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Polyphasic taxonomic analysis of Bifidobacterium animalis and Bifidobacterium lactis reveals relatedness at the subspecies level: reclassification of Bifidobacterium animalis as Bifidobacterium animalis subsp. animalis subsp. nov. and Bifidobacterium lactis as Bifidobacterium animalis subsp. lactis subsp. nov.

The taxonomic standing of Bifidobacterium lactis and Bifidobacterium animalis was investigated using a polyphasic approach. Sixteen representatives of both taxa were found to be phenotypically similar and shared more than 70% DNA-DNA relatedness (76-100%), which reinforces the conclusions of previous studies in which B. lactis and B. animalis were considered to be one single species. However, the results of protein profiling, BOX-PCR fingerprinting, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), and atpD and groEL gene sequence analysis demonstrate that representatives of B. animalis and B. lactis constitute two clearly separated subgroups; this subdivision was also phenotypically supported based on the ability to grow in milk. Given the fact that B. lactis Meile et al. 1997 has to be considered as a junior synonym of B. animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974, our data indicate that the latter species should be split into two new subspecies, i.e. Bifidobacterium animalis subsp. animalis subsp. nov. (type strain R101-8T=LMG 10508T=ATCC 25527T=DSM 20104T=JCM 1190T) and Bifidobacterium animalis subsp. lactis subsp. nov. (type strain UR1T=LMG 18314T=DSM 10140T=JCM 10602T).

Animals↗

Phenotypic and genomic analyses of human strains belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve.

A numerical analysis based on phenotypic characteristics (89 enzymatic tests and 49 carbohydrate acidification tests), in which experimental strips from Biomerieux-API, La Balme les Grottes, France, were used, was performed to characterize 82 new isolates belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. A total of 72 strains were isolated from child or adult feces, and the other strains were obtained from human vaginas and bronchi. In this study we also included 38 type and reference strains that were representative of all species of the genus Bifidobacterium and 6 strains belonging to the genus Lactobacillus. DNA-DNA relationships between B. longum and B. infantis were determined by using 19 strains related to these species, as determined by the numerical analysis. The degree of DNA binding was determined by the S1 nuclease method. The phenotypic study revealed that there were six main clusters, which were subdivided into nine subclusters. Subcluster Va contained the type strains of B. longum and B. infantis. The DNA-DNA relatedness values of some of the new isolates were very similar to the DNA-DNA relatedness values of the type strain of B. longum. On the basis of these data, it was difficult to isolate B. infantis strains and then to define B. infantis as a single species separated from B. longum. Subclusters IVb to IVf comprised reference strains of B. breve. Cluster III and subcluster Ia were not identified.

Actinomycetales Infections↗

Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum.

The relationships between Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis were examined by means of carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). The levels of DNA-DNA hybridization among the strains of B. infantis, B. longum and B. suis used in this study were 67-81% under optimal conditions (42 degrees C) and 63-85% under stringent conditions (52 degrees C). Although the strains showed varied carbohydrate-fermentation patterns, the three species were divided into three types, namely the infantis type, the longum type and the suis type, by ribotyping and RAPD-PCR. On the basis of these results, strains of B. infantis, B. longum and B. suis were recognized as distinct groups within a single species. It is concluded that B. infantis and B. suis should be unified as B. longum, the latter species being divided into three biotypes, the infantis type, the longum type and the suis type, by molecular methods.

Bifidobacterium↗

Species identification of genus Bifidobacterium based on partial HSP60 gene sequences and proposal of Bifidobacterium thermacidophilum subsp. porcinum subsp. nov.

Sequence homology of partial 60 kDa heat-shock protein (HSP60) genes was analysed for 50 Bifidobacterium strains that represent 12 Bifidobacterium species and subspecies with validly published names. Sequence similarities were 96.5-100 % within the same species, 95.5-97 % at the subspecies level and 80-96 % (mean, 88 %) at the interspecies level among the 10 Bifidobacterium species. Hence, the HSP60 gene was a more accurate tool for species identification within the genus Bifidobacterium than 16S rDNA. Two new Bifidobacterium strains isolated from piglet faeces were shown to be closely related to the thermophilic bifidobacterial group, based on 16S rDNA sequence analysis: strain P3-14(T) (=AS 1.3009(T)=LMG 21689(T)) exhibited 97.9 % similarity to Bifidobacterium boum JCM 1211(T), 97.2 % similarity to Bifidobacterium thermacidophilum AS 1.2282(T) and 97 % similarity to Bifidobacterium thermophilum JCM 1207(T). However, higher levels of DNA-DNA relatedness (83 %) and HSP60 gene sequence similarity (97 %) were determined between B. thermacidophilum AS 1.2282(T) and strain P3-14(T), indicating a closer relationship between them. The new strains differed from B. thermacidophilum AS 1.2282(T) in some phenotypic characteristics, such as growth at a lower temperature (46.5 degrees C), as well as different sugar-fermentation patterns. Hence, a novel Bifidobacterium subspecies, Bifidobacterium thermacidophilum subsp. porcinum subsp. nov., is designated.

Bifidobacterium↗

Bifidobacterium inopinatum sp. nov. and Bifidobacterium denticolens sp. nov., two new species isolated from human dental caries.

In a previous investigation of bifidobacteria isolated from human dental caries (V. Scardovi and F. Crociani, Int. J. Syst. Bacteriol. 24:6-20, 1974), 40 strains were assigned to the new species Bifidobacterium dentium. In this study we examined 70 new strains of bifidobacteria isolated from dental caries. The morphological characteristics, biochemical reactions, fermentation patterns, end products from glucose metabolism, protein electrophoretic patterns, levels of DNA hybridization, and DNA G+C contents of these organisms revealed that they belong to three different taxa. One of these taxa was identified as B. dentium. The other two are described as the following new Bifidobacterium species in this paper: Bifidobacterium inopinatum (type strain, DSM 10107) and Bifidobacterium denticolens (type strain, DSM 10105). The two new species differ from other Bifidobacterium species in their morphological characteristics (especially B. inopinatum, with its very small coccoid cells), in their carbohydrate fermentation patterns (most strains ferment dextran, and B. inopinatum does not ferment galactose), and in their DNA base compositions (especially B. inopinatum).

Bacterial Proteins↗

Comparative sequence analysis of the tuf and recA genes and restriction fragment length polymorphism of the internal transcribed spacer region sequences supply additional tools for discriminating Bifidobacterium lactis from Bifidobacterium animalis.

The relationship between Bifidobacterium lactis and Bifidobacterium animalis was examined by comparative analysis of tuf and recA gene sequences and by restriction fragment length polymorphism analysis of their internal 16S-23S transcribed spacer region sequences. The bifidobacterial strains investigated could be divided into two distinct groups within a single species based on the tuf, recA, and 16S-23S spacer region sequence analysis. Therefore, all strains of B. lactis and B. animalis could be unified as the species B. animalis and divided into two subspecies, Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis.

Animals↗

Use of modified Lactobacillus selective medium and Bifidobacterium iodoacetate medium for differential enumeration of Lactobacillus acidophilus and Bifidobacterium spp. in powdered nutritional products.

Modified Lactobacillus selective agar (APT agar + sodium acetate and glacial acetic acid; mLBS) was compared to selective modified Lactobacillus selective medium (LBS agar + tomato juice and acetic acid; mLSM) and nonselective de Man Rogosa Sharpe (MRS) agar for the enumeration of Lactobacillus acidophilus in probiotic-containing powdered nutritional products. The mLBS agar was equivalent to MRS agar and superior to the mLSM agar for enumerating L. acidophilus in products stored in sealed cans at 22 degrees C. When samples were analyzed for L. acidophilus concentration after high temperature storage in sealed cans or storage in open cans at high relative humidity, the mLBS and MRS agars were highly correlated (r2 = 0.93). Modified Bifidobacterium iodoacetate medium (12.5 mg iodoacetic acid/liter; mBIM) was compared to MRS agar + bile, cysteine, and dicloxacillin (MRS + BCD) for enumerating Bifidobacterium infantis or Bifidobacterium lactis in the nutritional products. The two media were equivalent for enumerating B. infantis in product stored at 22 degrees C in sealed cans. However, the two media were poorly correlated (r2<0.50) for enumeration of B. infantis and B. lactis in products stored in sealed cans at high temperature or in open cans at high relative humidity. The mLBS medium has potential industry application as a relatively inexpensive, convenient differential enumeration method for L. acidophilus. The mBIM medium cannot be recommended as a sole medium for enumeration of probiotic Bifidobacterium spp. in powdered nutritional products stored under high temperature and/or high relative humidity conditions.

Agar↗

Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum.

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli-Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium.

Amino Acid Sequence↗

PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation.

A PCR-ELISA method was extended for detection of most common Bifidobacterium species in humans and applied to a feeding trial including administration of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide (GOS)-containing syrup as probiotic and prebiotic preparations, respectively. For PCR-ELISA, oligonucleotide probes based on 16S rDNA sequences were designed and tested for specificity and sensitivity with nine different bifidobacterial species followed by analysis of faecal samples. Bifidobacteria were monitored for their fluctuations during and after the feeding trial. Bifidobacterium longum was the most common species found in the faecal samples, followed by B. adolescentis and B. bifidum. During ingestion of the probiotic B. lactis Bb-12, the strain appeared in the faeces but was absent again one week after finishing of the trial. The species that were observed in the faecal samples taken prior to the feeding experiments persisted also in samples derived from the pre-feeding and feeding periods. The most consistent change observed was the decrease in the relative amount of B. longum in the test group ingesting either B. lactis Bb-12 alone or in combination with GOS-syrup. Since the amounts of B. longum increased again in the post-feeding sample with these subjects, it may suggest that to some extent B. lactis Bb-12 is able to transiently replace B. longum.

Animals↗

Bifidobacterium lactis Meile et al. 1997 is a subjective synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974.

Bifidobacterium lactis 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. lactis should be considered as a junior subjective synonym of B. animalis.

Bifidobacterium↗

Different utilization of glucose and raffinose in Bifidobacterium breve and Bifidobacterium animalis.

Growth of 2 strains of Bifidobacterium breve and 4 strains of Bifidobacterium animalis was evaluated with glucose, raffinose or a mixture of glucose and raffinose as substrates. All strains of B. animalis and one strain of B. breve grew more slowly on glucose than on raffinose or the mixture of glucose and raffinose; one strain of B. breve grew more rapidly on the mixture of glucose and raffinose than on raffinose while 2 strains of B. animalis grew faster on raffinose than on the mixture of raffinose and glucose. Both strains of B. breve utilized glucose and raffinose simultaneously. In contrast, all strains of B. animalis strains displayed atypical growth with rapid utilization of raffinose followed by slow utilization of glucose. The cell morphology of all strains of B. animalis was affected by the sugar used for cultivation: tiny and rather spherical cells were observed on glucose while on media with raffinose the cells were large and had the species-specific shape. Description of preferential utilization of various saccharides by bifidobacteria can contribute to the development of new synbiotic preparations and new cultivation media for bifidobacteria.

Animals↗

Electron microscopic, biochemical and physiological studies of Bifidobacterium pseudolongum SS-24 and Bifidobacterium thermophilum SS-19.

Comparative studies of physiology, biochemical characteristics, and morphology by electron microscopy were conducted on Bifidobacterium pseudolongum SS-24 isolated from dogs and Bifidobacterium thermophilum SS-19 isolated from swine. Both B. pseudolongum and B. thermophilum grow unusually rapidly in the rumen fluid medium of Scott and Dehority, and reached a maximum of optical density after only 6 to 7 h of incubation. B. pseudolongum and B. thermophilum showed similar patterns of results for 21 biochemical characteristics tested, with a difference found only for N-acetyl-glucosaminidase. Scanning electron micrographs revealed that B. pseudolongum produced extensive amounts of extracellular material. The cell walls of B. pseudolongum and B. thermophilum were totally different. Transmission electron micrographs of ruthenium red-stained preparations of B. pseudolongum showed a very thick (ca. 0.2 microns) Gram-positive cell wall, whereas B. thermophilum was found to have a thin (ca. 0.05 microns) Gram-positive cell wall.

Animals↗

Microbiological effects of consuming a synbiotic containing Bifidobacterium bifidum, Bifidobacterium lactis, and oligofructose in elderly persons, determined by real-time polymerase chain reaction and counting of viable bacteria.

BACKGROUND: Because of changes in gut physiology, immune system reactivity, and diet, elderly people are more susceptible to gastrointestinal infections than are younger adults. The gut microflora, which provides a natural defense against invading microorganisms, changes in elderly people with the development of potentially damaging bacterial populations, which may lead to alterations in bacterial metabolism and higher levels of infection. METHODS: A randomized, double-blind, controlled feeding trial was done with 18 healthy elderly volunteers (age, >62 years) using a synbiotic comprising Bifidobacterium bifidum BB-02 and Bifidobacterium lactis BL-01 (probiotics) together with an inulin-based prebiotic (Synergy 1; Orafti). Real-time PCR was employed to quantitate total bifidobacteria, B. bifidum, and B. lactis in fecal DNA before, during, and after synbiotic consumption. Counting all viable anaerobes, bifidobacteria, and lactobacilli and identification of bacterial isolates to species level was also done. RESULTS: Throughout feeding, both bifidobacteria species were detected in fecal samples obtained from all subjects receiving the synbiotic, with significant increases in the number of copies of the 16S rRNA genes of B. bifidum, B. lactis, and total bifidobacteria, compared with the control week and the placebo group. At least 1 of these species remained detectable in fecal samples 3 weeks after feeding in individuals that had no fecal B. bifidum and/or B. lactis in the control week, indicating that the probiotics persisted in the volunteers. Counting of viable organisms showed significantly higher total numbers of fecal bifidobacteria, total numbers of lactobacilli, and numbers of B. bifidum during synbiotic feeding. CONCLUSION: Synbiotic consumption increased the size and diversity of protective fecal bifidobacterial populations, which are often very much reduced in older people.

Aged↗

Structural studies of cell wall polysaccharides from Bifidobacterium breve YIT 4010 and related Bifidobacterium species.

The chemical compositions of the cell walls obtained from 8 strains in 5 species of Bifidobacterium were analyzed. These cell walls were shown to be composed of peptidoglycan and polysaccharide moieties. Some variations with respect to contents of neutral sugars and content of phosphorus were observed with some cell wall preparations from the same species. The neutral polysaccharides in cell walls of 4 strains of Bifidobacterium (B. bifidum YIT 4007, B. breve YIT 4010, B. infantis YIT 4025, and B. longum ATCC 15707) were purified and their chemical structures were analyzed. One of these polysaccharides, obtained from B. breve YIT 4010, was analyzed in detail by GLC, 1H- and 13C-NMR spectroscopic analyses, methylation, Smith degradation and acetolysis, and the results suggested the following structure for the repeating unit of the polysaccharide: (Formula: see text).

Amino Acids↗

Bifidobacterium ruminantium sp. nov. and Bifidobacterium merycicum sp. nov. from the rumens of cattle.

Among several hundred bifidobacteria isolated from bovine rumens, eight strains were recognized primarily on the basis of DNA-DNA hybridization results as members of two new distinct DNA homology groups. We studied the morphology, oxygen, carbon dioxide, temperature, and pH requirements, fermentation patterns, end products of glucose fermentation, biochemical reactions, protein electrophoretic patterns, isozyme patterns, DNA homology relationships, and guanine-plus-cytosine contents of these organisms, and we propose that these two groups of strains should be considered new species, Bifidobacterium ruminantium (type strain, strain ATCC 49390) and Bifidobacterium merycicum (type strain, strain ATCC 49391).

Animals↗

Transfer of Bifidobacterium inopinatum and Bifidobacterium denticolens to Scardovia inopinata gen. nov., comb. nov., and Parascardovia denticolens gen. nov., comb. nov., respectively.

Bifidobacterium inopinatum Crociani et al. 1996 and Bifidobacterium denticolens Crociani et al. 1996 have distinct phenotypic characteristics and low G+C contents compared with other bifidobacteria. In the 16S rRNA phylogenetic tree, these two species grouped in an independent subcluster. In our previous work, partial heat-shock protein 60 (HSP60) gene-sequence analysis also indicated that these two species had distinct taxonomic positions. In this work, the complete HSP60 genes of five representative bacterial strains were sequenced by using an inverse PCR method. The complete sequence similarities turned out to be at the same level as those of the partial genes, thus confirming the result based on partial sequence analysis. On the basis of all the evidence mentioned above, it is proposed that these two species should be transferred to two new genera as Scardovia inopinata gen. nov., comb. nov., and Parascardovia denticolens gen. nov., comb. nov.

Bacterial Typing Techniques↗

[Architectonics of Bifidobacteria populations: submicroscopic aspect of cell cohesion in Bifidobacterium adolescentis and Bifidobacterium bifidum].

Populations (cultures) of Bifidobacterium adolescents and B. bifidum, growing on artificial liquid and agar media, are presented by highly ordered mycelial structures. The topography of them depends on mutual arrangement of polymorphic cells and the way of their daughter cells separation after division. Evidences obtained by scanning electron microscopy (SEM) of total preparations and by transmission electron microscopy (TEM) of ultrathin sections correlate well. These data showed the existence of morphologically varied intercellular contacts (coherence) that ensure the stability of such microbial consortia during adaptation to ambient conditions. Intercellular contacts with the aid of different extracellular structures--microfibrillae, knob-like juts, cell wall evaginations, and capsuleform stuff(glycocalix)--are the result of genetically determined self-regulating development of microbial populations as multicellular systems.

Adaptation, Physiological↗