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Reclassification of Lactobacillus maltaromicus (Miller et al. 1974) DSM 20342^T and DSM 20344 and Carnobacterium piscicola (Collins et al. 1987) DSM 20730^T and DSM 20722 as Carnobacterium maltaromaticum comb. nov.

¹ Università degli Studi di Milano, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Milano, Italy
² Sezione Microbiologia Agraria Alimentare e Ecologica, Milano, Italy

Correspondence
Diego Mora
diego.mora{at}unimi.it

	ABSTRACT

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Phenotypic and genotypic characterizations of Lactobacillus maltaromicus strains DSM 20342^T and DSM 20344 provided evidence for the reclassification of this species in the genus Carnobacterium. Moreover, phenotypic and genotypic comparisons made between L. maltaromicus and Carnobacterium piscicola highlighted that these two species should be considered synonyms. For these reasons, the species Carnobacterium maltaromaticum comb. nov. (type strain DSM 20342^T=ATCC 27865^T=CCUG 30142^T=CIP 103135^T=JCM 1154^T=LMG 6903^T=NRRL B-14852^T) is proposed to accommodate L. maltaromicus and C. piscicola.

	MAIN TEXT

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Modern taxonomic methods have led to numerous reclassifications and rearrangements of bacterial strains, species and genera. In this context, the genus Carnobacterium was created by the reclassification of atypical Lactobacillus species isolated from vacuum-packed meat and fish (Collins et al., 1987). The genus Carnobacterium was created to group heterofermentative, rod-shaped lactic acid bacteria that produce L-lactic acid from glucose and are characterized by the presence of meso-diaminopimelic acid in their cell-wall composition. At the time of writing, the genus Carnobacterium comprised seven species, Carnobacterium alterfunditum, Carnobacterium funditum (Franzmann et al., 1991), Carnobacterium divergens (Holzapfel & Gerber, 1983; Collins et al., 1987), Carnobacterium gallinarum, Carnobacterium mobile (Collins et al., 1987), Carnobacterium inhibens (Jöborn et al., 1999) and Carnobacterium piscicola (Hiu et al., 1984; Shaw & Harding, 1985). Collins et al. (1991) inferred the phylogenetic relationships among Lactobacillus species, Carnobacterium species and related lactic acid bacteria on the basis of 16S rDNA sequence data. In that study, Collins and colleagues suggested the revision of the taxonomic position of Lactobacillus maltaromicus due to the high 16S rDNA sequence similarity (100 % sequence similarity for a comparison based on 1340 nt of 16S rDNA sequence) detected between this species and C. piscicola, formerly Lactobacillus piscicola (Hiu et al., 1984). L. maltaromicus was first described by Miller et al. (1974) as a new lactic acid bacteria isolated from milk and producing a malty-like flavour and aroma.

In this study, a phenotypic and genotypic comparison among L. maltaromicus strains DSM 20342^T and DSM 20344 (Miller et al., 1974) and C. piscicola strains DSM 20730^T and DSM 20722 was carried out with the aim of clarifying the taxonomic position of these two species. Specifically, the aforementioned Lactobacillus and Carnobacterium species were compared by evaluating their carbohydrate fermentation patterns, by determining whether meso-diaminopimelic acid was present in their cell-wall composition, and by determining the nature of the enantiomeric form of the lactic acid produced by their metabolism. Moreover, all the strains were characterized genetically by restriction analysis of their amplified 16S rDNA, by amplification of the internal transcribed spacers between their 16S and 23S rDNA and by evaluation of their DNA–DNA relatedness.

L. maltaromicus strains DSM 20342^T and DSM 20344, C. piscicola strains DSM 20730^T and DSM 20722, and C. divergens DSM 20623^T and C. gallinarum DSM 4847^T were maintained routinely at 4 °C after growth at 30 °C for 12 or 24 h in TSBY medium (30 g trypticase soy broth l^-1, 3 g yeast extract l^-1, pH 7). For long-term maintenance, stock cultures were stored in 20 % (v/v) glycerol/80 % (v/v) TSBY medium at -80 °C.

The carbohydrate fermentation patterns obtained using the API CH50 system (bioMérieux), with incubation at 30 °C for 12–24 h, were very similar for the L. maltaromicus and C. piscicola strains. The strains were able to ferment glycerol, ribose, galactose, D-glucose, D-fructose, D-mannose, methyl -D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, sucrose, trehalose and -gentibiose. A weak positive reaction was detected for methyl -D-glucopyranoside. Lactose was fermented by the L. maltaromicus strains, while a weak positive reaction was observed for the C. piscicola strains. Starch and mannitol were fermented by the C. piscicola strains, while a weak positive reaction was detected for the L. maltaromicus strains. Neither species fermented erythritol, D-/L-arabinose, D-/L-xylose, adonitol, methyl -D-xylopyranoside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, melibiose, inulin, melezitose, D-raffinose, xylitol, D-turanose, D-lyxose, D-tagatose, D-/L-fucose, D-/L-arabitol, gluconate, 2-ketogluconate nor 5-ketogluconate.

All Lactobacillus and Carnobacterium strains tested produced the L(+) enantiomeric form of lactic acid, as determined by using the D-L lactic acid kit (Roche). Moreover, the presence of meso-diaminopimelic acid was detected in the cell wall of all the strains tested using the method of Hancock (1994). Genotypic characterizations, based on a randomly amplified polymorphic DNA (RAPD) fingerprinting analysis, a 16S–23S rDNA intergenic spacer analysis and a restriction analysis of amplified 16S rDNA, were carried out for L. maltaromicus strains DSM 20342^T and DSM 20344 and for C. piscicola DSM 20730^T in comparison with the two closest phylogenetic neighbours of these species, C. divergens DSM 20623^T and C. gallinarum DSM 4847^T (Collins et al., 1991). DNA extraction, PCR and restriction protocols were performed as described previously (Mora et al., 1998, 2000); RAPD fingerprinting analysis was carried out with primer OPI-02mod (5'-GCTCGGAGGAGAGG-3').

The amplified 16S–23S rDNA intergenic spacer analysis showed an identical electrophoretic profile for the L. maltaromicus and C. piscicola strains (Fig. 1), which was characterized by a main fragment of 480 bp and secondary fragments ranging in size from 510 to 700 bp. C. divergens DSM 20623^T and C. gallinarum DSM 4847^T were easily distinguished from the L. maltaromicus and C. piscicola strains due to the presence of main amplification fragments of 310 and 420 bp, respectively, in their electrophoretic profiles. Likewise, the L. maltaromicus and C. piscicola strains showed identical HaeIII/HinfI restriction profiles for their amplified 16S rDNA (Fig. 2), while distinct patterns were obtained for C. divergens DSM 20623^T and C. gallinarum DSM 4847^T. The high level of similarity of the ribosomal locus detected for the L. maltaromicus and C. piscicola strains was also confirmed by RAPD fingerprinting analysis, as shown in Fig. 3.

View larger version (86K): [in this window] [in a new window]   Fig. 1. 16S–23S rDNA intergenic spacer amplification patterns of L. maltaromicus, C. piscicola, C. gallinarum and C. divergens. Lanes: 1, L. maltaromicus DSM 20342T; 2, L. maltaromicus DSM 20344; 3, C. piscicola DSM 20730T; 4, C. gallinarum DSM 4847T; 5, C. divergens DSM 20623T; M, 100 bp ladder (MBI Fermentans).

View larger version (110K): [in this window] [in a new window]   Fig. 2. HaeIII/HinfI restriction analysis of amplified 16S rDNA of L. maltaromicus, C. piscicola, C. gallinarum and C. divergens. Lanes: 1, L. maltaromicus DSM 20342T; 2, C. piscicola DSM 20730T; 3, C. piscicola DSM 20722; 4, C. gallinarum DSM 4847T; 5, C. divergens DSM 20623T; M, GeneRuler 100 bp ladder plus (MBI Fermentans); M', GeneRuler 100 bp DNA ladder (MBI Fermentans).

View larger version (98K): [in this window] [in a new window]   Fig. 3. RAPD fingerprinting analysis of L. maltaromicus, C. piscicola, C. gallinarum and C. divergens. Lanes: 1, L. maltaromicus DSM 20342T; 2, L. maltaromicus DSM 20344; 3, C. piscicola DSM 20730T; 4, C. gallinarum DSM 4847T; 5, C. divergens DSM 20623T; M, GeneRuler 100 bp DNA ladder plus (MBI Fermentans); M', GeneRuler 1 kb ladder (MBI Fermentans).

In conclusion, the genotypic and phenotypic comparisons carried out among L. maltaromicus and C. piscicola strains provide evidence for the reclassification of L. maltaromicus DSM 20342^T and DSM 20344 in the genus Carnobacterium. Moreover, the results of DNA–DNA reassociation analyses highlight that L. maltaromicus and C. piscicola belong to the same species. On the basis of the results presented here, we propose the reclassification of L. maltaromicus DSM 20342^T and DSM 20344 (Miller et al., 1974) and C. piscicola DSM 20730^T and DSM 20722 (Hiu et al., 1984; Collins et al., 1987) as Carnobacterium maltaromaticum.

Description of Carnobacterium maltaromaticum comb. nov.
Basonym: Lactobacillus maltaromicus Miller et al. 1974; Carnobacterium piscicola Collins et al. 1987.

Carnobacterium maltaromaticum (malt.a.ro.mat.ic'um. N.L. neut. n. maltum -i malt; L. adj. aromaticus -a -um aromatic, fragrant; N.L. neut. adj. maltaromaticum possessing a malt-like aroma).

The description includes data compiled by Hiu et al. (1984) and Collins et al. (1987), and those generated in this study. Asporogenous, Gram-positive rods of varying length, which occur singly or in chains. Cells are non-motile, and catalase- and oxidase-negative. Facultatively anaerobic. L(+)-Lactic acid, ethanol and acetate are produced heterofermentatively. Gas production is weak and frequently undetectable. Growth occurs in MRS, TSBY and brain–heart infusion media. Growth occurs at 4 and 15 °C, but not at 45 °C; optimum growth occurs between 28 and 32 °C. Arginine and aesculin are hydrolysed. Nitrate is not reduced to nitrite. Acid is produced from glycerol, ribose, galactose, D-glucose, D-fructose, D-mannose, lactose, methyl -D-mannopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, sucrose, trehalose, -gentibiose, mannitol and starch. The peptidoglycan is of the meso-diaminopimelic acid direct type. Major cellular fatty acids are straight-chain saturated and monounsaturated acids, with tetradecanoic, hexadecanoic and 9- and 10-octadecenoic acids predominating. G+C content of the DNA ranges from 33·7 to 36·4 mol%.

The type strain of Carnobacterium maltaromaticum is DSM 20342^T (=ATCC 27865^T=CCUG 30142^T=CIP 103135^T=JCM 1154^T=LMG 6903^T=NRRL B-14852^T).

	Note added in proof

TOP ABSTRACT MAIN TEXT Note added in proof REFERENCES

 
Since this article was accepted for publication, Carnobacterium viridans has been described (Holley et al., 2002).

	REFERENCES

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Collins, M. D., Farrow, A. E., Phillips, B. A., Ferusu, S. & Jones, D. (1987). Classification of Lactobacillus divergens, Lactobacillus piscicola, and some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genus, Carnobacterium. Int J Syst Bacteriol 37, 310–316.[CrossRef]

Collins, M. D., Rodrigues, U., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez-Murcia, A., Phillips, B. A., Williams, A. M. & Wallbanks, S. (1991). Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77, 5–12.[CrossRef]

Franzmann, P. D., Höpfl, P., Weiss, N. & Tindall, B. J. (1991). Psychrotrophic, lactic acid-producing bacteria from anoxic waters in Ace Lake, Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov. Arch Microbiol 156, 255–262.[CrossRef][Medline]

Hancock, I. C. (1994). Analysis of cell wall constituents of Gram-positive bacteria. In Chemical Methods in Prokaryotic Systematics, pp. 63–84. Edited by M. Goodfellow & A. G. O'Donnell. New York: Wiley.

Hiu, S. F., Holt, R. A., Sriranganathan, N., Seidler, R. J. & Freyer, J. L. (1984). Lactobacillus piscicola, a new species from salmonid fish. Int J Syst Bacteriol 34, 393–400.

Holley, R. A., Guan, T. Y., Peirson, M. & Yost, C. K. (2002). Carnobacterium viridans sp. nov., an alkaliphilic, facultative anaerobe isolated from refrigerated, vacuum-packed bologna sauce. Int J Syst Evol Microbiol 52, 1881–1885.[Abstract]

Holzapfel, W. H. & Gerber, E. S. (1983). Lactobacillus divergens sp. nov., a new heterofermentative Lactobacillus species producing L(+)-lactate. Syst Appl Microbiol 4, 522–534.

Jöborn, A., Dorsch, M., Olsson, J. C., Westerdahl, A. & Kjelleberg, S. (1999). Carnobacterium inhibens sp. nov., isolated from the intestine of Atlantic salmon (Salmo salar). Int J Syst Bacteriol 49, 1891–1898.[CrossRef][Medline]

Miller, A., III, Morgan, M. E. & Libbey, L. M. (1974). Lactobacillus maltaromicus, a new species producing a malty aroma. Int J Syst Bacteriol 24, 346–354.[CrossRef]

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Mora, D., Fortina, M. G., Parini, C., Daffonchio, D. & Manachini, P. L. (2000). Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationship between pediocin AcH/PA-1 producing and non-producing strains. Microbiology 146, 2027–2038.[Abstract/Free Full Text]

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