Effect of dietary supplementation of Bifidobacterium and Lactobacillus strains in Apis mellifera L. against Nosema ceranae.

Nosema ceranae is a widespread microsporidium of European honeybee Apis mellifera L. affecting bee health. The ban of Fumagillin-B (dicyclohexylammonium salt) in the European Union has driven the search for sustainable strategies to prevent and control the infection. The gut microbial symbionts, associated to the intestinal system of vertebrates and invertebrates and its impact on host health, are receiving increasing attention. In particular, bifidobacteria and lactobacilli, which are normal inhabitants of the digestive system of bees, are known to protect their hosts via antimicrobial metabolites, immunomodulation and competition. In this work, the dietary supplementation of gut bacteria was evaluated under laboratory conditions in bees artificially infected with the parasite and bees not artificially infected but evidencing a low natural infection. Supplemented bacteria were selected among bifidobacteria, previously isolated, and lactobacilli, isolated in this work from healthy honeybee gut. Four treatments were compared: bees fed with sugar syrup (CTR); bees fed with sugar syrup containing bifidobacteria and lactobacilli (PRO); bees infected with N. ceranae spores and fed with sugar syrup (NOS); bees infected with N. ceranae and fed with sugar syrup containing bifidobacteria and lactobacilli (NP). The sugar syrup, with or without microorganisms, was administered to bees from the first day of life for 13 days. N. ceranae infection was carried out individually on anesthetised 5-day-old bees. Eight days after infection, a significant (P<0.05) lower level of N. ceranae was detected by real-time PCR in both NP and PRO group, showing a positive effect of supplemented microorganisms in controlling the infection. These results represent a first attempt of application of bifidobacteria and lactobacilli against N. ceranae in honeybees.

Bifidobacterium aesculapii sp. nov., from the faeces of the baby common marmoset (Callithrix jacchus).

Six Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase-positive bacterial strains with a peculiar morphology were isolated from faecal samples of baby common marmosets (Callithrix jacchus). Cells of these strains showed a morphology not reported previously for a bifidobacterial species, which resembled a coiled snake, always coiled or ring shaped or forming a 'Y' shape. Strains MRM 3/1(T) and MRM 4/2 were chosen as representative strains and characterized further. The bacteria utilized a wide range of carbohydrates and produced urease. Glucose was fermented to acetate and lactate. Strain MRM 3/1(T) showed a peptidoglycan type unique among members of the genus Bifidobacterium. The DNA base composition was 64.7 mol% G+C. Almost-complete 16S rRNA, hsp60, clpC and rpoB gene sequences were obtained and phylogenetic relationships were determined. Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1(T) and MRM 4/2 had the highest similarities to Bifidobacterium scardovii DSM 13734(T) (94.6%) and Bifidobacterium stellenboschense DSM 23968(T) (94.5%). Analysis of hsp60 showed that both strains were closely related to B. stellenboschense DSM 23968(T) (97.5% similarity); however, despite this high degree of similarity, our isolates could be distinguished from B. stellenboschense DSM 23968(T) by low levels of DNA-DNA relatedness (30.4% with MRM 3/1(T)). Strains MRM 3/1(T) and MRM 4/2 were located in an actinobacterial cluster and were more closely related to the genus Bifidobacterium than to other genera in the family Bifidobacteriaceae. On the basis of these results, strains MRM 3/1(T) and MRM 4/2 represent a novel species within the genus Bifidobacterium, for which the name Bifidobacterium aesculapii sp. nov. is proposed; the type strain is MRM 3/1(T) ( = DSM 26737(T) = JCM 18761(T)).

The potential of bifidobacteria as a source of natural folate.

AIMS: To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate-free and complex folate-containing media. METHODS AND RESULTS: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 µg per 100 g dry matter (DM). The highest value of 9295 µg per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 µg per 100 g DM. Ten strains grew in a synthetic folate-free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate-containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5-CH(3) -H(4) folate and H(4) folate were strain dependent. 5-CH(3) -H(4) folate dominated in most strains. CONCLUSIONS: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate-trophic probiotics or part of the natural microbiota.

Proposal to reclassify the three biotypes of Bifidobacterium longum as three subspecies: Bifidobacterium longum subsp. longum subsp. nov., Bifidobacterium longum subsp. infantis comb. nov. and Bifidobacterium longum subsp. suis comb. nov.

In the year 2002, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium suis were unified into a single species, Bifidobacterium longum, preserving the former species names through the creation of the three biotypes 'longum', 'infantis' and 'suis'. Consequently, the use of the species names B. infantis and B. suis was to be discontinued. The above taxonomic rearrangement of B. longum was based on DNA-DNA hybridizations and 16S rRNA and HSP60 gene sequence analysis. However, a variety of other genotypic techniques including ribotyping, amplified rDNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD)-PCR, BOX-PCR, PCR-denaturing gradient gel electrophoresis (DGGE), comparison of the recA, tuf and ldh gene sequences, plasmid profiling and considerable variation in carbohydrate fermentation patterns as well as results of starch and PAGE electrophoresis experiments clearly discriminate former B. longum, B. infantis and B. suis strains. In the present paper we compile this published information and propose the description of Bifidobacterium longum subsp. longum subsp. nov., Bifidobacterium longum subsp. infantis comb. nov. and Bifidobacterium longum subsp. suis comb. nov. The International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms is in favour of this proposal. The type strains of Bifidobacterium longum subsp. longum subsp. nov., subsp. infantis comb. nov. and subsp. suis comb. nov. are E194b (variant a)T (ATCC 15707T=DSM 20219T), S12T (=ATCC 15697T=DSM 20088T) and Su859T (ATCC 27533T=DSM 20211T), respectively.

Occurrence of the family bifidobacteriaceae in human dental caries and plaque.

Despite the constant presence of members of Bifidobacteriaceae in the human oral cavity, few studies of their occurrence in this habitat have been made. The presence and the distribution in human plaque and dental caries of Bifidobacterium dentium, Scardovia inopinata and Parascardovia denticolens, all belonging to the family Bifidobacteriaceae, were studied. The identification to species level was based on morphological and growth type features, fermentation tests, polyacrylamide gel electrophoresis of the soluble proteins and DNA-DNA homology. The three species, isolated on propionic acid trypticase phytone yeast extract medium, were present in 13 out of 19 and in 11 out of 15 subjects examined for dental caries and plaque, respectively. S. inopinata was the species most frequently isolated in dental caries, whereas B. dentium was more numerous in dental plaque. The prevalence of P. denticolens was similar in the two habitats. In conclusion, the prevalence of bifidobacteria species (B. dentium, P. denticolens, S. inopinata) in relation to non-bifidobacteria isolates was significantly different in caries versus plaque samples (p<0.0001; chi2 test).

Bifidobacterium animalis protects intestine from damage induced by zinc deficiency in rats.

We investigated the potential beneficial effects of Bifidobacterium animalis on intestinal damage using zinc-deficient (ZD) rats as a model for intestinal alterations. The ZD rats were fed diets containing 1 mg Zn/kg for 20 (ZD(20)) or 40 (ZD(40)) d to induce damage that differed in severity. Subgroups of these rats, the ZD(20) + B and ZD(40) + B groups, received a suspension of B. animalis (3.5 x 10(8) colony forming units) daily for the last 10 d. Another subgroup, the ZD(40) + B + 7 d group, was fed the ZD diet for 7 d after the B. animalis treatment period. Zinc deficiency induced ulcerations, edema, inflammatory cell infiltration and dilatation of blood vessels in duodenum, jejunum and ileum, with increasing severity between 20 and 40 d of zinc deficiency. The mucosa of the ZD(20) + B group was well preserved, and most of the morphologic alterations induced by zinc deficiency were normalized in the ZD(40) + B group. The high fecal concentrations of B. animalis in the ZD(40) + B and ZD(40) + B + 7 d groups indicate that these bifidobacteria survived passage through the gastrointestinal tract and proliferated. Electron microscopy confirmed the elevated numbers of bifidobacteria in cecum. Treatment with B. animalis resulted in greater epithelial cell proliferation and disaccharidase activities in the ZD(40) + B group compared with the ZD(40) group. These findings indicate that B. animalis can protect the intestine from alterations induced by zinc deficiency, suggesting that this bacterium may play a role in intestinal mucosal defense.

Effect of growth temperature on the biosynthesis of cell wall proteins from Bifidobacterium globosum.

Seventy strains of Bifidobacterium globosum isolated from gastrointestinal tracts of different animals were studied. Strains were grown at temperatures ranging from 25 to 46.5 degrees C in order to examine changes both in the expression of bifidobacterial outer proteins (BIFOPs) and in their hydrophobic properties. It was observed that the expression of BIFOPs found on the cell-surface changes according to growth temperature, with quantitative and/or qualitative variations. Generally speaking, it was observed that BIFOP expression at low-growth temperature was considerably attenuated, while at medium- and high-growth temperature it increased. Furthermore, at high-growth temperatures, the presence of a new common protein was detected in all the strains studied. Cells from B. globosum strains grown under different temperature conditions were studied in terms of their cellular hydrophobicity properties. At medium-growth temperature, the cell hydrophobicity was strictly correlated with BIFOP expression, while at low and high-growth temperatures, the presence of BIFOP only partially influenced the hydrophobic features.

Influence of aminoacid requirement on the growth of Bifidobacterium globosum strains.

A general procedure has been devised for the determination of amino acid requirements in Bifidobacterium globosum strains, based upon identification of individual amino acids singularly deprived of the defined synthetic medium. In the plasmid-positive and plasmid-negative clones of RU 809 and T 19 strains, we found a correlation between the presence of plasmid and L-leucine auxotrophy. This characteristic is not shared by the other 145 strains, 26 of which are plasmid-positive, of the B. globosum species.

L-leucine auxotrophy in Bifidobacterium globosum.

The enzymes involved in the biosynthetic pathway of L-leucine were studied in plasmid-negative and plasmid-positive clones derived from the RU 809 strain of the Bifidobacterium globosum species. The growth of plasmid-positive clones in synthetic medium required L-leucine. We have shown that no detectable activity of the beta-isopropylmalate dehydrogenase enzyme was present in plasmid-positive clones, whereas detectable and significant activity of this enzyme was found in plasmid-negative clones. The lack of activity of the beta-isopropylmalate dehydrogenase enzyme is considered responsible for the L-leucine auxotrophy in the plasmid-positive clones.

Bacteria in biopsies of human hypochlorhydric stomach: a scanning electron microscopy study.

A long-lasting condition of hypochloridria leads to a bacterial growth both in the gastric lumen and biopsies of human stomach. Some of these bacteria are probably involved in gastric carcinogenesis, due to their capacity of nitrosation. This study was carried out on biopsies taken during endoscopy from both gastric antrum and the body of patients with or without hypochloridria. Scanning electron microscopy observation shows that bacteria, other than Helicobacter pylori, found in hypochloridria, can be located not only over but also into and under the mucus layer covering the gastric epithelium. In such areas, mechanical and biochemical damage may occur.

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).

Degradation of complex carbohydrates by Bifidobacterium spp.

Two hundred and ninety strains of 29 species of bifidobacteria from human and animal origin were surveyed for their ability to ferment complex carbohydrates. The substrates fermented by the largest number of species were D-galactosamine, D-glucosamine, amylose and amylopectin. Many of the species isolated from animal habitats showed reduced fermentation activity. Bifidobacterium dentium strains fermented gum guar and gum locust bean; porcine gastric mucin was fermented only by B. bifidum, B. infantis was the only species to ferment D-glucuronic acid; strains of B. longum fermented arabinogalactan and the gums arabic, ghatti and tragacanth; alpha-L-fucose was fermented by strains of B. breve, B. infantis and B. pseudocatenulatum. A key to the differentiation of Bifidobacterium species of human origin is provided.

Characterization of the plasmid pVS809 from Bifidobacterium globosum.

A plasmid from a B. globosum strain was cut with 38 restriction enzymes and a physical map was constructed. Out of a total of 121 clones from curing experiments, plasmid was lost in 58% and 100% for acridine orange and ethidium bromide curing agent respectively. The plasmid does not exist as a chromosomal integrated form. An attempt to determine phenotypic characters encoded by the plasmid was made by electrophoretic analyses of the total proteins.

Murein types in Bifidobacterium ruminantium, Bifidobacterium merycicum and Bifidobacterium saeculare.

The murein types of strains belonging to Bifidobacterium ruminantium, Bifidobacterium merycicum and Bifidobacterium saeculare were determined. B. ruminantium was found to posses a different type of murein from B. adolescentis. B. merycicum and B. saeculare have the same type of murein that is shared by many other species of the genus.

Bifidobacterial cell wall proteins (BIFOP) in Bifidobacterium globosum.

Nearly 150 strains of Bifidobacterium globosum were isolated from faeces of calf, chicken, lamb, rabbit and rat, from sewage, from rumen content and from human infant faeces between 1962 and 1973 and scored by SDS-PAGE for the presence of cell-wall-related proteins, i.e. BIFOP (bifid outer proteins); their apparent molecular masses ranged from 94.5 to 34 kDa and were designated A to L. Purified preparations from six of these ten proteins were employed to produce polyclonal rabbit antisera for use in immunoblots to investigate the interrelationships of the major antigens, A, B and C (94.5-85.5 kDa) and their distribution in strains of various origin. Two antigens differently migrating (or polymorphic forms) reacted with anti-BIFOP F serum (called F- and F+); the identity of BIFOP E with respect to these antigens was studied with anti-E serum. Only one antigen in all strain preparations reacted to anti-BIFOP H serum, which was raised against an antigen purified from a 13.5-MDa plasmid-bearing strain from rumen.

Phase variations in Bifidobacterium animalis.

Strains isolated from rabbit, chicken, and rat feces and from sewage and fermented milk products, all identified as Bifidobacterium animalis, were found to show phase variations in colony appearance and in cellular morphology. The rate of transition in a switching system from opaque to transparent colonies and vice versa was determined. Differences in protein components and in penicillin-binding proteins (PBPs) of the cells from different colony types are shown.

Survival of bifidobacteria from human habitat in acidified milk.

Some industrial preparations from milk, such as yogurt, contain bifidobacteria as an additional probiotic element. The acidic environment of these products affects the viability of the bifidobacteria. The survival in acidic environment of one-hundred and ten bifidobacterial strains from human habitat was tested.

Identification of bifidobacteria from fermented milk products.

Six samples of fermented milk preparations were examined for the presence of bifidobacteria. Identification was based on fermentation tests, genetic relatedness studies and electrophoretic analysis. Contrary to label information, Bifidobacterium animalis was the only species present.

Different electrophoretic patterns of cellular soluble proteins in Bifidobacterium animalis.

Twenty-nine strains of Bifidobacterium animalis, a species found only in animal habitats, were studied. Strains from known origins such as rat, rabbit and chicken feces and strains isolated from extrabody environments such as sewage and fermented milk products were examined. The intestinal origins of strains isolated from sewage and fermented milk products were determined by means of the comparison of electrophoretograms of cellular soluble proteins. Unknown origins of strains were recognized as being rabbit or chicken intestinal tract.

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).