C:/ncn/ijs57

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1969-1972
DNA-DNA reassociation and phenotypic data
indicate synonymy between Aeromonas
enteropelogenes Schubert et al. 1990 and
Aeromonas trota Carnahan et al. 1991
Geert Huys,1 Rik Denys1 and Jean Swings1,2 Microbiology1 andBCCM4/LMG BacteriaCollection2 , GhentUniversity, K. L.
Author for correspondence : Geert Huys. Tel : j32 9 2645249. Fax : j32 9 2645346.
Mainly on the basis of phylogenetic and genotypic evidence, it has been
suggested previously that the species Aeromonas enteropelogenes Schubert
et al. 1990 is identical to the species Aeromonas trota Carnahan et al. 1991.
Probably because the description of A. enteropelogenes preceded the proposal
of A. trota by only a few months, DNA-DNA hybridizations were never
performed between representative strains of these two taxa. In the present
study, new DNA-DNA hybridizations between the type strain of A.
enteropelogenes, LMG 12646T (l DSM 6394T), and reference strains of A. trota,
including its type strain LMG 12223T (l ATCC 49657T), showed a genomic
relatedness of 81-99 %. In addition, phenotypic characterization revealed that
the two type strains exhibited identical API 20E and API 50CHE biochemical
profiles and were both susceptible to ampicillin and carbenicillin. Collectively,
our new DNA reassociation and phenotypic data confirm previous taxonomic
data that indicate that the taxa A. enteropelogenes and A. trota are
synonymous members of the same Aeromonas species. Although the species
name A. enteropelogenes has nomenclatural priority, the authors would like
to discourage the use of this name because the name A. trota has been cited
much more frequently. The preferential use of A. trota in future publications
may be the best option to avoid ambiguity in the description of ampicillin-
susceptible aeromonads and to secure nomenclatural continuity in Aeromonas
literature.
Keywords : Aeromonas enteropelogenes, Aeromonas trota, DNA-DNA hybridizations
Until the 1990s, it was generally assumed that all A. trota proposal, Schubert et al. (1990) proposed the members of the genus Aeromonas were uniformly name Aeromonas enteropelogenes for a psychrophilic resistant to ampicillin and carbenicillin. Because ampi- group of A. sobria-like organisms, all of which were cillin resistance is encoded chromosomally in Aero- recovered from human stools in India. Although monas, this property is an ideal stable discriminative originally described as two different species, phylo- feature for the selective isolation of aeromonads, genetic evidence presented by Collins et al. (1993) led e.g. by using ampicillin\dextrin agar (Havelaar et to the conclusion that A. enteropelogenes and A. trota al., 1987). In 1991, however, the use of ampicillin- were identical, since the 16S rRNA sequences of their containing media for screening faecal samples was type strains displayed 100 % similarity. The latter seriously questioned by the description of Aeromonas authors suggested that additional DNA-DNA hybrid- trota as the first ampicillin-susceptible Aeromonas ization studies should be performed in order to species (Carnahan et al., 1991). The majority of the determine conclusively the genuine taxonomic related- strains belonging to A. trota were isolated from faecal ness between these species but, to date, these data are specimens collected in southern and south-eastern Asia still not available. In subsequent studies, fatty acid and were initially considered as an Aeromonas sobria- analysis (G. Huys, unpublished results) and amplified like group. A few months before the publication of the fragment length polymorphism analysis (Huys et al., 01996 # 2002 IUMS Printed in Great Britain Table 1. DNA-DNA relatedness between A. enteropelogenes and A. trota reference strains
.
Results are mean percentages from four determinations. Reciprocal hybridizations showed a maximum standard deviation of p6% whereas repeated experiments exhibited a maximum standard deviation of p3%.
DNA hybridization with labelled reference DNA from strain :
1. A. enteropelogenes LMG 12646T 1996 ; Huys & Swings, 1999) further supported the was prepared using a combination of the protocols view that A. enteropelogenes and A. trota cannot be of Marmur (1961) and Pitcher et al. (1989) as de- separated on phenotypic or genotypic grounds. In this scribed previously (Goris et al., 1998). Hybridization context, the present study was undertaken to provide experiments were performed using the fluorometric decisive DNA-DNA reassociation data for A. trota microplate method (Ezaki et al., 1989) with modifi- and A. enteropelogenes and to assess their phenotypic cations by Goris et al. (1998) at an optimal renatu- and antibiotic profiles using a uniform methodology ration temperature of 45 mC in 50% formamide.
Despite the vast amount of phylogenetic, genotypic The following type or reference strains were obtained and phenotypic information generated so far regarding from the BCCM\LMG Bacteria Collection: A. entero- the taxonomic relationship between A. trota and A.
pelogenes LMG 12646T (l ATCC 49803T l DSM enteropelogenes (Collins et al., 1993 ; Huys et al., 1996 ; 6394T l Sanyal J11T), A. trota LMG 12223T G. Huys, unpublished results), a conclusive DNA- (l ATCC 49657T), LMG 13080 (l ATCC 49659), DNA reassociation study including representative strains of these two species has never been performed.
(l ATCC 49658), Aeromonas caviae LMG 3775T In this regard, however, it should be mentioned again (l ATCC 15468T) and A. sobria LMG 3783T (l CIP that the descriptions of the two species were published 74.33T). Additional information on these strains can within a few months of each other, obviously within be found in an earlier paper (Huys et al., 1996).
too short a period to allow cross-referencing. In Strains were cultured aerobically on Trypticase soy contrast to the proposal of A. trota (Carnahan et al., agar containing 3 % (w\v) Trypticase soy broth (BBL) 1991), the description of A. enteropelogenes (Schubert and 1n5% (w\v) bacteriological agar no. 1 (Oxoid) at et al., 1990) was based on a rather limited phenotypic 28 mC for 24 h. A. enteropelogenes (n l 1) and A. trota and DNA-DNA hybridization study. In 1990, at least (n l 4) strains were characterized biochemically using eight Aeromonas species were validated, but only three API 20E and API 50CHE microbial identification of these were included as reference taxa in the latter strips according to the manufacturer's instructions proposal. Similar to the 16S rRNA sequencing study (bioMe!rieux). For each isolate, antimicrobial suscep- of Collins et al. (1993), the current investigation tibilities were assessed for six agents using the disc included just the type strain of A. enteropelogenes, diffusion method (Bauer et al., 1966) with the modi- LMG 12646T, which was received as strain DSM fication that Mueller-Hinton medium was replaced by 6394T and which is equivalent to strain Sanyal J11T Iso-Sensitest agar (medium CM471, Oxoid) and IS originally described by Schubert et al. (1990). Together broth (medium CM473, Oxoid) and that strains were with the type strain and three reference strains of incubated at 28 mC. The following antibiotic discs A. trota, strain LMG 12646T was included in a DNA- (Oxoid) were applied using an ST6090 disc dispenser DNA hybridization study that demonstrated that the (Oxoid) : ampicillin (25 µg), carbenicillin (100 µg), two taxa were very highly related, as shown by a DNA kanamycin (25 µg), tetracycline (30 µg), rifampicin hybridization value of 86-98 % between the type (30 µg) and nalidixic acid (30 µg). Isolates were classi- strains, LMG 12223T and LMG 12646T, and by an fied based on the quantitative interpretation criteria overall range of DNA relatedness of 81-99 % among recommended by the NCCLS (National Committee strains of the two taxa (Table 1). In comparison, the for Clinical Laboratory Standards, 1993). For the type strains of A. enteropelogenes and A. trota exhi- DNA-DNA reassociation study, total genomic DNA bited 40-49 % relatedness with type strains of the non- International Journal of Systematic and Evolutionary Microbiology 52
Synonymy of A. enteropelogenes and A. trota related species A. caviae (hybridization group 4) and In conclusion, the DNA-DNA hybridization data A. sobria (hybridization group 7) (Table 1). Collec- presented in the current study clearly indicate that the tively, the new DNA-DNA hybridization data are in named taxa A. trota and A. enteropelogenes belong to perfect congruence with the above-mentioned studies the same genomic species based on the high DNA suggesting that the taxa A. enteropelogenes and relatedness determined between their type strains. In A. trota are members of the same genomic Aeromonas addition, phenotypic characterization suggested that these strains could not be separated by a single test outof 60 features and both displayed susceptibility to Phenotypically, the type strain of A. enteropelogenes ampicillin and carbenicillin. Together with previous and the type and reference strains of A. trota could not phylogenetic (Collins et al., 1993) and genotypic (Huys be separated by any of the 60 different tests included in et al., 1996) evidence, these new results prompt the the API 20E and API 50CHE systems. All A. entero- authors to conclude that the species names A. trota and pelogenes and A. trota strains included in this study A. enteropelogenes are subjective synonyms. According displayed the following phenotypic profile. Positive to Rule 24b of the International Code of Nomenclature reactions were found for oxidase, β-galactosidase, of Bacteria (Lapage et al., 1992), the name A. entero- arginine dihydrolase, lysine decarboxylase, citrate pelogenes has priority in the bacteriological literature utilization and indole and gelatinase tests, whereas over the name A. trota because it was announced in negative reactions were obtained with ornithine decar- Validation List no. 38 (Schubert et al., 1991) whereas boxylase, Voges-Proskauer, production of H#S, urease the latter name was included in the later Validation and tryptophan deaminase. Acid was produced from List no. 40 (Carnahan et al., 1992). On the other hand, the following substrates : N-acetyl glucosamine, starch, searches in international scientific citation databases cellobiose, -fructose, galactose, -glucose, gluco- reveal that the name A. trota has so far been used much nate, glycerol, glycogen, maltose, -mannose, man- more frequently than the name A. enteropelogenes. A nitol, ribose and trehalose. No acid was produced search of the Web of Science citation database (In- from adonitol, -arabinose, -arabinose, -arabitol, stitute for Scientific Information, Thomson Scientific) -arabitol, arbutin, dulcitol, erythritol, aesculin, in the period following the publication of the two -fucose, -fucose, β-gentiobiose, 2-ketogluconate, original species descriptions revealed 34 references to 4-ketogluconate, inositol, inulin, lactose, -lyxose, A. trota but only six to A. enteropelogenes. Strikingly, melibiose, melezitose, methyl α--glucoside, methyl all references that include the name A. enteropelogenes --mannoside, methyl β-xyloside, -raffinose, rham- cover its suspected synonymy with A. trota and do not nose, salicin, sorbitol, -sorbose, sucrose, -tagatose, report unique properties of the organism as such. For -turanose, xylitol, -xylose and -xylose. When com- this reason, it is our opinion that nomenclatural paring only the common tests, our phenotypic results continuity in Aeromonas literature can be most opti- were in good agreement with the characteristics re- mally secured when the use of A. trota is further ported in the original descriptions of A. enteropelo- promoted to describe ampicillin-susceptible aero- genes (Schubert et al., 1990) and A. trota (Carnahan monads. To this end, it is anticipated that other et al., 1991). Solely on the basis of data from the workers may find additional arguments in order to literature, Carnahan (1993) noted previously that the formulate a Request for an Opinion from the Judicial two species shared similar phenotypic profiles, a find- Commission to overrule nomenclatural priority.
ing that is now supported using the same methodfor all strains included.
Acknowledgements
The high phenotypic relatedness between A. entero-pelogenes and A. trota was also reflected by their G. H. is a post-doctoral fellow of the Fund for Scientific antibiotic susceptibility profiles. According to the Research-Flanders (Belgium) (FWO-Vlaanderen).
NCCLS interpretation criteria, all tested strains werefound to be susceptible to ampicillin, carbenicillin, References
kanamycin, tetracycline, rifampicin and nalidixic acid.
The remarkable susceptibility of A. enteropelogenes Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. (1966).
Antibiotic susceptibility testing by a standardized single disk method.
strain LMG 12646T to ampicillin and carbenicillin, Am J Clin Pathol 45, 493-496.
which was not reported in the original description of Carnahan, A. M. (1993). Aeromonas taxonomy : a sea of change. Med
this species by Schubert et al. (1990), agrees with the Microbiol Lett 2, 206-211.
typical susceptibility profile of A. trota (Carnahan et Carnahan, A. M., Chakraborty, T., Fanning, G. R., Verma, D., Ali,
al., 1991) and again illustrates the phenotypic closeness A., Janda, J. M. & Joseph, S. W. (1991). Aeromonas trota sp. nov., an
between the two taxa. Recently, the strong need to ampicillin-susceptible species isolated from clinical specimens. J Clin clarify the taxonomic confusion over the synonymy Microbiol 29, 1206-1210.
between A. trota and A. enteropelogenes was empha- Carnahan, A. M., Chakraborty, T., Fanning, G. R., Verma, D., Ali,
sized in the studies of Khan et al. (1999) and Delamare A., Janda, J. M. & Joseph, S. W. (1992). Aeromonas trota. In
Validation of the Publication of New Names and New Combinations
et al. (2000), in which it was reported that two species Previously Effectively Published Outside the IJSB, List No. 40. Int J Syst within the genus Aeromonas (A. trota and A. entero- Bacteriol 42, 191-192.
pelogenes) respectively shared a highly similar aero- Collins, M. D., Martinez-Murcia, A. J. & Cai, J. (1993). Aeromonas
lysin gene and an unusual tolerance to high salt levels.
enteropelogenes and Aeromonas ichthiosmia are identical to Aeromonas trota and Aeromonas veronii, respectively, as revealed by small-subunit Khan, A. A., Nawaz, M. S., Khan, S. A. & Cerniglia, C. E. (1999).
rRNA sequence analysis. Int J Syst Bacteriol 43, 855-856.
Identification of Aeromonas trota (hybridization group 13) by amplifi- Delamare, A. P. L., Costa, S. O. P., Da Silveira, M. M. & Echever-
cation of the aerolysin gene using polymerase chain reaction. Mol Cell rigaray, S. (2000). Growth of Aeromonas species on increasing
Probes 13, 93-98.
concentrations of sodium chloride. Lett Appl Microbiol 30, 57-60.
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D.,
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
Seeliger, H. P. R. & Clark, W. A. (editors) (1992). International Code
deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- of Nomenclature of Bacteria (1990 Revision). Bacteriological Code.
dilution wells as an alternative to membrane filter hybridization in Washington, DC : American Society for Microbiology.
which radioisotopes are used to determine genetic relatedness among Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
bacterial strains. Int J Syst Bacteriol 39, 224-229.
acid from micro-organisms. J Mol Biol 3, 208-218.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998).
National Committee for Clinical Laboratory Standards (1993).
Evaluation of a microplate DNA-DNA hybridization method com- Performance standards for antimicrobial disk susceptibility tests, 5th edn.
pared with the initial renaturation method. Can J Microbiol 44,
Approved standard M2-A5. Vilanove, PA : National Committee for Havelaar, A. H., During, M. & Versteegh, J. F. M. (1987). Ampicillin-
dextrin agar for the enumeration of Aeromonas species in water by
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid
membrane filtration. J Appl Bacteriol 62, 279-287.
extraction of bacterial genomic DNA with guanidium thiocyanate. Lett
Appl Microbiol 8, 151-156.
Huys, G. & Swings, J. (1999). Evaluation of a fluorescent amplified
fragment length polymorphism (FAFLP) methodology for the geno-
Schubert, R. H. W., Hegazi, M. & Wahlig, W. (1990). Aeromonas
typic discrimination of Aeromonas taxa. FEMS Microbiol Lett 177,
enteropelogenes species nova. Hyg Med 15, 471-472.
Schubert, R. H. W., Hegazi, M. & Wahlig, W. (1991). Aeromonas
Huys, G., Coopman, R., Janssen, P. & Kersters, K. (1996). High-
enteropelogenes. In Validation of the Publication of New Names and New resolution genotypic analysis of the genus Aeromonas by AFLP Combinations Previously Effectively Published Outside the IJSB, List fingerprinting. Int J Syst Bacteriol 46, 572-580.
no. 38. Int J Syst Bacteriol 41, 456-458.
International Journal of Systematic and Evolutionary Microbiology 52

Source: http://www.cellbiol.ru/files/pdfdb/c0t/diferences/Huys_2002.pdf