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1 Instituto de Investigaciones Biomédicas Fundación Pablo Cassará, Saladillo 2452, Buenos Aires (1440), Argentina
2 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina
3 Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Health Services, Richmond, CA, USA
Correspondence
Jorge Zorzopulos
zorzopul{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005.
A phylogenetic tree showing that members of the genus Enterobacter are intertwined with members of other genera is available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), it has been reported that the genus Klebsiella is heterogeneous and composed of species which form three clusters that include members of other genera.
Kluyvera is a genus of small rod-shaped bacteria, thus conforming to the general definition of the family Enterobacteriaceae (Holt et al., 1994). Bacteria of this genus are mainly grouped in four known species, Kluyvera ascorbata, Kluyvera cryocrescens, Kluyvera cochleae and Kluyvera georgiana (Farmer et al., 1981; Müller et al., 1996).
The present study was undertaken to gain insight into the phylogenetic relationships among species within the genus Kluyvera and between the genus Kluyvera and related genera within the family Enterobacteriaceae, using 16S rRNA gene-based trees, DNA–DNA hybridization analysis and phenotypic characterization.
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METHODS |
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Biochemical studies.
Enterobacter intermedius and Kluyvera strains were characterized phenotypically using a battery of 43 biochemical tests in conventional media. These tests included: triple-sugar iron agar reactions; pigmentation (25 °C); motility; production of cytochrome oxidase, nitrate reductase and indole; growth in KCN broth; urea hydrolysis (Christensen's); utilization of malonate, citrate (Simmon's), acetate and mucate; production of 
-galactosidase (ONPG) and phenylpyruvic acid (phenylalanine deaminase); lysine decarboxylase, ornithine decarboxylase and arginine dihydrolase (Møeller's) activities; elaboration of acetylmethylcarbinol (Voges–Proskauer); degradation of gelatin, corn oil (lipase), DNA and polypectate (25 °C); and aesculin hydrolysis (broth). Carbohydrate fermentation reactions were performed in extract broth against 1 % solutions of the following carbohydrate or carbohydrate-like compounds: adonitol, amygdalin, L-arabinose, D-arabitol, cellobiose, dulcitol, methyl 
-D-glucopyranoside, D-glucose, glycerol, myo-inositol, lactose, maltose, D-mannitol, melibiose, raffinose, L-rhamnose, D-sorbitol, salicin, sucrose, trehalose and D-xylose. All of these tests have been described previously (Abbott et al., 1992, 2003; Janda et al., 1996). Unless otherwise specified, tests were incubated at 35 °C for 2–4 days (7 days for carbohydrate fermentation and extracellular enzymes) and final results were recorded. Biochemical reactions presented in Tables 1 and 2 are at 48 h incubation.
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).
DNA–DNA hybridization.
The genetic relatedness among members of the genus Kluyvera was determined by DNA–DNA hybridization on nylon membranes (Johnson, 1991). Serial dilutions of cell suspensions of equal OD570 values in denaturation solution (0·5 M NaOH/1·0 M NaCl) were applied to nylon membranes (Pall Biodyne). The membranes were then neutralized with 1·5 M NaCl/0·5 M Tris/HCl (pH 8) and fixed by UV radiation for 15 min. Pre-hybridization (2 h) was performed at 65 °C in a buffer containing 0·15 M NaCl, 1 % SDS and 0·3 % non-fat dried milk, and hybridization (18 h) was performed at 65 °C in a buffer containing 0·03 M NaCl, 1 % SDS and 0·3 % non-fat dried milk. The hybridization probe was chromosomal DNA from the indicated strain digested with AluI endonuclease and 32P-labelled with the Random Primer DNA Labelling System (Gibco, Life Technologies). The results were scored first by autoradiography and then by cutting the spots and measuring the radiation in a scintillation counter (Beckman). Hybridization levels were calculated as described by Johnson (1991).
Sequencing of the 16S rRNA gene and phylogenetic analysis.
DNA from bacterial strains was purified (Sambrook et al., 1989), and the 16S rRNA genes were amplified by PCR using the bacterial primers 27f (5'-AGAGTTTGATCMTGGCTCAG-3'), corresponding to positions 8–27 of forward Escherichia coli numbering, and 1492r (5'-GGTTACCTTGTTACGACTT-3'), corresponding to positions 1510–1492 of reverse Escherichia coli numbering. The following temperature programme was used: 94 °C for 5 min, 30 cycles of 94 °C for 60 s, 60 °C for 60s and 72 °C for 60 s, followed by a final 7 min incubation at 72 °C. The PCR product was purified using GFX-PCR DNA and a gel band purification kit (Amersham Biosciences) and sequenced completely by using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer) and an ABI Prism 377 DNA Sequencer (Perkin-Elmer). The obtained 16S rRNA gene sequences were aligned with those of type strains of bacterial genera related to the genus Kluyvera available in the EMBL and Ribosomal Database Project libraries (Maidak et al., 1997) by using the CLUSTAL W program (Thompson et al., 1994) with default parameters and optimized using a multiple sequence alignment editor (Galtier et al., 1996). Phylogenetic trees were constructed by both the neighbour-joining distance method (Kimura two-parameter model and jumble option) and the parsimony character method, using programs contained in the PHYLIP package (Felsenstein, 1989). The stability of the relationships was assessed by bootstrapping (1000 replicates), with programs included in the same package. The sequence of Aeromonas hydrophila ATCC 7966T (GenBank/EMBL/DDBJ accession no. X74677) was used as an outgroup to establish the root of the tree.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) showed that Enterobacter intermedius ATCC 33110T is remarkably similar to the type strain of K. cochleae and that both strains are Voges–Proskauer-positive, differing from the type strains of the other Kluyvera species. Furthermore, Table 2 shows that this remarkable similarity is not exclusive to the type strains but can be extended to other available independently isolated strains of these two species. The DNA relatedness among the type strains of species in the genus Kluyvera and the type strain of Enterobacter intermedius was also studied (Table 3). As can be observed, this DNA relatedness was higher than the current minimal standard (70 % relatedness) accepted for the phylogenetic definition of a species (Wayne et al., 1987; Stackebrandt & Goebel, 1994) when labelled DNA from Enterobacter intermedius was used as a probe. However, the DNA relatedness was about 59 % when labelled DNA from K. cochleae was used as a probe. This difference may be due to the presence of extrachromosomal DNA in one or both of the reference strains. In agreement with this, Fig. 2 shows that cells of the type strain of Enterobacter intermedius have several high-copy-number plasmids, while the cells of the type strain of K. cochleae apparently do not have extrachromosomal elements. Using chromosomal DNA of the type strain of Enterobacter intermedius extracted from gels, the DNA relatedness with the DNA of the type strain of K. cochleae increased to more than 70 % (not shown). Thus, DNA–DNA hybridization assays indicate that the type strains of these bacterial species are included within the same species.
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). DNA–DNA hybridization assays indicate that K. cochleae and K. intermedia are heterotypic synonyms. Rule 42 of the Bacteriological Code requires the oldest legitimate epithet be retained. Thus, the oldest legitimate epithet for these taxa is that of Izard et al. (1980). Therefore, K. cochleae Müller et al. 1996 is a later synonym of K. intermedia (Izard et al. 1980) Pavan et al. 2005.
It is also worth noting that the 16S rRNA gene-based tree presented here strongly suggests that members of the genera Kluyvera and Buttiauxella, as defined by phenotypic assays, are monophyletic, in agreement with the conclusion of a previous report (Spröer et al., 1999). In contrast, members of the genus Enterobacter are intertwined with members of other genera (see Fig. A, available as supplementary material in IJSEM Online), a fact also suggested by trees constructed using groE genes (Harada & Ishikawa, 1997). This indicates the need for an extensive revision of the phenotypic criteria to classify bacteria within the genus Enterobacter.
There is a good correlation between the results of DNA–DNA hybridization studies presented previously (Farmer et al., 1981) and the results of the phylogenetic analysis presented herein. In both cases, Klebsiella, Enterobacter and Citrobacter species are close relatives of Kluyvera species. Salmonella, Escherichia, Shigella and Erwinia species are intermediate and Proteus and Yersinia species are the most distant relatives among the species analysed in both studies. In the Farmer et al. (1981) analysis, the large heterogeneity of the Enterobacter species observed in our study was also evident.
In conclusion, the results presented herein indicate that there is good agreement between the grouping of species of the genus Kluyvera by 16S rRNA gene-based phylogenetic analysis and phenotypic clustering, and that strains of Enterobacter intermedius should be reclassified as proposed since they are by phylogenetic, phenotypic and DNA–DNA relatedness criteria members of the genus Kluyvera. On the other hand, it is clear from this study that extensive studies are necessary to bring about coherence within the genus Enterobacter.
Description of Kluyvera intermedia comb. nov.
Kluyvera intermedia (in.ter.me'di.a. L. adj. intermedia intermediate).
Basonym: Enterobacter intermedius Izard et al. 1980.
The description is that given by Izard et al. (1980). Some characteristics are as follows. Cells are straight rods, 0·5–0·7x2–3 µm, Gram-negative, motile by scant peritrichous flagella. Facultatively anaerobic and chemo-organotrophic, having both a respiratory and a fermentative type of metabolism. Colonies are circular, convex, greyish and smooth on nutrient agar, with growth at 30 and 37 °C. Catalase-positive. Oxidase-negative. Nitrate reductase-positive. Indole-negative. Voges–Proskauer-positive. Acid produced from amygdalin, L-arabinose, cellobiose, dulcitol, D-glucose, glycerol, lactose, maltose, D-mannitol, melibiose, raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, trehalose and D-xylose. Negative for urea hydrolysis. ONPG-positive. Grows in KCN broth. Utilizes citrate and acetate. Does not utilize adonitol, arabitol, erythritol or myo-inositol. Does not degrade gelatin, DNA (DNase-negative) or corn oil (lipase-negative), but degrades mucate. Arginine dihydrolase-negative. Does not form phenylpyruvic acid. Does not produce pigment at 25 °C. Polypeptate-positive. Isolated from molluscs, surface water, soil and a variety of human samples including stool, blood, wounds, bile and a gall bladder.
The type strain is ATCC 33110T (=CIP 79.27T=LMG 2785T=CCUG 14183T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Abbott, S. L., Cheung, W. K. W. & Janda, J. M. (2003). The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol 41, 2348–2357.
Drancourt, M., Bollet, C., Carta, A. & Rousselier, P. (2001). Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov. Int J Syst Evol Microbiol 51, 925–932.[Abstract]
Farmer, J. J., III, Fanning, G. R., Huntley-Carter, G. P., Holmes, B., Hickman, F. W., Richard, C. & Brenner, D. J. (1981). Kluyvera, a new (redefined) genus in the family Enterobacteriaceae: identification of Kluyvera ascorbata sp. nov. and Kluyvera cryocrescens sp. nov. in clinical specimens. J Clin Microbiol 13, 919–933.
Felsenstein, J. (1989). PHYLIP – Phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Galtier, N., Gouy, M. & Gautier, C. (1996). SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12, 543–548.
Harada, H. & Ishikawa, H. (1997). Phylogenetical relationship based on groE genes among phenotypically related Enterobacter, Pantoea, Klebsiella, Serratia and Erwinia species. J Gen Appl Microbiol 43, 355–361.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. & Williams, S. T. (1994). Bergey's Manual of Determinative Bacteriology, 9th edn. Williams & Wilkins.
Izard, D., Gavini, F. & Leclerc, H. (1980). Polynucleotide sequence relatedness and genome size among Enterobacter intermedium sp. nov. and the species Enterobacter cloacae and Klebsiella pneumoniae. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Orig Reihe C 1, 51–60.
Janda, J. M., Abbott, S. L., Khashe, S., Kellogg, G. H. & Shimada, T. (1996). Further studies on biochemical characteristics and serological properties of the genus Aeromonas. J Clin Microbiol 34, 1930–1933.[Abstract]
Johnson, J. L. (1991). DNA reassociation experiments. In Nucleic Acid Techniques in Bacterial Systematics, p. 329. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey, M. J. & Woese, C. R. (1997). The RDP (Ribosomal Database Project). Nucleic Acids Res 25, 109–111.
Müller, H. E., Brenner, D. J., Fanning, G. R., Grimont, P. A. D. & Kämpfer, P. (1996). Emended description of Buttiauxella agrestis with recognition of six new species of Buttiauxella and two species of Kluyvera: Buttiauxella ferragutiae sp. nov., Buttiauxella gaviniae sp. nov., Buttiauxella brennerae sp. nov., Buttiauxella izardii sp. nov., Buttiauxella noackiae sp. nov., Buttiauxella warmboldiae sp. nov., Kluyvera cochleae sp. nov., and Kluyvera georgiana sp. nov. Int J Syst Bacteriol 46, 50–63.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Spröer, C., Mendrock, U., Swiderski, J., Lang, E. & Stackebrandt, E. (1999). The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae. Int J Syst Bacteriol 49, 1433–1438.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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