‘Aeromonas intestinalis’ and ‘Aeromonas enterica’ isolated from human faeces, ‘Aeromonas crassostreae’ from oyster and ‘Aeromonas aquatilis’ isolated from lake water represent novel species Academic research paper on "Biological sciences"

NEW SPECIES

'Aeromonas intestinalis' and 'Aeromonas enterica' isolated from human faeces, 'Aeromonas crassostreae' from oyster and 'Aeromonas aquatilis' isolated from lake water represent novel species

M. J. Figueras1, F. Latif-Eugenín , F. Ballester2, I. Pujol2, D. Tena3, K. Berg4, M. J. Hossain5, R. Beaz-Hidalgo and M. R. Liles5

1) Unidad de Microbiología, Facultad de Medicina y Ciencias de la Salud, IISPV, Universidad Rovira i Virgili, 2) Laboratorio de Referencia de Cataluña Sud. Hospital Universitario St. Joan de Reus, Reus, Spain, 3) Section of Microbiology, University Hospital of Guadalajara, Guadalajara, Spain, 4) Department of Food and Environmental Sciences, University of Helsinki, Finland and 5) Department of Biological Sciences, Auburn University, Auburn, Alabama, USA

Abstract

Four Aeromonas strains from clinical and environmental samples differed from known species on the basis of rpoD gene sequence. Multilocus phylogenetic analysis and in silico DNA-DNA hybridization confirmed them as four new species even though their I6S rRNA gene sequence similarity with their closest relatives was >98.7%, as occurred for other Aeromonas spp.

©2016 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Keywords: Aeromonas, faeces, new species, oysters, taxonomy, water

Original Submission: 8 October 2016; Revised Submission: 15 November 2016; Accepted: 18 November 2016 Article published online: 24 November 2016

Corresponding author: M. J. Figueras, Unidad de Microbiología, Facultad de Medicina y Ciencias de la Salud, IISPV, Universidad Rovira i Virgili, Sant Lloren^ 21, 43201 Reus, Spain E-mail: nariajose.figueras@urv.cat

The genus Aeromonas belongs to the Family Aeromonadaceae and includes oxidase-positive, facultatively anaerobic, Gramnegative bacilli [1,2]. To date, 32 species are recognized, and I9 so far have been implicated in human diarrhoea, bacteraemia or wound infections, and are considered human opportunistic pathogens [2-5]. New data have demonstrated that Aeromonas spp. are true enteropathogens [5].

Recently a group of four Aeromonas strains could not be assigned to any species. Two of them, II78CTand II3634T, were recovered from patients with diarrhoea from two Spanish hospitals; stool samples were collected in plastic containers. Strain II78CT was isolated, in the absence of other microbes, from the diarrhoeic faeces of a 45-year-old man with vomiting and fever over 2 months. Strain I 13634T was recovered from a 32-year-old woman with abdominal pain lasting several days. One (AOSE3-I4AT) of the other two strains was isolated from Crassostrea gigas harvested from Alfacs Bay (River Ebro Delta, Spain), and the other (AE207T) was isolated from Lake Pyhajarvi water (Finland).

The phylogenetic tree constructed with the rpoD gene, which is a gene we have described earlier as a first-line tool for the identification of Aeromonas spp. [2,6], indicated that the four strains formed independent branches from the rest of species, and this was confirmed with the tree constructed with the concatenated sequences of six housekeeping genes (rpoD, gyrB, gyrA, recA, dnaJ and dnaX, 3558 bp) (Fig. 1), all performed as previously described [3]. The interspecies I6S rRNA gene sequence (1367 bp) similarity of the four strains with the types of their closest Aeromonas species was >98.7% (Supplementary Fig. SI). This is in agreement with the high values of >98.7 to I00% observed among several accepted Aeromonas species [2,3,6,7]. The closest species to clinical strain II78CT was A. jandaei (98.85% similarity), while for strain II3634T they were A. salmonicida (99.86%), A. bestiarum (99.7I%) and A. piscícola (99.7I%). Strain AOSE3-I4AT grouped with A. encheleia (99.93% similarity) and AE207T with A. tecta (99.50% similarity) (Supplementary Fig. SI). The genomes ofthe four new species were sequenced, and the in silico DNA-DNA hybridization (isDDH) values with the genomes of the type strains of the closest species was calculated as described elsewhere [3]. The four strains showed isDDH values of <70%, which confirmed them as four new species (Fig. I and Supplementary Fig. SI).

New Microbe and New Infect 2017; 15: 74-76

© 2016 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) http://dx.doi.org/ I0.I0I6/j.nmni.20l6.l 1.019

Figueras et al. New Aeromonas species 75

— A. veronii CECT 4257T -A. aquatilis AE207T

-A. australiensis CECT 8023T

-A. allosaccharophila CECT 4199T

A. finlandiensis CECT 8028T

-A. sobria CECT 4245T

A. fluvialis CECT 7401T A. jandaei CECT 4228T

— A. lacus CECT 8024T

-A. cavernicola DSM 24474T

A. salmonicida CECT 894T

-A. popoffii CECT 5176

~ A. enterica 113634T

-A. piscícola CECT 7443T A. bestiarum CECT 4227T -A. trota CECT 4255T

A. hydrophila CECT 839T -A. dhakensis CECT 5744T

-A. bivalvium CECT 7113T

-A. crassostreae AOSE3-14A1

-A. molluscorum CECT 5864T

-A. rivuli CECT 7518T

-A. simiae IBS-S6874T

-A. diversa CECT 4254T

-A. schubertii CECT 4240T

— A. caviae CECT 838T A. sanarellii CECT 7402T A. taiwanensis CECT 7403T -A. media CECT 4232T -A. rivipollensis DSM24593T A. intestinalis 1178C1

-A. lusitana CECT 7828T

-A. tecta CECT 7082T

A. eucrenophila CECT 4224T

-A. encheleia CECT 4342T

-A. aquatica CECT 8025T

FIG. 1. Neighbour-joining phylogenetic tree obtained with concatenated sequences of six housekeeping genes (rpoD, gyrB, gyrA, recA, dnaJ and dnaX,

3558 bp) showing position of four new Aeromonas spp. (bold) relative to 32 Aeromonas spp. Numbers at nodes represent bootstrap percentages

obtained by repeating analysis 1000 times. Scale bar = 0.01 estimated substitution per site. In silico DNA-DNA hybridization (isDDH) represents

genetic similarity obtained between genome of each of four new species and genomes of their closest neighbour species.

76 New Microbes and New Infections, Volume 15 Number C, January 2017

The clinical strains II78CT and 113634т were initially identified with MicroScan W/A and Vitek II, respectively. In both cases, the identifications obtained were erroneous and masked these new species under the species A. hydrophila and A. sobria, respectively.

More than one phenotypical characteristic distinguished the four new species from their closest relatives. The most important were the nonacid production from D-sucrose but production from lactose by strain II78CT, the production of acid from L-fucose by II3634T, the use DL-lactate by AOSE3-I4AT and the mannitol-negative reaction of strain AE207T that also showed to be susceptible to the vibriostatic agent O/I29.

The names proposed are as follows: 'Aeromonas intestinalis' (in.tes.ti.na'lis, N.L. fem. adj. intestinalis, 'pertaining to the intestine'), 'Aeromonas enterica' (en.te'ri.ca, Gr. n. enteron, 'gut, bowel, intestine'; L. fem. suff. -ica, suffix used with the sense 'pertaining to'; N.L. fem. adj. enterica, 'pertaining to intestine'), 'Aeromonas crassostreae' (crass.os'tre.ae, N.L. gen. n. crassos-treae, 'of the oyster genus Crassostrea') and 'Aeromonas aquatilis' (a.qua'ti.lis, L. fem. adj. aquatilis 'aquatic, growing in water').

Nucleotide sequence accession number

The GenBank/European Molecular Biology Laboratory/DNA Data Bank of Japan accession numbers of the I6S rRNA gene sequences of strains II78CT (= CECT 8980T = LMG 29048T), II3634T (= CECT 8981T = LMG 29049T), AOSE3-I4AT (= CECT 8982T = LMG 29050T) and AE207T (= CECT 8026T = LMG 267I4T) are LT630759, LT630760, LT63076I and LT630765, respectively. The rpoD, gyrB, gyrA, recA, dnaJ and dnaX of the other strains of the four novel species have also been deposited under the accession numbers LT6307I0-LT6307I2 and LT6307I6, LT6307I7-LT6307I9 and LY630723, LT630724-LT630726 and LT630730, LT63073I-LT630733 and LT630737, LT630738-LT630740 and LT630744, LT630745 - LT630747 and LT63075I, respectively.

Deposit in a culture collection

All the type strains have been deposited in the Culture Collections of Spain (CECT) and Belgium (LMG): 'A. intestinalis' II78CT (= CECT 8980T = LMG 29048T), 'A. enterica' II3634T

(= CECT 898IT = LMG 29048T), 'A. aquatilis' AE207T (= CECT 8026T = LMG 267I4T) and 'A. crassostreae' AOSE3-I4AT (= CECT 8982T = LMG 29050T).

Acknowledgements

This study was supported by the projects JPIW20I3-095-C03-03 of MINECO (Spain) and AQUAVALENS of the Seventh Framework Program (FP7/2007-20I3) grant agreement 311846 from the European Union. We thank A. Oren, Hebrew University of Jerusalem, for supervising and correcting etymology of the species names.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/I0.I0I6/j.nmni.20I6.II.0I9.

Conflict of Interest

None declared.

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