Bifidobacterium lactis Meile et al Is a Subjective Synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974

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1 Microbiol. Immunol., 44(10), , 2000 Bifidobacterium lactis Meile et al Is a Subjective Synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974 Yimin Cai*,', Mitsuharu Matsumoto2, and Yoshimi Benno3 `National Grassland Research Institute, Tochigi , Japan, 2Fundamental Research Laboratory, Kyodo Milk Industry Company Ltd., Tokyo , Japan, and 'Japan Collection of Microorganisms, The Institute of Physical and Chemical Research, Wako, Saitama , Japan Received June 10, 2000; Accepted July 24, 2000 Abstract: Bifidobacterium lactic JCM 10602T (T- type strain) and Bifidobacterium animalis JCM 1190T were found to be phenotypically similar. These strains were subjected to investigation of their genetic relationships. The 16S rrna sequence of B. animalis JCM 1190T was aligned with that of other Bifidobacterium species. B. animalis and B. lactis were the most closely related species in the phylogenetic tree and showed a high similarity in sequences (98.8%). The levels of DNA-DNA hybridization between the type strains of B. lactis and B. animalis ranged from 85.5 to 92.3%, showing that they represent a single species. It is proposed that B. lactic should be considered as a junior subjective synonym of B. animalis. Key words: Bifidobacterium animalis, Bifidobacterium lactic, 16S rrna, DNA-DNA hybridization, Synonym On the basis of DNA-DNA hybridization data, the taxon Bifidobacterium animalis was separated from other Bifidobacterium species and established for strains previously assigned mainly to the species Bifidobacterium longum (15). A new species, B. lactis, isolated from fermented milk was proposed by enzyme profiles, utilization of carbohydrates, the G + C content of DNA and a high tolerance towards oxygen (10). From 16S rrna sequence analysis, these two species exhibited a high similarity in sequences (10, 12). During the course of characterization and identification of bifidobacteria obtained from some fermented milk, a bifidobacterial strain, Bb 12, isolated from a commercial yoghurt sample was found to be phenotypically similar to B. animalis JCM 1190' and B. lactis JCM 10602T. In order to determine its taxonomic status, this strain was studied by 16S rrna sequence analysis and DNA-DNA hybridization experiments, which propose that B. lactis is a later subjective synonym of B. animalis. Materials and Methods *Address correspondence to Dr. Yimin Cai, National Grassland Research Institute, Nishinasuno, Tochigi , Japan. Fax: cai@ngri.affrc.go.jp Strains studied. B. lactis JCM (=DSM ) was obtained from Kyodo Milk Industry Company, Ltd. (Tokyo, Japan). Strain Bb 12 was isolated from a fermented starter of yoghurt (Chr Hansen, Denmark) on BL agar (Nissui-seiyaku Ltd., Tokyo) incubated at 37 C for 2 days under anaerobic conditions. B. animalis JCM 1l90T (=ATCC 25527`) and other strains of Bifidobacterium were obtained from the Japan Collection of Microorganisms (JCM). Morphological, physiological and biochemical tests. The bifidobacteria strains were cultivated to full growth in lactobacilli MRS agar (Difco Laboratories, Detroit, Mich., U.S.A.) at 37 C under anaerobic conditions. Gram staining and morphology were determined as described by Mitsuoka (11). Carbohydrate fermentation tests were carried out with a semi-automatic system for bacterial identification (1). The enzyme activity tests were performed using the API ZYM System (API Products, Basingtoke, U.K.) according to the instructions of the manufacturer. 16S rdna sequencing. The 16S rdna sequence coding region of strain Bb 12 was amplified by polymerase chain reaction (PCR) and performed in a Takara PCR ThermalCycler (Takara Shuzo Co., Ltd., Ohtsu, Japan) as described by Suzuki et al (18). The sequences of the PCR products were determined directly using an ALFexpressTM Aut Cycle" Sequence Kit (Pharmacia Abbreviations: JCM, Japan Collection of Microorganisms; PCR, polymerase chain reaction. 815

2 816 Y. CAI ET AL Biotech, Calif., U.S.A.) with the primers as described by Suzuki et al (18). Analyses of DNA sequence reactions were performed with an ALFexpress DNA sequencer (Pharmacia Biotech, Uppsala, Sweden). The sequence of strain Bb 12 was aligned with the published sequence of bifidobacteria from DDBJ, GenBank, and EMBL. Escherichia coli (J01695) was used as an out-group organism. Nucleotide substitution rates (K... values) were calculated (6), and a phylogenetic tree was constructed using the neighbor-joining method (14). The topology of the tree was evaluated by bootstrap analysis of the sequence data with Clustal W software (20) based on 100 random resamplings. DNA base composition and DNA-DNA hybridization. DNA was extracted from cells harvested from MRS broth culture which had been incubated for 8 hr at 37 C. It was purified by the procedure of Saitou and Miura (13). DNA base composition was determined by the method of Tamaoka and Komagata (19) using high-performance liquid chromatography following enzymatic digestion of DNA to deoxyribonucleosides. The equimolar mixture of four deoxyribonucleotides in a Yamasa GC Kit (Yamasa Shoyu Co., Ltd., Choshi, Japan) was used as the quantitave standard. Microplate hybridization was performed according to Ezaki et al (3) using black Maxisorp Fluoro Nunk microplates (Nunk A/S, Roskilde, Denmark). The membrane filter hybridization was performed as described by the method of Ezaki et al (3) using the photobiotin labeling system. Results and Discussion Strain Bb 12 is a Gram-positive, nonmotile, nonspore-forming, obligately anaerobic and irregularly rodshaped cell. From the morphological and biochemical Table 1. Carbohydrate fermentation patterns of Bifidobacterium lactis and related microorganisms All strains produced acid from glucose and galactose-positive, but failed to produce acid from melezitose, inositol and gluconate. ~--, positive; -, negative; w, weakly positive. JCM, Japan Collection of Microorganisms; Superscript T, type strain. JCM 1190T =ATCC 25527T; JCM 10602T=DSM 10140T.

3 BIFIDOBACTERIUM LACTIS IS A SYNONYM OF B. ANIMALIS 817 Table 2. Enzymatic activities of Bifidobacteriurn lactic JCM 10602T and related microorganisms Tests were performed using the API 20S ZYM system, and readings were determined at 4 hr. Quantity of hydrolyzed substrate: +, > 20 nmol; (+), nmol; ±, 6-10 nmol; and -, <5 nmol. All strains were lipase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, (3-glucosidase, N-acetyl-(3-glucosamidase, a- mannosidase, and a-fucosidase-negative. JCM 10602"=DSM 10140"; JCM 1190T=ATCC 25527T. properties, this isolate was considered to be a typical Bifidobacterium species. The carbohydrate fermentation patterns of strain Bb 12 were similar to B. lactis JCM 10602T, B. animalis JCM 1190T, B. animalis JCM 1253, B. animalis JCM 7117 and B. animalis JCM 7124, as shown in Tables 1 and 2. With the API ZYM systems, no differences were seen between these strains. But these strains were different from other Bifidobacterium type strains in some carbohydrate fermentation and enzymatic activity patterns. Meile et al (10) reported that the nonutilization of starch by B. lactis is a major difference from B. animalis. However, in our study, both strains were unable to utilize starch, which is consistent with the data of Lauer et al (9). This is unequivocal because the variable character was only based on starch fermentation not affecting genomic relatedness. Results of the present study have shown that strain Bb 12 isolated from fermented milk was found to be phenotypically similar with B. animalis JCM 1190' as well as B. lactis JCM , and that it could not be identified to the species level on the basis of phenotypic characteristics. Therefore, we pursued the study of these strains to define their genetic relatedness. The 16S rrna sequence of strain Bb 12 consisted of 1495 bases, and has been deposited in the DDBJ database under accession number AB Figure 1 shows a phylogenetic tree based on the neighbor-joining method. Phylogenetic analysis placed strain Bb 12, B. lactis JCM 10602T, and B. animalis JCM 1190T in the same cluster comprising B. magnum ATCC 27540T, B. cuniculi ATCC 27916T and B. pseudolongum subsp. globosum ATCC 25865". This cluster was recovered in 100% of the bootstrap trees. B. animalis is the most closely related species to B. lactis and strain Bb 12 in the phylogenetic tree, and these three strains show a high similarity in sequences (>98.8%). DNA base composition and DNA-DNA hybridization data are shown in Table 3. B. lactis JCM 10602T, strain Bb 12, B. animalis JCM 11901, B. animalis JCM 1253, B. animalis JCM 7117 and B. animalis JCM 7124 have a G+C content of 60.8 to 62.1 mol%. These strains show 82.4 to 98.1 % DNA hybridization value similarity, showing that they belong to a single species, and they have DNA homology values of less than 18.8% to B. longum JCM 1217'. It is now accepted that taxonomy based on a molecular methods, mainly on DNA homology and 16S rrna sequences, should be used to establish the phylogenetic and taxonomic position of bacterial species (16, 17). The 16S rrna phylogenetic definition of a species would generally include strains with approximately 70% or greater DNA-DNA relatedness (16). In the present study, the tree topology indicated that B. animalis JCM 1190" forms a cluster only with B. lactis JCM 10602' and strain Bb 12, and that B. magnum ATCC 27540T, B. globosum ATCC 25865' and B. cuniculi ATCC 27916' to be somewhat closely associated with this cluster. In order to clearly underline the species status of B.

4 818 Y. CAI ET AL Fig. 1. Phylogenetic tree derived from a 16S rrna sequence of the genus Bifidobacterium. The tree was created using the neighbor-joining method and K,u, values. The numbers on the tree indicate bootstrap values for the branch points. B., Bifidobacterium. *Nucleotide accession number for the 16S rrna gene. lactis, Meile et al (10) hybridized its EcoRI-restricted and uniformally labeled chromosomal DNA with the same amounts of EcoRI-cut DNA isolated from both B. animalis and B. longum. They observed 27% relatedness with B. animalis and less than 10% relatedness with B. longum, and supported the status of B. lactis as a new species based on the Southern hybridization data. However, in our study, using both microplate and membrane filter methods, the DNA-DNA hybridization results demonstrated that B. lactis JCM 10602T had a high level of DNA relatedness (>85.5%) with B. animalis JCM 1190'. More recently, protocols for DNA hybridization in microplates have been developed, and these have been successfully used to determine genetic relatedness among bacterial strains (8) and for evidence of new species (2, 5). This method can be an alternative to radioisotope and membrane filter hybridization methods (3). A comparison between two variants of the microplate DNA-DNA hybridization method and initial renaturation method was done by Goris et al (4) with 4 sets of bacteria. The authors concluded that both methods were in very good correlation, and that the microplate method could be applied as a reliable taxonomic tool because of its high speed in generating data in combination with the limited amount of DNA needed. The number of hybridization reactions can easily be extended to provide a more complete overview of the genetic relatedness of a bacterial group. In the present study, the two different DNA-DNA hybridization methods resulted in different relative binding ratios, but sim-

5 BIFIDOBACTERIUM LACTIS IS A SYNONYM OF B. ANIMALIS 819 Table 3. DNA base compositions and levels of DNA relatedness of Bifidobacterium species JCM, Japan Collection of Microorganisms. The superscript T indicates type strain. ilar percent fluorescence intensity, showing that B. lactis JCM 10602' and B. animalis JCM 1190' are a uniform species. Klein et at (7) also described that the DNA- DNA relatedness was the closest between B. lactis JCM 10602' and B. animalis JCM 1190'. Since B. lactis was described some 20 years later (10) than B. animalis, B. lactis should be reclassified as B. animalis. On the basis of phenotypic and genotypic characteristics, it is concluded that B. lactis Meile et al is a junior synonym of B. animalis (Mitsuoka) Scardovi and Trovatelli The name B. lactis has no taxonomic standing. References 1) Benno, Y A semi-automatic system for bacterial identification. Riken Rev. 12: ) Cai, Y., Okada, H., Mori, H., Benno, Y., and Nakase, T Lactobacillus paralimentarius sp. nov., isolated from sourdough. Int. J. Syst. Bacteriol. 49: ) Ezaki, T., Hashimoto, Y., and Yabuuchi, E Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacteria strains. Int. J. Syst. Bacteriol. 39: ) Goris, J., Suzuki, K., Vos, P., Nakase, T., and Kersters, K Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can. J. Microbiol. 44: ) Hou, X., Kawamura, Y., Sultana, F., Shu, S., Hirose, K., Goto, K., and Ezaki, T Description of Arthrobacter creatinolyticus sp. nov., isolated from human urine. Int. J. Syst. Bacteriol. 48: ) Kimura, M., and Ohta, T On the stochastic model for pro- estimation of mutation distance between homologous teins. J. Mol. Evol. 2: ) Klein, G., Pack, A., Bonaparte, C., and Renter, G Taxonomy and physiology of probiotic lactic acid bacteria. Int. J. Food Microbiol. 41: ) Kusunoki, S., and Ezaki, T Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscesses (Kubica et al.) to species status: Mycobacterium abscessus comb. nov. Int. J. Syst. Bacterial. 42: ) Lauer, E., and Kandler, O DNA-DNA homology, murein types and enzyme patterns in the type strains of the genus Bifidobacterium. Syst. Appl. Microbiol. 4: ) Meile, L., Ludwig, W., Rueger, U., Gut, C., Kaufmann, P., Dasen, G., Wenger, S., and Teuber, M Bifidobacteriurn lactis sp. nov., a moderately oxygen tolerant species isolated from fermented milk. Syst. Appl. Microbiol. 20: ) Mitsuoka, T The world of intestinal bacteria. In The isolation and identification of anaerobic bacteria, a color atlas of anaerobic bacteria. Sobun Press, Tokyo. 12) Miyake, T., Watanabe, K., Watanabe, T., and Oyaizu, H Phylogenetic analysis of the genus Bifidobacterium and related genera based on 16S rdna sequences. Microbiol. Immunol. 42: ) Saitou, H., and Miura, K Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72: ) Saitou, N., and Nei, M The neighbor-joining methods: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: ) Scardovi, V., and Trovatelli, L.D Bifidobacterium animalis (Mitsuoka) comb. nov. and the "minimum" and "subtile" group of new bifidobacteria found in sewage. Int. J. Syst. Bacteriol. 24: ) Stackebrandt, E., and Goebel, B.M 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: ) Stackebrandt, E., and Teuber, M Molecular taxonomy and phylogenetic position of lactic acid bacteria. Biochemie

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