Indication for Co-evolution of Lactobacillus johnsonii with its hosts

ABSTRACT: BACKGROUND: The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. RESULTS: A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e., chickens, humans or mice. CONCLUSIONS: Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.     

Development trends in taxonomy,with special reference to fungi

Taxonomy is a traditional subject, but it still receives attention and has become a topic of much discussion in recent years. Many of these discussions have raised concerns about the future of taxonomy, especially with regard to the workforce responsible for the discovery of new species in the context of declining biodiversity. Previous discussions were based on the taxonomic data of plants and animals, but the status of fungal taxonomy has not been mentioned. Fungi have one of the highest levels of biodiversity among all living organisms, second only to insects. The discussion of the future of taxonomy without the inclusion of fungal data is incomplete. Here, we present the results of analyses based on all new fungal taxa published since 1753. Fungal taxonomy is an ever‐growing area of study with increasing numbers of new taxa being described and growing numbers of fungal taxonomists. Compared with plants and most animal groups, there has been a much sharper increase in the rate at which new fungal taxa are being described. Furthermore, the number of taxonomists studying fungi has increased at a faster speed than those studying plants or animals. This indicates that fungal taxonomy is a prosperous subject and a dynamic area for scientific studies, and that it deserves much more attention and support. The study of fungal taxonomy will deepen our understanding of the biodiversity of our planet.     

Probiotic meat products and human nutrition

Raw cured and ripened meat products have been traditionally manufactured using the fermentation of native or added carbohydrates by lactic acid bacteria found in meat or in its environment. The commercial application of probiotic microorganisms in dry fermented meat products is not yet common. Probiotic bacterial strains that can be used in the manufacturing of dry fermented meat products should be capable of surviving in conditions found in fermented products; furthermore, they should dominate other microorganisms found in the finished product. The initial number of microorganisms in sausage filling or on the surface of ham or loin cannot be reduced as in milk pasteurization, for example. Therefore, the choice of appropriate microorganisms is important. Probiotic meat products are a relatively new and not very well recognized field of meat industry, but the most important issue is to find a compromise between technological aspects, safety, quality and health-beneficial effects of food. Therefore, the object of this review is on the one hand to analyze technological possibilities and quality parameters of probiotic meat products, and on the other hand to discuss risks and benefits of probiotic meat used in human nutrition.     

Accessing the inaccessible: molecular tools for bifidobacteria

Bifidobacteria are an important group of the human intestinal microbiota that have been shown to exert a number of beneficial probiotic effects on the health status of their host. Due to these effects, bifidobacteria have attracted strong interest in health care and food industries for probiotic applications and several species are listed as so-called "generally recognized as safe" (GRAS) microorganisms. Moreover, recent studies have pointed out their potential as an alternative or supplementary strategy in tumor therapy or as live vaccines. In order to study the mechanisms by which these organisms exert their beneficial effects and to generate recombinant strains that can be used as drug delivery vectors or live vaccines, appropriate molecular tools are indispensable. This review provides an overview of the currently available methods and tools to generate recombinant strains of bifidobacteria. The currently used protocols for transformation of bifidobacteria, as well as replicons, selection markers, and determinants of expression, will be summarized. We will further discuss promoters, terminators, and localization signals that have been used for successful generation of expression vectors.     

The bacterial species dilemma and the genomic-phylogenetic species concept

The number of species of Bacteria and Archaea (ca 5000) is surprisingly small considering their early evolution, genetic diversity and residence in all ecosystems. The bacterial species definition accounts in part for the small number of named species. The primary procedures required to identify new species of Bacteria and Archaea are DNA-DNA hybridization and phenotypic characterization. Recently, 16S rRNA gene sequencing and phylogenetic analysis have been applied to bacterial taxonomy. Although 16S phylogeny is arguably excellent for classification of Bacteria and Archaea from the Domain level down to the family or genus, it lacks resolution below that level. Newer approaches, including multilocus sequence analysis, and genome sequence and microarray analyses, promise to provide necessary information to better understand bacterial speciation. Indeed, recent data using these approaches, while meagre, support the view that speciation processes may occur at the subspecies level within ecological niches (ecovars) and owing to biogeography (geovars). A major dilemma for bacterial taxonomists is how to incorporate this new information into the present hierarchical system for classification of Bacteria and Archaea without causing undesirable confusion and contention. This author proposes the genomic-phylogenetic species concept (GPSC) for the taxonomy of prokaryotes. The aim is twofold. First, the GPSC would provide a conceptual and testable framework for bacterial taxonomy. Second, the GPSC would replace the burdensome requirement for DNA hybridization presently needed to describe new species. Furthermore, the GPSC is consistent with the present treatment at higher taxonomic levels.     

Towards a prokaryotic genomic taxonomy

One of the most interesting developments in the field of modern-day microbiology is the ever increasing number of whole-genome sequences that is publicly available. There is an increasing interest in the use of these genome sequences to assess evolutionary relationships among microbial taxa, as it is anticipated that much additional taxonomic information can be extracted from these sequences. In a first part of the present review, mechanisms that are responsible for the evolution of genomes will be discussed. Subsequently, we will give an overview of approaches that are presently available to assess the taxonomic relationships between prokaryotic species based on complete genome sequences, followed by a brief discussion of the potential implications of these novel approaches for bacterial taxonomy in general and our thinking about the bacterial species concept in particular.     

Proteomics for routine identification of microorganisms

The invention of MALDI-TOF-MS enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. Besides 'true' proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. This approach has since been developed as a diagnostic tool readily available for routine, high-throughput analysis of microbial isolates from clinical specimens by intact-cell mass spectrometry (ICMS), the direct analysis of whole bacterial cell without a preceding fractionation or separation by chromatography or electrophoresis. ICMS exploits the reproducibility of mass fingerprints for individual bacterial and fungal strains as well as the high similarity of mass fingerprints within a species. Comparison of mass spectral data to genomic sequences emphasized the validity of peak patterns as taxonomic markers. Supported by comprehensive databases, MALDI-TOF-MS-based identification has been widely accepted in clinical laboratories within only a few years.     

The use of bacterial spore formers as probiotics

The field of probiosis has emerged as a new science with applications in farming and aqaculture as alternatives to antibiotics as well as prophylactics in humans. Probiotics are being developed commercially for both human use, primarily as novel foods or dietary supplements, and in animal feeds for the prevention of gastrointestinal infections, with extensive use in the poultry and aquaculture industries. The impending ban of antibiotics in animal feed, the current concern over the spread of antibiotic resistance genes, the failure to identify new antibiotics and the inherent problems with developing new vaccines make a compelling case for developing alternative prophylactics. Among the large number of probiotic products in use today are bacterial spore formers, mostly of the genus Bacillus. Used primarily in their spore form, these products have been shown to prevent gastrointestinal disorders and the diversity of species used and their applications are astonishing. Understanding the nature of this probiotic effect is complicated, not only because of the complexities of understanding the microbial interactions that occur within the gastrointestinal tract (GIT), but also because Bacillus species are considered allochthonous microorganisms. This review summarizes the commercial applications of Bacillus probiotics. A case will be made that many Bacillus species should not be considered allochthonous microorganisms but, instead, ones that have a bimodal life cycle of growth and sporulation in the environment as well as within the GIT. Specific mechanisms for how Bacillus species can inhibit gastrointestinal infections will be covered, including immunomodulation and the synthesis of antimicrobials. Finally, the safety and licensing issues that affect the use of Bacillus species for commercial development will be summarized, together with evidence showing the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case by basis.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

Computer Program Designed to Follow Fluctuations in Microbial Populations and Its Application in a Study of Chesapeake Bay Microflora

A computer program has been developed which performs cluster analysis of microorganisms using methods of numerical taxonomy. The program is designed to group related strains, identify the groups by reference to known strains, and calculate a hypothetical median organism (HMO) for each group. The HMO serves to condense taxonomic information and provides a tag for each strain cluster. Every strain in a group is compared with the HMO established for that group. A representative strain for the group is obtained by selection of the strain showing highest similarity to the HMO. New data sets can be compared with data sets of previous analyses. Hence, the occurrence of the same taxonomic groups within separate data sets can be determined. Quantitative or qualitative differences in distribution of taxonomic groups within or between data sets can be measured. The output from the computer is a graphical display, using an on-line plotter; thus, the investigator is provided with visual comparison of data sets. Results obtained from a study applying the computer program in an analysis of taxonomic data obtained for 43 bacterial strains isolated from Chesapeake Bay indicate the usefulness of this method of taxonomic analysis in microbial ecology.     

Host and geographical factors influence the thermal niche of enteric bacteria isolated from native Australian mammals

The thermal profiles of 118 bacterial strains, representing six species of the family Enterobacteriaceae, isolated from a variety of native Australian mammals were determined under in vitro conditions. Each of the bacterial species had a unique thermal profile and differed in their minimum or maximum temperature for growth and in their response to changing temperatures. The taxonomic classification of the host from which the bacterial strains were isolated explained a significant amount of the variation in thermal profile among strains of a species. Host effects were detected at all taxonomic levels: order, family, genus, and species. The locality (State or Territory) or climate zone from which the strain was collected explained a significant amount of the variation in the thermal profile of Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae strains. Genetically similar strains, as determined by allozyme profiles, had similar thermal profiles for the bacterial species Hafnia alvei and Escherichia coli. The results of this study indicate that there are potentially many aspects of host biology that may determine the thermal profile of these bacteria.     

The unholy trinity: taxonomy, species delimitation and DNA barcoding

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this 'DNA barcoding' initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the 'DNA barcoding' initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the 'DNA barcoding' initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings--Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of 'DNA barcoding'.     

红菇科可食真菌的若干分类问题

红菇科Russulaceae包含大量全球广泛采食的野生食用菌,同时也有一定数目的毒菌。该科特别是红菇属的分类是大型真菌分类的难点。近年来DNA数据大量应用于红菇科的分类,更新了属的界定和概念,发现了大量新物种,为食用菌和毒菌的识别和鉴定带来了可用的名称。然而,DNA证据并不总是与形态证据吻合,这又为食用菌和毒菌的识别和名称的使用带来了困扰和不便。本文针对乳菇属、多汁乳菇属和红菇属中的重要食用菌类群,回顾了近年来的分类研究进展,分析了研究背后的数据实情和存在的分类问题。认为：在食用菌和毒菌的确定上,依靠物种复合群共有的形态特征更具有可操作性;依据DNA序列进行的劈分式分类和依靠少数样品的特征及DNA序列上的少量差异发表新种的做法可能产生不便于使用的后果;在乳菇属和红菇属中,“BLAST相似度低的即为新种”的分类实践存在错误风险;充分结合历史资料和各个类群的特点,确定物种划分的阈值,才能有望解决红菇科真菌的分类问题。     

The 3Cs provide a novel concept of bacterial species: messages from the genome as illustrated by Salmonella

A key issue troubling bacterial taxonomy and systematics is the lack of a biological species definition. Criteria to be used for defining bacterial species on genetic and biological bases should be able to reveal clear-cut boundaries among clusters of bacteria. To date, DNA–DNA re-association assays and ribosomal RNA sequence comparison have been useful in determining relative evolutionary distances among bacteria but the data are continuous and thus cannot define bacterial clusters as taxonomic units to be called species. Using Salmonella as models, we have looked for definite genetic and biologic uniqueness of clusters of bacteria. Based on our findings that each Salmonella lineage has a unique genome structure shared by strains of the same lineage but not overlapping with strains of other Salmonella lineages, we conclude that this is a result of genetic isolation following divergence of the bacteria. We propose that there should be genetic boundaries between different species of bacteria at the genomic level, which awaits further genomic information for validation.     

Evaluating the Impact of Different Sequence Databases on Metaproteome Analysis: Insights from a Lab-Assembled Microbial Mixture

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge.This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.     

Implications of poor taxonomy in conservation

Red Lists are legal instruments that guide decisions within the context of conservation. Such lists can indicate the most appropriate conservation actions, leading to choices of priority areas for conservation, or create alternatives to ensure the preservation of threatened species. However, if Red Lists are effective tools in the conservation of threatened species, then it is important that we have guaranteed the quality of taxonomic delimitation of these taxa. Here we discuss a case of taxonomic confusion, emphasizing the implications of misidentification in Red Lists. To avoid mistakes in species conservation, we advocate that a minimum level of taxonomic knowledge is needed to ensure success in preserving target species. Therefore, complementarity of taxonomy and conservation guarantee stronger conservation actions.     

Implication of chromosomal analysis for the taxonomy of parasitic wasps (hymenoptera)

A review of principles for application of the morphology of the karyotype in the taxonomy of parasitic wasps is given. Specific character of the use of chromosomal characteristics at different taxonomic levels is determined. In the taxonomy of hymenopterans, the data on the morphology of the karyotype are the most important at the species level. By the taxonomic level and the degree of morphological isolation, closely related species of parasitic wasps, differing in the structure of chromosomal sets, can be subdivided into the following groups: well-distinguishable species; species with indistinct differences in the appearance (proper sibling species); morphologically identical populations; intrapopulation forms; specimens with spontaneous chromosomal mutations. It is suggested that chromosomal studies in the taxonomy of hymenopterans should be used as a method of express analysis of outdoor populations and laboratory strains of these insects.