Application of molecular methods to classification and identification of bacteria of the genus <Emphasis Type="Italic">Bifidobacterium</Emphasis>

The molecular methods currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach for taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research in the field of molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.     

基因序列在蚤蝇科分子系统学研究中的应用

概述基因序列在双翅目蚤蝇科分子系统学研究中的应用。对蚤蝇科已测序的分类单元和基因序列进行了总结,12S rDNA和16S rDNA应用最广泛,涉及蚤蝇科17个属;获得基因序列最多的是Melaloncha属。蚤蝇科分子系统学研究内容为高级阶元系统发育分析、物种鉴定和隐存种发现。今后蚤蝇科分子系统学研究应增加蚤蝇标本的种类与数量,选择标准化基因。     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

钝头蛇属Pareas（蛇亚目：钝头蛇科）系统分类研究进展

钝头蛇属（Pareas）蛇类形态高度保守,种间形态差异微弱。以分子系统学方法为中心的整合分类方法的应用,为本类群分类难题的解决作出了重要贡献,近8年有9个新种得以描述,其中,仅在近3年就增加了7个新种,并有6个同物异名被恢复。本文依据最新研究成果,对近期钝头蛇属蛇类系统分类研究成果进行了综述,并斟酌中文种名,整理了物种名录,并编制了分类检索表。截止目前,钝头蛇属共有26种,其中在我国有分布的18种,中国特有种9种。同时,对研究中存在的问题进行了探讨,并对下一步工作提出了建议：本属物种多样性估计过低、标本采集覆盖范围不足、证据使用不甚全面,大范围密集采样以及系统发育基因组学方法的应用有助于本类群系统关系的最终解决。     

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.     

Insights into the taxonomy, genetics and physiology of bifidobacteria

Despite the generally accepted importance of bifidobacteria as probiotic components of the human intestinal microflora and their use in health promoting foods, there is only limited information about their phylogenetic position, physiology and underlying genetics. In the last few years numerous molecular approaches have emerged for the identification and characterization of bifidobacterial strains. Their use, in conjunction with traditional culturing methods, has led to a polyphasic taxonomy which has significantly enhanced our knowledge of the role played by these bacteria in the human intestinal ecosystem. The recent adaptation of culture-independent molecular tools to the fingerprinting of intestinal and food communities offers an exciting opportunity for revealing a more detailed picture of the true complexity of these environments. Furthermore, the availability of bifidobacterial genome sequences has advanced knowledge on the genetics of bifidobacteria and the effects of their metabolic activities on the intestinal ecosystem. The release of a complete Bifidobacterium longum genome sequence and the recent initiative to sequence additional strains are expected to open up a new era of comparative genomics in bifidobacterial biology. Moreover, the use of genomotyping allows a global comparative analysis of gene content between different bifidobacterial isolates of a given species without the necessity of sequencing many strains. Genomotyping provides useful information about the degree of relatedness among various strains of Bifidobacterium species and consequently can be used in a polyphasic identification approach. This review will deal mainly with the molecular tools described for bifidobacterial identification and the first insights into the underlying genetics involved in bifidobacterial physiology as well as genome variability.     

Applyed aspects of molecular biology of Bifidobacteria and Lactobacillus

The data obtained in the molecular genetic studies of the representatives of the genera Bifidobacterium and Lactobacillus are given. At present the genomes of Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus plantarum sized 2,256.640, 1,993.564 and 3,308.274 base pairs respectively, were completely sequenced. The use of different methods of gene indication and gene typing essentially widened the existing notions of the species variety of these bacteria, made it possible to change the classification of known species and to find out new ones, vegetating in different biotopes of the human body. Molecular genetic approaches also ascertained the taxonomic position of production strains of lactic-acid bacteria used in food industry, helped determine the kinetics of the passage of probiotic strains of bifidobacteria and lactobacilli along the gastrointestinal tract, as well as determine the duration of their preservation in the intestine after oral administration. In addition, the data on the evaluation of the therapeutic effect of some preparations, probiotics and synbiotics, with determination of the population level of endogenic and orally introduced exogenic bifidobacteria and lactobacilli are presented.     

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.     

昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

Genetic heterogeneity and functional properties of intestinal bifidobacteria

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.     

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.     

Phylogeny of the genus Aphis Linnaeus, 1758 (Homoptera: Aphididae) inferred from mitochondrial DNA sequences

Aphis is the largest aphid genus in the world and contains several of the most injurious aphid pests. It is also the most reluctant aphid genus to any comprehensive taxonomic treatment: while most species are easily classified into "species groups" that form well defined entities, numerous species within these groups are difficult to tell apart morphologically and identification keys remain ambiguous and mostly rely on host plant affiliation. In this paper, we used partial sequences of COI/COII and CytB genes to reconstruct the first phylogeny of Aphis and discuss the present systematics. The monophyly of the subgenus Bursaphis and of the tree major species groups, Black aphid, Black backed aphid and frangulae-like species was recovered by all phylogenetic analyses. However our data suggested that the nominal subgenus was not monophyletic. Relationships between major species groups were often ambiguous but "Black" and "Black backed" species groups appeared as sister clades. The most striking result of this study was that our molecular data met the same limits as the morphological characters used in classifications: mitochondrial DNA did not allow the differentiation of species that are difficult to identify. Further, interspecies relationships within groups of species for which taxonomic treatment is difficult stayed unresolved. This suggests that species delineation in the genus Aphis is often ambiguous and that diversification might have been a rapid process.     

Revision of the systematics of the genus Calliptamus Serville 1831, (Orthoptera: Acrididae: Calliptaminae) in Algeria using morphological,chemical, and genetic data

Summary. The genus Calliptamus contains swarming orthopterans that cause serious damage in Algerian agricultural systems. However, it remains difficult to identify species within this genus; a thorough understanding of the group’s systematics and the utilization of novel taxonomic criteria are needed. We used morphological analysis along with two other methods of species identification – chemotaxonomy with cuticular compounds and DNA barcoding involving the COI gene – to classify 81 individual grasshoppers collected at two different sites in the Sétif region (northeastern Algeria). The chemotaxonomic analyses yielded ambiguous results, but DNA barcoding allowed us to differentiate two Calliptamus species found in Algeria: Calliptamus barbarus (Costa 1836), and Calliptamus wattenwylianus (Pantel 1896). Several morphological criteria used in identification keys appear to reflect differences among morphotypes rather than differences between species, and their taxonomic specificity is not supported by the barcoding data. The number of spines on the hind tibia is the only morphological criterion that reflected genetic differences between species; it is thus considered to be a taxonomically useful feature for identifying species in this genus.     

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.     

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.     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

Characteristics of the soluble antigens of bifidobacteria

The immunological study of aqueous buffer extracts obtained from 45 strains of bifidobacteria belonging to the species B. bifidum, B. longum, B. adducens, B. breve, B. infantis and B. parvulorum was made. This study revealed 3 levels of the immunological specificity of soluble bifidobacterial proteins: common to the genus Bifidobacterium, common to a limited number of strains belonging to one or several species of bifidobacteria and strain-specific.     

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.