Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.     

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

Use of specific primers based on the 16S-23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples

Effective methods for the identification and enumeration of lactic acid producing bacteria (LAB) cells are important for the quality control and assurance of probiotic products. In this study, we designed a polymerase chain reaction (PCR) primer set from the sequence in 16S-23S internal transcribed spacer (ITS) region and used it for the specific detection of Bifidobacterium adolescentis, one of the Bifidobacterium species used in probiotics. Specificity of the PCR primers, i.e., bits-1/bits-2, was assured by assay strains of B. adolescentis, other Bifidobacterium species, and strains of non-Bifidobacterium spp. Coupled with the use of a known primer set specific for Bifidobacterium species, Bifidobacterium strains and B. adolescentis could be identified from LAB strains in fermented dairy products and human fecal samples.     

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.     

双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

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.     

Antagonistic Action of Lactobacilli and Bifidobacteria in Relation to Staphylococcus aureus and Their Influence on the Immune Response in Cases of Intravaginal Staphylococcosis in Mice

The antibacterial activity of Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2 strains or their various compositions in relation to Staphylococcus aureus in vitro and on models of experimental intravaginal staphylococcosis of mice was determined. It was found that under the influence of these strains and their various compositions, the in vitro growth of Staph. aureus was inhibited, and the number of colonies of Staph. aureus plated from the vagina of infected mice was significantly reduced. The antibacterial activity of these strains separately and in compositions correlated with their ability to improve the performance of the immune response. These strains were the most effective in the following compositions: Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2. Strains of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. bifidum VK2, and B. longum VK1 are prospective components of future probiotic drugs efficient in treating staphylococcosis and for immunity correction.     

Assessment of cell surface properties and adhesion potential of selected probiotic strains

Aim:  To evaluate the physicochemical cell surface and adhesive properties of selected probiotic strains for human use.
Methods and Results:  Probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus paracasei , Lactobacillus rhamnosus GG, Lactobacillus brevis , Lactobacillus casei , Leuconostoc mesenteroides and Pediococcus acidilactici were tested for the physicochemical properties of cell surfaces and the adhesion abilities against foodborne pathogens. Bif .  longum B6 (53·6%) and Lact .  rhamnosus GG (46·5%) showed the highest hydrophobicity, while the least affinity to xylene was observed in Ped .  acidilactici (10·4%). Bifidobacterium longum B6 showed the strongest coaggregation phenotype with Listeria monocytogenes (53·0%), Shigella boydii (42·0%) and Staphylococcus aureus (45·9%). Lactobacillus rhamnosus GG had the strong binding ability to Caco-2 cells and effectively inhibited the adhesion of L .  monocytogenes , Salmonella Typhimurium, Sh .  boydii and Staph .  aureus to Caco-2 cells. The hydrophobicity was highly correlated with coaggregative abilities and competitive inhibition, suggesting a good relationship between in vitro adhesion and in vivo colonization.
Conclusion:  The results suggest that Bif .  longum B6 and Lact .  rhamnosus GG can be candidate probiotics available for human consumption.
Significance and Impact of the Study:  Because the use of probiotic strains has been more concerned with their beneficial effects in the GI tract, it is essential to examine the potential of probiotic strains based on the physicochemical properties in terms of bacterial-binding and adhesion capabilities.     

Evaluation of the PCR method for identification of Bifidobacterium species

AIMS: Bifidobacterium species are known for their beneficial effects on health and their wide use as probiotics. Although various polymerase chain reaction (PCR) methods for the identification of Bifidobacterium species have been published, the reliability of these methods remains open to question. METHODS AND RESULTS: In this study, we evaluated 37 previously reported PCR primer sets designed to amplify 16S rDNA, 23S rDNA, intergenic spacer regions, or repetitive DNA sequences of various Bifidobacterium species. CONCLUSIONS: Ten of 37 experimental primer sets showed specificity for B. adolescentis, B. angulatum, B. pseudocatenulatum, B. breve, B. bifidum, B. longum, B. longum biovar infantis and B. dentium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that published Bifidobacterium primer sets should be re-evaluated for both reproducibility and specificity for the identification of Bifidobacterium species using PCR. Improvement of existing PCR methods will be needed to facilitate identification of other Bifidobacterium strains, such as B. animalis, B. catenulatum, B. thermophilum and B. subtile.     

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.     

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Antiproliferative effects of homogenates derived from five strains of candidate probiotic bacteria.

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including Lactobacillus rhamnosus GG, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophilus. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of L. rhamnosus GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.     

Screening of species-specific lactic acid bacteria for veal calves multi-strain probiotic adjuncts

The selection of promising specific species of lactic acid bacteria with potential probiotic characteristics is of particular interest in producing multi species-specific probiotic adjuncts in veal calves rearing. The aim of the present work was to select and evaluate in?vitro the functional activity of lactic acid bacteria, Bifidobacterium longum and Bacillus coagulans strains isolated from veal calves in order to assess their potential use as multi species-specific probiotics for veal calves. For this purpose, bacterial strains isolated from faeces collected from 40 healthy 50-day-calves, were identified by RiboPrinter and 16s rRNA gene sequence. The most frequent strains belonged to the species B. longum, Streptococcus bovis, Lactobacillus animalis and Streptococcus macedonicus. Among these, 7 strains were chosen for testing their probiotic characteristics in?vitro. Three strains, namely L. animalis SB310, Lactobacillus paracasei subsp. paracasei SB137 and B. coagulans SB117 showed varying individual but promising capabilities to survive in the gastrointestinal tract, to adhere, to produce antimicrobial compounds. These three selected species-specific bacteria demonstrated in?vitro, both singularly and mixed, the functional properties needed for their use as potential probiotics in veal calves.     

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.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

健康蒙古族人肠道中乳酸菌和双歧杆菌多样性

【背景】越来越多的研究发现人类的诸多疾病与肠道菌群失衡有关。乳酸菌和双歧杆菌属于肠道中的有益菌,在不同人群肠道中的多样性不尽相同。【目的】在种水平上分析健康蒙古族人群肠道菌群中乳酸菌和双歧杆菌的多样性。【方法】以27名健康蒙古族志愿者为研究对象,其中14名来自中国内蒙古,13名来自蒙古国。首次采用乳酸菌和双歧杆菌的特异性引物扩增与PacBioSMRT三代测序技术相结合,在种水平上探讨志愿者肠道中乳酸菌和双歧杆菌的丰度和生物多样性,并进一步分析性别、BMI(Bodymassindex)值和地域对上述两者可能的影响,以及优势菌种之间的相关性。【结果】在种的水平上,27名志愿者肠道样品中共鉴定到68个乳酸菌和11个双歧杆菌,其中平均相对含量在1%以上的乳酸菌有8个,包括唾液链球菌(Streptococcus salivarius,36.41%)、瘤胃乳酸杆菌(Lactobacillus ruminis,17.94%)、德氏乳杆菌(Lactobacillus delbrueckii,3.11%)、罗氏乳杆菌(Lactobacillus rogosae,2.23%)、轻型链球菌(Streptococcus mitis,2.18%)、阴道乳杆菌(Lactobacillus vaginalis,2.02%)、魏斯氏乳杆菌(Weissella confusa,1.54%)和鼠李糖乳杆菌(Lactobacillus rhamnosus,1.09%);双歧杆菌有5个,包括青春双歧杆菌(Bifidobacterium adolescentis,39.88%)、长双歧杆菌(Bifidobacterium longum,27.15%)、链状双歧杆菌(Bifidobacterium catenulatum,26.30%)、两歧双歧杆菌(B. bifidum,3.92%)和角双歧杆菌(Bifidobacterium angulatum,1.71%),聚类分析分为链状双歧杆菌和青春双歧杆菌2个主要的类群。分析结果显示:性别、BMI值和地域均未能显著影响志愿者肠道中乳酸菌和双歧杆菌的菌群结构(P0.05),但男性和女性之间、中国内蒙古地区和外蒙古国的志愿者之间的个别乳酸菌菌种相对含量存在显著差异(P0.05)。对样品中的优势乳酸菌和双歧杆菌进行Spearman相关性分析发现,乳酸菌和双歧杆菌彼此之间相关性较为密切,不同菌种间相关性不尽相同,与具体的菌种有关。【结论】首次采用PacBio SMRT测序技术在种的水平揭示了健康蒙古族人肠道中乳酸菌和双歧杆菌菌种多样性,为在种水平上解析肠道中乳酸菌和双歧杆菌多样性提供了新的研究思路和实施方案。