeromonas species, except for A. schubertii, by their inability to produce indole, gas from glucose and acid from D-mannitol. The monkey isolates differ from A. schubertii by their ability to produce acid from D-cellobiose and D-sucrose and by their pyrazinamidase activity. The two isolates could be differentiated from one another by some phenotypic features: according to the data obtained by using API 50CH strips, strain IBS S6874T produced acid from arbutin, but did not hydrolyse aesculin after 48 h incubation, in contrast to strain IBS S6652.
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Description of Aeromonas simiae sp. nov.
Aeromonas simiae (si'mi.ae. Gr. n. simia monkey; N.L. adj. simiae from monkeys).
Strains display the following characteristics that are typical of the genus Aeromonas: Gram-negative, straight, motile rods with a polar flagellum, cytochrome oxidase-positive, chemoorganotrophic with both oxidative and fermentative metabolisms, catalase-positive, reduce nitrate to nitrite without production of gas and resistant to vibriostatic agent O/129 (150 µg). Unable to grow in nutrient broth with 6 % NaCl. Optimal growth occurs at 30 °C after 24 h on trypticase soy agar or sheep-blood agar, but strains also grow at 42 °C after 24 h. Unable to produce indole, gas from glucose or 
-haemolysis on sheep-blood agar. Voges–Proskauer- and ornithine decarboxylase-negative; lysine decarboxylase-, arginine dihydrolase- and pyrazinamidase-positive. Hydrolysis of aesculin and acid production from arbutin are variable. By fermentation, acid is produced from glycerol, ribose, galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, D-cellobiose, D-sucrose, D-maltose, D-trehalose, starch, glycogen and gluconate, but not from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, melibiose, inulin, melezitose, raffinose, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, 2-ketogluconate, 5-ketogluconate, salicin or lactose. Major characteristics that differentiate the species from other Aeromonas species are shown in Table 1
. Susceptible to cephalothin, but resistant to ampicillin.
The type strain, IBS S6874T (=CIP 107798T=CCUG 47378T), was isolated from the faeces of a monkey (Macaca fascicularis) that was kept in the Centre for Primatology, Louis Pasteur University, Strasbourg, France. This strain has all of the above-described properties for the species; in addition, aesculin is not hydrolysed after 48 h incubation and acid is produced from arbutin.
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ACKNOWLEDGEMENTS |
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