Lactobacillus tucceti sp. nov., a new lactic acid bacterium isolated from sausage

Following the application of several molecular techniques strain R 19c, isolated from sausage by Reuter in 1970 and deposited at the DSMZ as Lactobacillus sp., has been identified as pertaining to a new species. It showed singular ISR-DdeI and ISR-HaeIII profiles that allowed its differentiation from 68 lactic acid bacteria reference strains analyzed. Phylogenetic analysis based on 16S rRNA gene sequences places this strain in the genus Lactobacillus within the Lactobacillus alimentarius group. Species L. versmoldensis is the closest phylogenetic neighbor with 96.3% sequence similarity. DNA-DNA hybridization experiments confirmed the independent status at species level of this strain. Species-specific primers for PCR detection of this new species have been developed. Phenotypically it can be distinguished from the closest relative L. versmoldensis by several traits such as the peptidoglycan type (L-Lys-Gly-D-Asp), acid production from L-rhamnose, D-mannitol and L-fucose and its inability to ferment d-galactose, d-melibiose and d-sucrose. The name Lactobacillus tucceti sp. nov. is proposed with strain R 19c(T) (=DSM 20183(T)= CECT 5920(T)) as the type strain.     

The genus Lactobacillus--a genomic basis for understanding its diversity

The genus Lactobacillus is a diverse group that includes many species used in food production and preservation. Some lactobacilli are considered probiotic, conferring health benefits upon the host. The heterogeneity of this genus poses challenges and opportunities when characterizing or exploiting individual strains. To date, 10 Lactobacillus genome sequences have been published, and at least 11 more sequencing projects are ongoing. These studies will dramatically improve one's understanding of metabolic processes, bioprocessing capabilities and potential roles in health and well-being of the Lactobacilli. This review describes the current status of Lactobacillus genome sequence projects, highlights the major findings and summarizes functional genomics or comparative genomics studies. The genomic basis for the unusual diversity of this genus is discussed, and the potential for comparative genomics to rigorously extend phylogenetic analysis of the Lactobacilli is described.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

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.     

Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.     

Lactobacillus uvarum sp. nov.--a new lactic acid bacterium isolated from Spanish Bobal grape must

Five strains isolated from grape musts in Spain in 1997, have been characterized by several molecular techniques, and three of them have been identified as pertaining to a new species. All strains are Gram-positive rods, aerotolerant and homofermentative bacteria that do not exhibit catalase activity. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the genus Lactobacillus, closely related to Lactobacillus mali. DNA-DNA hybridization experiments confirmed that strain 71 belongs to the lately described species L. satsumensis, strain 88 belongs to L. mali and the other three isolates have an independent status at species level. Restriction analysis of the amplified 16S rRNA gene (16S-ARDRA), internal spacer region (ISR) analysis, random amplified polymorphism DNA (RAPD) and ribotyping were performed in order to establish genotypic similarities and differences between the new species and their closest species. The three isolates can be genetically differentiated from their closest relatives by RAPD analysis and ribotyping. Phenotypically, they can be distinguished by several traits such as their ability to grow at pH 3.3 and NaCl 5% (w/v) and by certain carbohydrate fermentations. The name L. uvarum sp. nov. is proposed. The type strain is 8T (=DSM 19971T = colección espa?ola de cultivos tipo (CECT) 7335T).     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

Indication and identification of Lactobacillus spp. with the use of polymerase chain reaction

AIM: The aim of the work is the development of laboratory test for indication and identification of Lactobacillus spp. by the polymerase chain reaction. MATERIALS AND METHODS: The work is developed on the base of the GenBank/EMBL data about genetic sequences of the Lactobacillus spp. The sequences of DNA were studied with the help of the ClustalW program. The strains of the Lactobacillus spp., which are the object of the research, have been registered in Russian collection of industrial microorganisms. RESULTS: The laboratory test of nested-PCR for indication and identification L. plantarum, L. fermentum, L. acidophilus, L. delbrueckii, L. casei, L. rhamnosus was performed. The specificity of the nested-PCR was correlated with the control analyses of monoculture Lactobacillus spp. and commercial products. CONCLUSION: The new developed laboratory nested-PCR test may be use in control system of milk foods enriched by probiotic microorganisms.     

An introduction to rod-shaped lactic-acid bacteria

The genus Lactobacillus is mainly found in the intestinal tract of healthy humans and animals as well as in fermenting vegetables or plant materials, such as silage. It has a moderate diffusion in meat products and is rarely found in wines and beers. On the other hand, rod-shaped lactic-acid bacteria are largely used in the preparation of a variety of foods and feed products. As starter cultures, they are omnipresent in cheeses and dairy manufacturing. Specific fermentation processes have been developed in order to encourage the growth of the desired species, some of which are fastidious organisms such as L. delbrueckii ssp. bulgaricus, L. helveticus and L. sanfrancisco. In addition, a promising and interesting perspective is the use of rod-shaped lactic-acid bacteria, primarily L. acidophilus, L. reuteri and L. salivarius as probiotic starters to preserve the natural biological equilibrium of the intestinal tract.     

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.     

Campylobacter bacteriophages and bacteriophage therapy

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.     

Summary of the diverse situation of similar biotherapeutic products in the selected countries (August 2010)

The WHO guidelines on evaluating similar biotherapeutic products (SBPs) were adopted by the Expert Committee on Biological Standardization in 2009. The fundamental messages of the guidelines are that a) generic approach is not suitable for licensing SBPs, b) only products that have been subjected to a comparability exercise and show similarity to the reference biotherapeutic product (RBP) in terms of their quality, safety and efficacy are defined as SBPs, and c) the products that are not shown to be similar to the originator products as indicated in the guidelines should neither be described as "similar" nor called SBPs. In view of these, the products which have not been subjected to a head to head comparison with the RBP should be referred to as another term, e.g. 'non-innovator' therapeutic products. In order to review the current situation in each country, a survey was planned in line with the implementation workshop of the guidelines in August 2010. The results show that the diversity of regulatory framework for licensing SBPs and the ambiguous use of the terms, 'similar' or 'generic', present considerable challenges for the future use of SBPs.     

Carnobacterium: positive and negative effects in the environment and in foods

The genus Carnobacterium contains nine species, but only C. divergens and C. maltaromaticum are frequently isolated from natural environments and foods. They are tolerant to freezing/thawing and high pressure and able to grow at low temperatures, anaerobically and with increased CO(2) concentrations. They metabolize arginine and various carbohydrates, including chitin, and this may improve their survival in the environment. Carnobacterium divergens and C. maltaromaticum have been extensively studied as protective cultures in order to inhibit growth of Listeria monocytogenes in fish and meat products. Several carnobacterial bacteriocins are known, and parameters that affect their production have been described. Currently, however, no isolates are commercially applied as protective cultures. Carnobacteria can spoil chilled foods, but spoilage activity shows intraspecies and interspecies variation. The responsible spoilage metabolites are not well characterized, but branched alcohols and aldehydes play a partial role. Their production of tyramine in foods is critical for susceptible individuals, but carnobacteria are not otherwise human pathogens. Carnobacterium maltaromaticum can be a fish pathogen, although carnobacteria are also suggested as probiotic cultures for use in aquaculture. Representative genome sequences are not yet available, but would be valuable to answer questions associated with fundamental and applied aspects of this important genus.     

Paratransgenesis: an approach to improve colony health and molecular insight in honey bees (Apis mellifera)?

The honey bee (Apis mellifera) is highly valued as a commercial crop pollinator and a model animal in research. Over the past several years, governments, beekeepers, and the general public in the United States and Europe have become concerned by increased losses of honey bee colonies, calling for more research on how to keep colonies healthy while still employing them extensively in agriculture. The honey bee, like virtually all multicellular organisms, has a mutually beneficial relationship with specific microbes. The microbiota of the gut can contribute essential nutrients and vitamins and prevent colonization by non-indigenous and potentially harmful species. The gut microbiota is also of interest as a resource for paratransgenesis; a Trojan horse strategy based on genetically modified symbiotic microbes that express effector molecules antagonizing development or transmission of pathogens. Paratransgenesis was originally engineered to combat human diseases and agricultural pests that are vectored by insects. We suggest an alternative use, as a method to promote health of honey bees and to expand the molecular toolbox for research on this beneficial social insect. The honey bees' gut microbiota contains lactic acid bacteria including the genus Lactobacillus that has paratransgenic potential. We present a strategy for transforming one Lactobacillus species, L. kunkeei, for use as a vector to promote health of honey bees and functional genetic research.     

Functional and safety aspects of enterococci in dairy foods

The genus Enterococcus like other LAB has also been featured in dairy industry for decades due to its specific biochemical traits such as lipolysis, proteolysis, and citrate breakdown, hence contributing typical taste and flavor to the dairy foods. Furthermore, the production of bacteriocins by enterococci (enterocins) is well documented. These technological applications have led to propose enterococci as adjunct starters or protective cultures in fermented foods. Moreover, enterococci are nowadays promoted as probiotics, which are claimed for the maintenance of normal intestinal microflora, stimulation of the immune system and improvement of nutritional value of foods. At the same time, enterococci present an emerging pool of opportunistic pathogens for humans as they cause disease, possess agents for antibiotic resistance, and are frequently armed with potential virulence factors. Because of this “dualistic” nature, the use of enterococci remains a debatable issue. However, based on a long history of safe association of particular enterococci with some traditional food fermentations, the use of such strains appears to bear no particular risk for human health. Abundance of knowledge as well as progress in molecular techniques has, however, enabled exact characterization and safety assessment of strains. Therefore, a balanced evaluation of both, beneficial and undesirable nature of enterococci is required. A clear understanding of their status may, therefore, allow their safe use as a starter, or a probiotic strain. The present review describes the broader insight of the benefits and risks of enterococci in dairy foods and their safety assessment.     

A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies

Background aimsAdvanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area.MethodsWe have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy “Plagencell network” for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years.ResultsThe cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <–150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years.ConclusionsOur data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.     

Fine-scale cospeciation between Brachycaudus and Buchnera aphidicola: bacterial genome helps define species and evolutionary relationships in aphids

Aphids harbour an obligatory symbiont, Buchnera aphidicola, providing essential amino acids not supplied by their diet. These bacteria are transmitted vertically and phylogenic analyses suggest that they have 'cospeciated' with their hosts. We investigated this cospeciation phenomenon at a fine taxonomic level, within the aphid genus Brachycaudus. We used DNA-based methods of species delimitation in both organisms, to avoid biases in the definition of aphid and Buchnera species and to infer association patterns without the presumption of a specific interaction. Our results call into question certain 'taxonomic' species of Brachycaudus and suggest that B. aphidicola has diversified into independently evolving entities, each specific to a 'phylogenetic' Brachycaudus species. We also found that Buchnera and their hosts simultaneously diversified, in parallel. These results validate the use of Buchnera DNA data for inferring the evolutionary history of their host. The Buchnera genome evolves rapidly, making it the perfect tool for resolving ambiguities in aphid taxonomy. This study also highlights the usefulness of species delimitation methods in cospeciation studies involving species difficult to conceptualize--as is the case for bacteria--and in cases in which the taxonomy of the interacting organisms has not been determined independently and species definition depends on host association.     

Phytochemistry of the Carnivorous Sundew Genus Drosera (Droseraceae) – Future Perspectives and Ethnopharmacological Relevance

Species of the carnivorous genus Drosera L. have long been a source of valuable natural products. The various phytochemicals characteristic of these species, particularly 1,4‐naphthoquinones and flavonoids, have contributed to the diverse utilization of sundews in traditional medicine systems worldwide. A growing number of studies have sought to investigate the comparative phytochemistry of Drosera species for improved sources of pharmaceutically important compounds. The outcomes of these studies are here collated, with emergent trends discussed in detail. Important factors which affect production of secondary metabolites in plants are critically examined, such as environmental influences and in vitro culture, and recommendations subsequently presented based on this. Explicitly, the current review aims to i) present an updated, comprehensive listing of the phytochemical constituents of the genus (including quantitative data where available), ii) summarize important factors which may influence the production of phytopharmaceuticals in plants, and iii) recommend guidelines for future research based on the above, including improved standardization and quality control. We have also included a section discussing future perspectives of research on Drosera spp. based on three different research lines i) the potential to produce much needed lead compounds for treatment of tuberculosis, ii) the potential role of anthocyanins in nitrogen transport, and iii) research into ‘Natural Deep Eutectic’ solvents produced by Drosera spp. in the droplets or ‘dew’ employed to capture insect prey.