Mining the microbiota of the neonatal gastrointestinal tract for conjugated linoleic acid-producing bifidobacteria

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml(-1) by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/microg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.     

Bifidobacteria from the Faeces of Piglets

S ummary . Ninety-five strains of bifidobacteria isolated from 52 specimens of piglet faeces collected at 19 farms were studied. The main phenotypic characters of the strains were determined; however their assignment to known species of the genus Bifidobacterium was based primarily on their deoxyribonucleic acid homology relationships following DNA-DNA hybridization tests. The majority of the strains were recognized as Bifidobacterium suis Matteuzzi et al. Some strains could not be assigned to any known species of the genus so they were allotted provisionally to 2 unassigned bacterial groups.     

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.     

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.     

猪源双歧杆菌的分离与鉴定

运用双歧杆菌选择性培养基,从１～４周龄乳猪的粪便中共分离纯化到５２个菌株。通过染色镜检、生化反应、代谢产物分析、抗生素敏感性试验研究表明这些菌株均与双歧杆菌属特征相符。根据糖发酵试验初步鉴定结果,其中４５个菌株为小猪双歧杆菌,５株为猪双歧杆菌,２株为嗜热双歧杆菌。部分菌株的急性毒性试验表明受试菌株对小白鼠无任何毒性副反应。     

Simultaneous identification of five bifidobacterium species isolated from human beings using multiple PCR primers

On the basis of 16S rRNA sequences, 5 species-specific forward primers were designed for the identification of 5 Bifidobacterium species isolated from human intestine, namely B. bifidum, B. adolescentis, B. infantis, B. breve and B. longum. As the 5 primers targeted at different sites of 16S rDNA, by using their mixture and a genus-specific reversed primer, the 5 Bifidobacterium species can be simultaneously identified in individual or in mixed culture through PCR amplification. The specificity of the primers was confirmed by the use of genomic DNAs from type strains of all 32 Bifidobacterium species and 6 other relatives. The 5-primer mixture was also applied to the identification of Bifidobacterium strains used commercially. The results turned out to be in accordance with those from conventional identification. This multiple-primer method provides a useful tool for rapid identification of the 5 Bifidobacterium species indicated.     

人类肠道双歧杆菌一新种的初步研究

在健康人群肠道双歧杆菌检索中,从中老年粪便先后分离出一种细胞形态呈环状双歧杆菌３株（编号为ＭＯ－５,ＭＯ－２及ＭＯ－１）。经生物学特征的常规鉴定,根据《伯杰系统细菌手册》第２卷１９８６＾［２］。及其他有关文献描述,该菌的生理生化特征与双歧杆菌属中的青春双歧杆菌相类似,但从该菌的细胞形态呈环状和水杨苷发酵反应的阴性结果,再与青春双歧杆菌相比有区别。故认为该菌可能归属于双歧杆菌属中一新种。暂称它为环状     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora.

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.     

Comparison of the Phospholipid Composition of Bifidobacterium and Lactobacillus Strains

Phospholipid composition of 10 Bifidobacterium strains of human intestinal origin and of 9 Lactobacillus strains was determined by quantitative two-dimensional thin-layer chromatography. Phospholipids of three Bifidobacterium strains from honey bees and of two strains from bovine rumen liquor were qualitatively investigated. Diphosphatidylglycerol and phosphatidylglycerol were present in strains of both genera. All Bifidobacterium strains contained as specific phospholipids a new polyglycerolphospholipid, compound 15, and its lyso derivatives, earlier detected in B. bifidum var. pennsylvanicus. Also, lyso compounds of diphosphatidylglycerol and alanyl phosphatidylglycerol were only present in this genus in variable amounts. Lysyl phosphatidylglycerol was the only ninhydrin-positive phospholipid in seven Lactobacillus strains. In L. delbrückii and L. helveticus it was absent and partially replaced by an unidentified ninhydrin-negative phospholipid. The differences in phospholipid composition between bifidobacteria and lactobacilli may be another argument to differentiate these two genera.     

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.     

荧光原位杂交法检测双歧杆菌

检验荧光原位杂交法在双歧杆菌属鉴定方面的调途。方法：采用对数生长期的８株双歧杆菌和１０析其他厌氧、需氧杆菌在相同的条件下分别与双歧杆菌属特异性１６ＳｒＲＮＡ寡核苷酸基因探针和细菌界通用１６ＳｒＲＮＡ寡核苷酸基因探针在载玻片上进行原位杂交,在荧光显微镜下观察杂交结果,拍摄同一视野的荧光显微镜照片和相关显微镜照片,计算杂交率。结果所用的双歧杆菌菌株均与两种基因探针杂交,在荧光显微镜下发校菌与黑暗背景对     

Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution

Bifidobacteria were consistently present in the faeces of both man and pigs but only occasionally in the faeces of cattle and sheep, and they were not isolated from faecal samples from other animals; total counts of bifidobacteria were obtained by membrane filtration with YN-17 medium, a modification of Resnick and Levin's YN-6 medium. Mannitol-fermenting strains of bifidobacteria were isolated from both human and animal faeces, but sorbitol-fermenting strains were obtained only from human samples. These sorbitol-fermenting strains were identified as either Bifidobacterium adolescentis or B. breve and their numbers were obtained by membrane filtration on Human Bifid Sorbitol agar (HBSA). Sorbitol-fermenting bifidobacteria are specific indicators of human faecal pollution of waters and wastewaters.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.     

Detection of Bifidobacterium species by enzymatic methods

The properties of Bifidobacterium strains of human origin were examined by three enzymic tests and the amounts of acetic and lactic acids produced were also quantified. It was evident that two strains of the American Type Culture Collection (ATCC) did not belong to the genus. Moreover, at least one strain of Bifidobacterium added to some milk preparations did not show distinctive characteristics of the genus. It was also shown that most of bifidobacteria studied produced alpha-galactosidase (EC 3.2.1.22) and alpha-glucosidase (EC 3.2.1.20). The presence of alpha-galactosidase could afford a rapid differentiation of bifidobacteria used in some dairy products since this enzyme was not detected in Lactobacillus strains studied.     

Pathway of glucose fermentation in relation to the taxonomy of bifidobacteria

Cell-free extracts of 17 strains of Bifidobacterium bifidum (Lactobacillus bifidus) were examined for the presence of aldolase, glucose-6-phosphate dehydrogenase, and fructose-6-phosphate phosphoketolase. All strains turned out to lack aldolase, an enzyme unique to glycolysis, and glucose-6-phosphate dehydrogenase, characteristic of the hexosemonophosphate pathway. In all strains, fructose-6-phosphate phosphoketolase could be demonstrated. It can be concluded that bifidobacteria ferment glucose via a pathway which is different from those found in members of the genus Lactobacillus. The results strengthen the previous suggestions that classification of the bifidobacteria in the genus Lactobacillus is not justified.     

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees

One hundred and eighty-seven fructose-6-phosphate phosphoketolase positive strains were isolated from the digestive tract of three different bumblebee species. Analyses of the partial 16S rRNA gene sequences of the representative strains showed only 92.8% and 92.5% similarity to Bifidobacterium coryneforme YIT 4092(T) and Bifidobacterium indicum JCM 1302(T), 92.2% similarity to Alloscardovia omnicolens CCUG 18650 and slightly reduced similarity of 91% to other members of the family Bifidobacteriaceae. On the other hand, analyses of the partial heat-shock protein 60 (hsp60) gene sequence revealed that the proposed type strain BLAPIII-AGV(T) was affiliated only to the 60 kDa chaperonin sequence of uncultured bacteria from human vagina (79-80%) and the hsp60 gene sequence of A. omnicolens CCUG 31649(T) (75.5%). The peptidoglycan type was A4α with an l-Lys-d-Asp interpeptide bridge. The polar lipids contained diphosphatidylglycerol, an unknown phospholipid, six glycolipids and two phosphoglycolipids. The major fatty acids were C(18:1), C(20:0) and C(18:0). These and other analyses indicated that the isolates represented a new genus within the family Bifidobacteriaceae. This observation was further substantiated by determination of the DNA G+C contents (46.1-47.1 mol%). Affinity of the strains to some scardovial genera (Aeriscardovia, Alloscardovia and Metascardovia) was also confirmed by their ability to grow under aerobic conditions. Besides the above mentioned differences, Bombiscardovia coagulans was found to differ from all scardovial genera in the ability to grow at temperatures as low as 5°C, which was another major phenotypically different characteristic of this new member of the family Bifidobacteriaceae. Hence, on the basis of phylogenetic analyses using partial 16S rRNA and hsp60 gene sequence data, and the temperature related phenotypic difference, we propose a novel taxa, B. coagulans gen. nov., sp. nov. (type strain=BLAPIII-AGV(T)=DSM 22924(T)=ATCC BAA-1568(T)).