Proposal to Recognize the Family Aeromonadaceae Fam. Nov. R

Home , Aeromonadaceae, Aeromonas

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1986, p. 473-477 Vol. 36, No. 3 0020-77 13/86/030473-05$02.00/0 Copyright 0 1986, International Union of Microbiological Societies

Proposal to Recognize the Family Aeromonadaceae fam. nov. R. R. COLWELL,l* M. T. MACDONELL,'? AND J. DE LEY2 Department of Microbiology, The University of Maryland, College Park, Maryland 20742l and Laboratorium voor Microbiologie en Microbiele Genetica, Faculteit der Wetenschappen, Rijksuniversiteit, B-9000 Gent, Belgium2

The genus Aeromonas is currently allocated, primarily on the basis of phenotypic expression, to the eubacterial family Vibrionaceae. However, a sizeable collection of molecular genetic evidence, including 16s ribosomal ribonucleic acid catalog, 5s ribosomal ribonucleic acid sequence, and ribosomal ribonucleic acid-deoxyribonucleic acid hybridization data, indicates that Aeromonas spp. possess an evolutionary history sufficiently different from that of the Vibrionaceae to warrant removal from this family. The phylogenetic locus (i.e., intermediate between the families Enferobacferiuceae and Vibrionaceae) and phylogenetic depth of the genus Aeromonas suggest that Aeromonas spp. represent a discrete family of eubacteria, for which the name Aeromonadaceae fam. nov. is proposed.

Kluyver and van Niel(10) proposed the genus Aeromonas Substantial molecular genetic evidence now exists which to accommodate enteric bacterium-like microorganisms suggests that species currently assigned to the genus Aero- which were associated with freshwater aquatic environ- monas (e.g., A. hydrophila, A. salmonicida, A. sobria, A. ments and were motile by means of polar flagella (14). The caviae, and A. media) possess a phylogenetic history suffi- partitioning of this group of bacteria was intended to portray ciently distinct from the histories of the families Enterobac- phylogenetic relationships. The identification of species teriaceae and Vibrionaceae to warrant exclusion of the comprising the genus Aeromonas has varied significantly genus Aeromonas from the Vibrionaceae (Table 1) and since the creation of the genus in 1936. As a consequence, elevation to the level of family. Support for such a change in the taxonomic history of the genus Aeromonas has been one the taxonomy of Aeromonas can be found in the results of of confusion and controversy (7, 8, 13-22), involving not deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid only definition of the species assigned to the genus but also (rRNA) competition studies (2); such data for species of the the taxonomic locus of the genus with respect to the families genera Vibrio (Beneckea) and Aeromonas and the family Enterobacteriaceae and Vibrionaceae as they are presently Enterobacteriaceae support our conclusion that Vibrio spp., defined (2,lO). A major problem faced in defining the precise Aeromonas spp. and enteric species share the relationships taxonomic position of Aeromonas hydrophila and related shown in Fig. 1, which is based on rRNA-DNA hybridiza-

TABLE 1. Family Vibrionaceae"

~ Genus Species composition Reference(s) Vibrio V. alginolyticus, V. campbellii, V. carchariae, V. cholerae, V. costicola, V. diazotrophicus, 3, 13 V.fluvialis, V.furnessii, V. gazogenes, V. harveyi, V. rnetschnikovii, V. natriegens, V. nereis, V. nigrapulchritudo, V.parahaemolyticus, V. orientalis, V. proteolyticus, V. splendidus, V. vulnificus Photobacterium P. angusturn, P. fischeri, P. leiognathi, P. logei, P. phosphoreurn 13 Listonella L. anguillarum, L. damsela, L. pelagica 13 Shewanella S. benthica, S. hanedai, S. putrifaciens 13 Plesiomonasb P. shigelloides Aeromonasc A. caviae, A. hydrophila, A. media, A. salmonicida, A. sobria 3, 13

a The following is a general description of the family Vibrionaceae: gram-negative straight or curved rods; motile by polar flagella; do not form endospores or microcysts; chemoorganotropic and facultatively anaerobic; capable of respiratory and fermentative metabolism; oxygen is a universal electron acceptor; do not denitrify; most are oxidase positive; all utilize D-glucose as a sole source of carbon and energy; most require 2 to 3% NaCl or seawater for optimum growth; primarily associated with aquatic habitats; several species are pathogenic to humans, fish, eels, and frogs; DNA base composition, 38 to 63 mol% guanine plus cytosine. Removal from the family Vibrionaceae has been recommended (13). We recommend elevation to the family level.

species sharing a similar evolutionary history is that these tion data (2). Figure 1 shows that the genus Aeromonas can species possess phenotypic properties defined, a priori, to be be considered to be more closely related to enteric species characteristic of each of two distinct eubacterial families, the than to the Vibrionaceae. Essentially the same intergeneric Vibrionaceae and the Enterobacteriaceae. pattern observed for DNA-rRNA competition experiments was obtained by using the results of 16s rRNA cataloging (23) (Fig. 2A), as well as through a comparative analysis of * Corresponding author. 5s rRNA sequences (11-13) (Fig. 2B). In fact, the primary t Present address: Center of Marine Biotechnology, The Univer- structure of 5s rRNAs prepared from Aeromonas spp. is sity of Maryland, College Park, MD 20742. sufficiently different from the structure of 5s rRNAs from

473 474 NOTES INT. J. SYST. BACTERIOL.

60 70 ao 90 100

Photobacterium sm.

1Vibrio sp~. Pasteurella wltocida

Aeromonas spp.

Interobacteriaceae

. 60 70 80 90 100

% rRNA-DNA BINDING FIG. 1. Dendrogram showing relationships among species of the families Enterobacteriaceae and Vibrionaceae and the genus Aeromonas. The relationships shown are based on DNA-rRNA competition data (2). species of the families Enterobacteriaceae and Vibrionaceae Furthermore, studies in which comparisons were made that minor differences in secondary structures are implied among the immunologic distances of glutamine synthetases (11). Secondary structure differences alone corroborate sig- and superoxide dismutases for species of the genera Vibrio, nificant separation in the evolutionary histories of Aeromo- Aeromonas, and Photobacterium and species of the Entero- nus spp. and both the Vibrionaceae and the Enterobacteri- bacteriaceae indicated that Aeromonas spp. are approxi- aceae. The determination of rRNA cistron similarities mately equidistant from both Vibrio and enteric species (1). among numerous named strains and type strains by DNA- On a basis of the information described above, we suggest rRNA hybridization revealed the existence of at least five that sufficient evidence exists to justify the conclusion that large rRNA superfamilies sensu De Ley (4) among the the species of the genus Aeromonas represent a phylogeneti- gram-negative bacteria (4-6, 25). Figure 3 shows the relat- cally distinct family of eubacteria. Therefore, we propose edness of the main large groups in superfamily I. All strains creation of the family Aeromonadaceae for these and similar of Aeromonas constitute a branch which is approximately species, including new species to be described elsewhere equidistant from the Enterobacteriaceae and the Vibrio- (J. D. Deming, M. T. MacDonell, W. S. Straube, and R. R. naceae. These data demonstrate that the present genus Colwell, manuscript in preparation). Aeromonas deserves to be elevated to family level. Description of Aeromonadaceae fam. nov. Aeromona- Results of DNA-DNA hybridization among strains of the duceae (Ae .ro .mo .na. da’ce.ae. M. L. fem. n. Aeromonas genera Aeromonas and Vibrio (24) indicate a very low level type genus of family; suffix -aceae to denote family; M.L. of relatedness between Aeromonas and Vibrio species, be- fem. pl. n. Aeromonadaceae Aeromonas family). Gram- tween which the average relative binding ratio is no greater negative straight or curved rods. Motile by means of polar than that observed between such distantly related strains as flagella. Do not form endospores or microcysts. Chemo- Pseudomonas aeruginosa and Vibrio spp. (i.e.,

FIG. 2. Evolutionary trees indicating relationships among species of the families Enterobacteriaceae and Vibrionaceae and the genus Aeromonas, based on rRNA homologies. (A) Phylogenetic relationships among procaryotic taxa based on 16s rRNA catalogs. Adapted from reference 23. Note the relationships among the enteric bacteria, vibrios, and aeromonads, suggesting that Aeromonas spp. share a more recent common ancestor with species of the Enterobacteriaceae than with species of the Vibrionaceae. Abbreviations: Aq., Aquaspirillum; P., Pseudomonas; R., Rhodospirillum; X., Xanthomonas; S-dep, sulfur-dependent group. (B) Evolutionary tree depicting phylogenetic relationships among the enteric bacteria, vibrios, photobacteria, and aeromonads, based on 5s rRNA sequence analyses. The tree was derived from evolutionary distance coefficients (9) by using the difference matrix clustering program KITSCH (PHYLIP phylogeny inference package; J. Felsenstein, University of Washington). For a detailed discussion of the taxonomy of the family Vibrionaceae as indicated by comparative sequence analysis of 5s rRNAs, see reference 13. Abbreviations: A., Aeromonas (except A. aerogenes, Aerobacter aerogenes); E., Escherichia; P., Photobacterium (except P. mirabilis, Proteus mirabilis; and P. shigelloides, Plesiomonas shigelloides); S. marcescens, Serratia marcescens; S. typhimurium, Salmonella typhimurium; V., Vibrio; Y ., Yersinia. Note that Listonella species appear as Vibrio. VOL. 36, 1986 NOTES 475

ENTEROBACTERIACEAE -Vibrio Photobacterium

P. aeruginosa I -I? cepacia I?diminuta

Nitrosomonas

Bacteroides Met hanococcus Cyanobacteria

A. 476 NOTES INT. J. SYST.BACTERIOL.

Tde) of rRNA-DNA heteroduplexes ('C) 65 70 75 80 m m 1 Pasteurellaceae 11 Enterobacteriaceae I

ISuperfamily II FIG. 3. Simplified rRNA cistron similarity dendrogram of rRNA superfamily I sensu De Ley (4). The results of more than 1,OOO DNA-rRNA hybridizations involving about 500 named and numbered strains (including 60 Aeromonas strains) are summarized and expressed as the temperature at which one-half of each hybrid was thermally denatured [Tm(e)]. The data for Alteromonas are from reference 25; the data for the Pusteurellaceae are from De Ley and Mannheim (manuscript in preparation), and the rest of the data are from De Ley and Tytgat (manuscript in preparation). tory and fermentative metabolism. Oxygen is a universal among marine and terrestrial enterobacteria by means of in vitro electron acceptor. Reduce nitrate. Do not denitrify. Most are DNAlribosornal RNA hybridization. Microbios Lett. 3: 11-20. oxidase positive. Most utilize D-glucose as a sole or principal 3. Baumann, P., and R. H. W. Schubert. 1984. Vibrionaceae, p. source of carbon and energy. Most utilize ammonium salts 516-549. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual as sole sources of nitrogen. A few have relatively simple of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. organic growth factor requirements. Primarily aquatic inhab- 4. De Ley, J. 1978. Modern molecular methods in bacterial taxon- itants found in freshwater and in association with aquatic omy: evaluation, application, prospects, p. 247-357. In Pro- animals; also found in sewage. Psychrophilic and meso- ceedings of the 4th International Conference on Plant Patho- philic; motile and nonmotile strains are included. Several genic Bacteria. INRA, Angers. species are pathogenic for humans, fish, eels, and frogs, as 5. De Ley, J. 1984. DNA:rRNA hybridizations in bacterial taxon- well as other vertebrates and invertebrates. The guanine- omy, p. 3-9. In A. Sanna and G. Morace (ed.), New horizons in plus-cytosine content of the DNA ranges from 40 to 63 microbiology. Elsevier/North-Holland Publishing Co., New mol%. York. The type genus is Aeromonas Kluyver and van Niel, 1936, 6. De Vos, P., M. Goor, M. Gillis, and J. De Ley. 1985. Ribosomal 398. ribonucleic acid cistron similarities of phytopathogenic Pseu- Aerornonas aer domonas species. Int. J. Syst. Bacteriol. 35169-184. The genus (Ae.ro.mo'nas. Gr. n. air, gas; 7. Eddy, B. P. 1960. Cephalotrichous, fermentative Gram-negative Gr. n. monas unit, monad; M.L. fem. n. Aeromonas gas- bacteria: the genus Aerornonas. J. Appl. Bacteriol. 25216-249. producing monad) is described in reference 16. 8. Eddy, B. P. 1962. Further studies on Aeromonas. I. Additional strains and supplementary biochemical tests. J. Appl. Bacteriol. 25: 137-146. Support for this research was provided in part by National Kimura, M. Science Foundation grant BSR-82-08418, by Office of Naval Re- 9. 1980. A simple method for estimating evolutionary search Contract N000-14-81-K0638, and by Agency for Interna- rates of base substitutions through comparative studies of tional Development Grant DPE-5542-GSS-4060-00. Support pro- nucleotide sequences. J. Mol. Evol. 16:111-120. vided by the University of Maryland Sea Grant College is also 10. Kluyver, A. J., and C. B. van Niel. 1936. Prospects for a natural appreciated. J.D.L. is indebted to the Fonds vodr Geneeskundig system of classification of bacteria. Zentralbl. Bakteriol. Wetenschappelijk Onderzoek and the Nationaal Fonds voor Parasitenkd. Infektionskr. Hyg. Abt. 1 orig. 94:369403. Wetenschappelijk Onderzoek (Belgium) for several research and 11. MacDonell, M. T., and R. R. Colwell. 1984. The nucleotide base personnel grants. sequence of Vibrionaceae 5s rRNA. FEBS Lett. 175183-188. We gratefully acknowledge P. R. Brayton for assistance with 12. MacDonell, M. T., and R. R. Colwell. 1984. The nucleotide preparation of figures. sequence of 5s ribosomal RNA from Vibrio marinus. Microbiol. Sci. 1:229-231. 13. MacDonell, M. T., and R. R. Colwell. 1985. The phylogeny of LITERATURE CITED the Vibrionaceae and recommendation for two new genera, Listonella and Shewanella. Syst. Appl. Microbiol. 6:171-182. Baumann, L., S. S. Bang, and P. Baumann. 1980. Study of 14. Macinnes, J. L., T. J. Trust, and J. H. Crosa. 1979. Deoxyribo- relationship among species of Vibrio, Photobacterium, and nucleic acid relationships among members of the genus Aero- terrestrial enterobacteria by an immulogical comparison of monas. Can. J. Microbiol. 25579-586. glutamine synthetase and superoxide dismutase. Curr. Micro- 15. McCarthy, D. H. 1975. Aeromonas proteolytica-a halophilic biol. 4133-138. aeromonad? Can. J. Microbiol. 21902-904. Baumann, L., and P. Baumann. 1976. Study of the relationship 16. Popoff, M. Y. 1984. Genus 111. Aeromonas Kluyver and van VOL. 36, 1986 NOTES 477

Niel 1936, 398, p. 545-548. In N. R. Krieg and J. G. Holt (ed.), proteolytica (Merkel et al. 1964) comb. nov. Zentralbl. Bergey’s manual of systematic bacteriology, vol. 1. The Wil- Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 orig. 211: liams & Wilkins Co., Baltimore. 409412. 17. Popoff, M. Y., C. Coynault, M. Kiredjian, andM. Lemelin. 1981. 22. Schubert, R. H. W. 1969. Zur Taxonomie von Aeromonas Polynucleotide sequence relatedness among motile Aeromonas salmonicida subsp. achromogenes (Smith 1963) Schubert 1967 species. Curr. Microbiol. 5109-114. und Aeromonas salmonicida subsp. masoucida Kimura 1969. 18. Popoff, M. Y., and M. Veron. 1976. A taxonomic study of the Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Aeromonas hydrophila-Aeromonas punctata group. J. Gen. Orig. 211:213-216. Microbiol. 94:ll-22. 23. Stakebrandt, E., and C. R. Woese. 1984. The phylogeny of 19. Schubert, R. H. W. 1960. Untersuchungen ueber die Merkmale prokaryotes. Microbiol. Sci. 1: 117-122. der Gattung Aeromonas. Zentralbl. Bakteriol. Parasitenkd. 24. Staley, T. E., and R. R. Colwell. 1973. Deoxyribonucleic acid Infektionskr. Hyg. Abt. 1 orig. 180:310-327. reassociation among members of the genus Vibrio. Int. J. Syst. 20. Schubert, R. H. W. 1964. Zur Taxonomie der Voges-Proskaure- Bacteriol. 23: 316-332. negativen “hydrophila-ahnlichen” Aeromonaden. Zentralbl. 25. Van Landschoot, A., and J. De Ley. 1983. Intra- and intergeneric Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. similarities of the rRNA cistrons of Alteromonas, Marinomonas 193:482490. (gen. nov.) and some other Gram-negative bacteria. J. Gen. 21. Schubert, R. H. W. 1969. Aeromonas hydrophila subsp. Microbiol. 129:3057-3074.