Akihito Endo

Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov.

A taxonomic study was made of the genus Leuconostoc. The species in the genus were divided into three subclusters by phylogenetic analysis based on the 16S rRNA gene sequences. The three subclusters were the Leuconostoc mesenteroides subcluster (comprising L. carnosum, L. citreum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis, L. mesenteroides and L. pseudomesenteroides), the L. fructosum subcluster (L. durionis, L. ficulneum, L. fructosum and L. pseudoficulneum) and the L. fallax subcluster (L. fallax). Phylogenetic trees based on the sequences of the 16S-23S rRNA gene intergenic spacer region, the rpoC gene or the recA gene indicated a good correlation with the phylogenetic tree based on 16S rRNA gene sequences. The species in the L. fructosum subcluster were morphologically distinguishable from the species in the L. mesenteroides subcluster and L. fallax as species in the L. fructosum subcluster had rod-shaped cells. In addition, the four species in the L. fructosum subcluster needed an electron acceptor for the dissimilation of d-glucose and produced acetic acid from d-glucose rather than ethanol. On the basis of evidence presented in this study, it is proposed that the four species in the L. fructosum subcluster, Leuconostoc durionis, Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc pseudoficulneum, should be transferred to a novel genus, Fructobacillus gen. nov., as Fructobacillus durionis comb. nov. (type strain D-24(T)=LMG 22556(T)=CCUG 49949(T)), Fructobacillus ficulneus comb. nov. (type strain FS-1(T)=DSM 13613(T)=JCM 12225(T)), Fructobacillus fructosus comb. nov. (type strain IFO 3516(T)=DSM 20349(T)=JCM 1119(T)=NRIC 1058(T)) and Fructobacillus pseudoficulneus comb. nov. (type strain LC-51(T)=DSM 15468(T)=CECT 5759(T)). The type species of the genus Fructobacillus is Fructobacillus fructosus gen. nov., comb. nov.. No significant physiological and biochemical differences were found between the species in the L. mesenteroides subcluster and L. fallax in the present study and thus L. fallax remains as a member of the genus Leuconostoc.

Honeybees and beehives are rich sources for fructophilic lactic acid bacteria.

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics.

Lactobacillus florum sp. nov., a fructophilic species isolated from flowers.

Three strains of fructophilic lactic acid bacteria were isolated from flowers in South Africa. The isolates formed a subcluster in the Lactobacillus buchneri phylogenetic group, closely related to Lactobacillus fructivorans, Lactobacillus homohiochii, Lactobacillus lindneri and Lactobacillus sanfranciscensis according to phylogenetic analysis based on the 16S rRNA gene sequences. Levels of DNA-DNA relatedness indicated that the three strains belonged to the same taxon and formed a genetically distinct group, well separated from their phylogenetic relatives. The three strains produced acids from only two of the 49 carbohydrates tested, i.e. D-glucose and D-fructose. D-Fructose was more rapidly fermented than D-glucose. Good growth was recorded on d-fructose or D-glucose in the presence of external electron acceptors. However, delayed growth was recorded on d-glucose without electron acceptors. The novel strains produced lactic acid, ethanol and acetic acid from D-glucose at a ratio of 1 : 0.8 : 0.2. These characteristics were distinct from other species of the genus Lactobacillus. Based on the data provided, the three isolates represent a fructophilic and novel species of the genus Lactobacillus, for which the name Lactobacillus florum sp. nov. is proposed. The type strain is F9-1(T) (=JCM 16035(T)=DSM 22689(T)=NRIC 0771(T)).

Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG(+) phenotype, lack of virulence and absence of a capsule

Aims:  To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice.

Bifidobacterium reuteri sp. nov., Bifidobacterium callitrichos sp. nov., Bifidobacterium saguini sp. nov., Bifidobacterium stellenboschense sp. nov. and Bifidobacterium biavatii sp. nov. isolated from faeces of common marmoset (Callithrix jacchus) and red-handed tamarin (Saguinus midas)

Five strains of bifidobacteria were isolated from faeces of a common marmoset (Callithrix jacchus) and a red-handed tamarin (Saguinus midas). The five isolates clustered inside the phylogenetic group of the genus Bifidobacterium but did not show high sequence similarities between the isolates and to known species in the genus by phylogenetic analysis based on 16S rRNA gene sequences. Sequence analyses of dnaJ1 and hsp60 also indicated their independent phylogenetic positions to each other in the Bifidobacterium cluster. DNA G+C contents of the species ranged from 57.3 to 66.3 mol%, which is within the values recorded for Bifidobacterium species. All isolates showed fructose-6-phosphate phosphoketolase activity. Based on the data provided, the five isolates represent five novel species, for which the names Bifidobacterium reuteri sp. nov. (type strain: AFB22-1(T) = JCM 17295(T) = DSM 23975(T)), Bifidobacterium callitrichos sp. nov. (type strain: AFB22-5(T) = JCM 17296(T) = DSM 23973(T)), Bifidobacterium saguini sp. nov. (type strain: AFB23-1(T) = JCM 17297(T) = DSM 23967(T)), Bifidobacterium stellenboschense sp. nov. (type strain: AFB23-3(T) = JCM 17298(T) = DSM 23968(T)) and Bifidobacterium biavatii sp. nov. (type strain: AFB23-4(T) = JCM 17299(T) = DSM 23969(T)) are proposed.

Taxonomic status of lactic acid bacteria in wine and key characteristics to differentiate species

Oenococcus oeni is the best malolactic bacterium adapted to low pH and the high SO2 and ethanol concentrations in wine. Leuconostoc mesenteroides and Leuconostoc paramesenteroides (now classified as Weissella paramesenteroides) have also been isolated from wine. Pediococcus damnosus is not often found in wine and is considered a contaminant of high pH wines. Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus have occasionally been isolated from wines. Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus hilgardii (previously Lactobacillus vermiforme), Lactobacillus fructivorans (previously Lactobacillus trichoides and Lactobacillus heterohiochii) and Lactobacillus fermentum have been isolated from most wines. Lactobacillus hilgardii and L. fructivorans are resistant to high acid and alcohol and have been isolated from spoiled fortified wines. Lactobacillus vini, Lactobacillus lindneri, Lactobacillus nagelii and Lactobacillus kunkeei have been described more recently. The latter two species are known to cause sluggish or stuck alcoholic fermentations in wine. Although Lactobacillus collinoides and Lactobacillus mali (previously Lactobacillus yamanashiensis) decarboxylate L-malic acid, they are more often found in cider and fruit juices. Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus diolivorans, Lactobacillus jensenii and Lactobacillus paracasei are seldomly isolated from wines. Some strains of Lactobacillus casei may be closer related to Lactobacillus paracasei or a distant relative, Lactobacillus zeae. Oenococcus kitaharae, isolated from compost is genetically closely related to Oenococcus oeni, but does not decarboxylate malate, prefers higher growth pH and is phenotypically well distinguished from O. oeni. This review summarises the current taxonomic status of malolactic bacteria and lists key phenotypic characteristics that may be used to identify the species.

Recommended minimal standards for description of new taxa of the genera Bifidobacterium, Lactobacillus and related genera.

Minimal standards for the description of new cultivable strains that represent novel genera and species belonging to the genera Bifidobacterium, Lactobacillus and related genera are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision): the description of novel species should be based on phenotypic, genotypic and ecological characteristics to ensure a rich polyphasic characterization. Concerning genotypic characterization, in addition to DNA G+C content (mol%) data, the description should be based on DNA-DNA hybridization (DDH), 16S rRNA gene sequence similarities and at least two housekeeping gene (e.g. hsp60 and recA) sequence similarities. DDH might not be needed if the 16S rRNA gene sequence similarity to the closest known species is lower than 97 %. This proposal has been endorsed by members of the Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms of the International Committee on the Systematics of Prokaryotes.

Fructophilic Lactobacillus kunkeei and Lactobacillus brevis Isolated from Fresh Flowers, Bees and Bee-hives

Two-hundred-and-thirty-six isolates were collected from fresh flowers, bees and bee-hives. Of these, 20 isolates preferred d-fructose as carbon source, produced lactic acid and acetic acid but trace amounts of ethanol and were classified as fructophilic. Poor growth was recorded when strains were incubated anaerobically in the presence of d-glucose as sole carbon source. Good growth was, however, recorded when d-glucose was metabolized in the presence of external electron acceptors such as fructose, pyruvate and oxygen. Nineteen of the strains were classified as Lactobacillus kunkeei and one as Lactobacillus brevis based on phenotypic characteristics, 16S rRNA sequences, recA sequences and DNA homology. This is the first description of a fructophilic strain of L. brevis.

Fructobacillus tropaeoli sp. nov., a fructophilic lactic acid bacterium isolated from a flower

A fructophilic lactic acid bacterium, designated strain F214-1(T), was isolated from a flower of Tropaeolum majus in South Africa. Based on phylogenetic analysis of 16S rRNA gene sequences, the strain formed a subcluster with Fructobacillus ficulneus and Fructobacillus pseudoficulneus and, based on recA gene sequences, the strain formed a subcluster with F. ficulneus. DNA-DNA hybridization studies showed that strain F214-1(T) was phylogenetically distinct from its closest relatives. Acid was produced from the fermentation of d-glucose, d-fructose and d-mannitol only. d-Fructose was the preferred sole carbon and energy source and was fermented more rapidly than d-glucose. Growth of the strain on d-glucose under anaerobic conditions was very weak but external electron acceptors such as oxygen and pyruvate enhanced growth on d-glucose. Lactic acid and acetic acid were produced from d-glucose in equimolar amounts. Ethanol was produced at very low levels, despite the strains obligately heterofermentative metabolism. Based on these data, strain F214-1(T) represents a novel species of fructophilic bacteria in the genus Fructobacillus, for which the name Fructobacillus tropaeoli sp. nov. is proposed. The type strain is F214-1(T) ( = JCM 16675(T)  = DSM 23246(T)).

Diversity of Lactobacillus and Bifidobacterium in feces of herbivores, omnivores and carnivores.

The Lactobacillus and Bifidobacterium population in the feces of 26 animals (16 species) were studied by culture-dependent and culture-independent techniques. Lactobacilli were detected from a few herbivores, all carnivores and some omnivores. Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus vaginalis and Lactobacillus ingluviei were the most dominant lactobacilli in carnivores. These species were, however, not predominant in herbivores and omnivores. Lactobacillus brevis, Lactobacillus casei, Lactobacillus parabuchneri, Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, usually present in raw plant material, were present in omnivores but not in carnivores. Bifidobacteria were detected in only four herbivores and two omnivores. Bifidobacterium pseudolongum was the only Bifidobacterium species detected in herbivores. Bifidobacteria detected in the two omnivores are phylogenetically not closely related to known species and are possible novel species in the genus.