Bacterial Spore Formers: Probiotics and Emerging Applications Chapter Abstracts

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Chapter 1
Ubiquity, Longevity, and Ecological Roles of Bacillus Spores
Wayne L. Nicholson

Our preceptions of bacterial spores have been undergoing recent dramatic changes, in part due to the upsurge of interest in the uses of spores of Bacillus spp. as probiotics in plant and animal health. In this chapter I will review the occurrence and diverse roles of Bacillus spp. and their endospores in the environment, with particular emphasis on the worldwide geographic distribution of spores, the ecology of spores in various niches, spore longevity, host-symbiont and host-parasite interactions among spores and higher organisms, and human exploitation of spores.

Chapter 2
Taxonomy and Systematics of Aerobic Endospore Forming Bacteria
Dagmar Fritze

The genus Bacillus sensu stricto presents itself today with about 90 species, and publications of new species appear with ever increasing frequency. Besides the well known Bacillus a small number of other Gram-positive, aerobic, endospore forming genera have been long-recognized and over the past eight years some twenty additional genera harbouring aerobic endospore-forming bacteria (AEFB) have been created. Together, these genera embrace more than 200 species. In terms of phylogenetic relationships, established on 16S rRNA sequence comparisons, these genera are classified in the order Bacillales together with a number of non-spore forming genera.

Chapter 3
Ecology of Bacillus Species in Soil
Andreas D.M. Felske

Are sporeforming bacteria of any significance in soil under normal conditions? This historical question is discussed here, in the light of recent culture-independent community analyses of soil DNA, that indicated hitherto unknown bacterial lineages as the predominant soil microbes. While early studies have suggested a prevalence of endospores, recent data indicate vegetative Bacillus cells among the predominant soil bacteria. Moreover, these abundant vegetative forms appeared to be hitherto unknown Bacillus spp., which were only recently accessible to cultivation. Here, a preliminary phylogenetic location of these new species is outlined and defined as the branch where almost half of all environmental 16S rDNA Bacillus clones from various studies accumulated. In contrast to the other predominant soil flora, this new soil Bacillus lineage appears to be at least, in part, cultivable. In future, this will allow seminal studies on specific physiological attributes of these new dominant soil Bacillus species. This review intends to draw more attention to this neglected group of abundant but at least, in part, cultivable soil bacteria.

Chapter 4
Pathogenic Bacilli: B. anthracis and Close Relatives
Marco R. Oggioni, Annalisa Ciabattini, Marco Cassone and Gianni Pozzi

Species of the genus Bacillus are ubiquitous, and with a few exceptions, non-pathogenic bacteria. The direct involvement in human and animal disease of Bacillus anthracis is the prime example on which Koch formulated his postulates. The contribution of this species and that of the other closely related species to human disease are reviewed in this article. This review confirms the extremely low pathogenic potential for most of these species with the obvious exception of B. anthracis and B. cereus. Altogether the other species of the genus Bacillus, being non-pathogenic and not harbouring drug resistance genes, appear to meet the requirements of the classification systems used for food and feed safety, including both the European QPS system (Qualified Presumption of Safety) and the American GRAS system (Generally Recognised As Safe).

Chapter 5
Sporulation in Bacillus subtilis and other bacteria
Imrich Barák

Bacterial endospores and the organisms that form them have been the focus of intense research interest for many decades. Of all spore-forming bacteria only Bacillus subtilis became a model to study the remarkable process of spore formation and the factors controlling this simple example of unicellular differentiation. This review summarizes important advances in our understanding of gene expression during spore formation with a particular emphasis on the developmental stages that lead to the initiation of sporulation and a survey of what is now known of this process in other Bacilli and Clostridia species.

Chapter 6
The Functional Architecture and Assembly of the Spore Coat
Adriano Henriques, Teresa Costa, Lígia O. Martins and Rita Zilhão

Bacterial endospores are shielded against noxious chemicals and peptidoglycan-breaking enzymes by a protein coat that is also required for efficient spore germination. In most Bacillus and Clostridia species, the coat obeys to a common architecture, with three main layers: an amorphous undercoat that contacts the underlying cortex peptidoglycan, a lamellar lightly-staining inner layer, and a striated electrodense outer layer. Most species to species structural variation relates to the presence or absence of exosporia and of various surface appendages. The coat is mainly composed of proteins whose assembly is controlled at several levels: i) by the time, level and site of expression of the various genes involved; ii) by the action of a group of morphogenetic proteins, that guide assembly of the structural components; iii) by various post-transcriptional and post-translational mechanisms. The result is a stable yet dynamic structure that senses and responds to nutritional cues. Transcriptional profiling studies, proteomics, cell biological techniques, and the structural characterization of selected components is illuminating the assembly process, and expanding the repertoire of tools to manipulate the composition, structure, and properties of the spore coat in view of specific applications in biotechnology or biomedicine.

Chapter 7
The Spore Surface
Adam Driks

Bacterial spores are encased in a protein coat and, for some species, an additional structure called the exosporium. The best-studied features of the spore, namely its resistance properties and the ability to revive after long periods of dormancy depend, to a large degree, on structures inside the spore. Less well-studied though is the spore surface. Among other functions, the spore surface is home to important enzymatic activities and may play essential roles in attachment and signaling to other organisms. Recent results stemming from basic studies of the coat reveal that the spore surface can be genetically manipulated, leading to important biotechnological applications. Finally, studies of the spore surface are providing clues to the dynamic nature of the spore and, as a result, shedding light on how the structural flexibility of the coat may be fundamental to proper spore function.

Section II. Spores as Probiotics

Chapter 8
Safety of Aerobic Endospore-Forming Bacteria
Niall Logan

The safety of aerobic endospore-forming bacteria to humans and other animals is a matter of increasing concern, and this occurs at a time of rapid changes in the classification and nomenclature of these organisms. Given the increasing interest in Bacillus species as probiotic supplements for both human and animal use, it seems pertinent to consider what is presently known about the safety issues that arise from their use. The pathogenicities of Bacillus anthracis and Bacillus cereus are well established, but less is generally known about the risks associated with other aerobic endospore-forming species and their products. This review provides a comprehensive survey of current knowledge about the involvement of species of Bacillus and related genera in human opportunistic infections, and the association of Bacillus enzymes with occupational asthma. Also covered are the issues of revised species nomenclature and product mislabelling, both of which are areas of potential misunderstanding or even abuse by commercial manufacturers of probiotic supplements.

Chapter 9
The Fate of Ingested Spores
Huynh A. Hong and Le Hong Duc

Bacillus spores when ingested are not treated simply as a food. Mounting studies, reported here, show that spores have a complex interaction with the GALT. The most important finding is that a proportion of ingested spores can germinate in the intestine and undergo limited rounds of growth before sporulating again. Spores can disseminate to the GALT and are likely to even persist briefly within phagocytic cells resident in the Peyers' Patches. This persistence may elicit cellular responses against spores as well as enhancing systemic humoral and local responses. Immune responses against spores when given at high dose is of course important when considering their use as probiotics. We propose in this chapter the novel idea that Bacillus spores, although not long term residents of the gut, may actually use the animal gut (including humans) as part of their natural life cycle.

Chapter 10
Mechanisms of Gene Transfer and the Spread of Antibiotic Resistance in Spore Forming Organisms in the GI Tract
Peter Mullany, Teresa Barbosa, Karen Scott and Adam Roberts

In this chapter we will consider the mechanisms used for gene transfer in the spore forming bacteria that normally inhabit and have access to the gastrointestinal tract (GIT), these being principally of the genera Bacillus and Clostridia. The most abundant group of anaerobes inhabiting the GIT are low % DNA G+C Gram-positive bacteria closely related to Clostridial species and which fall in the Clostridial Cluster XIVa subphylum. Very little is known about the ability of these bacteria to sporulate, although some isolates have recently been found to contain homologues of sporulation genes (P. Louis, per. comun.). Since these bacteria may yet be shown able to form spores, gene transfer between them will also be considered. We will consider both the genetic elements responsible for mediating gene transfer and the environmental influences on gene transfer.

Chapter 11
Bacillus Spores as Probiotic Products for Human Use
Sonia Senesi

Increasingly, published scientific literature on probiotics supports the concept that consumption of exogenously supplied live bacteria can positively influence human health. The worldwide interest in probiotics has opened new perspectives in the application of microorganisms as biotherapeutic tools and favoured the expanding marketing of probiotic-based products. Lactic acid-producing bacteria belonging to the genus Lactobacillus and Bifidobacterium are the most studied probiotic organisms and the most frequently marketed for human use as commercial products. Spores of the genus Bacillus are used as probiotics especially in Europe and in Southeast Asia, where most of them are currently marketed to treat and prevent a broad variety of gastrointestinal disorders in man. Studies addressing the mechanism by which spores of Bacillus spp. exert probiotic activities are so far limited, although an increasing number of studies are proving encouraging. This chapter will focus on Bacillus-based probiotics marketed for human use particularly the taxonomic position, characterisation, efficacy and safety of the Bacillus strains present in the commercial formulations.

Chapter 12
Production and Probiotic Effects of Natto
Tomohiro Hosoi and Kan Kiuchi

This review summarizes the production of natto and probiotic effects of Bacillus subtilis (natto). Natto is a popular food made by fermenting cooked soybeans with Bacillus subtilis (natto) in Japan. Natto has a characteristic aroma and stickness and its quality is affected by the type of soybean and B. subtilis (natto) strains. Processing methods such as the soaking and steaming of soybeans, inoculation of B. subtilis (natto) spores, packaging, and fermentation are all important for the highest quality natto. Natto contains numerous nutrients originating from both soybeans as well as from intact cells and metabolites of B. subtilis (natto) and many of these have physiological activity. B. subtilis, B. subtilis (natto), and natto are considered to have the potential as probiotics. Although B. subtilis is not a predominant bacterium in the human intestine, characteristics of the cells such as spore formation, resistance of the spore to oxygen and acidity seem to be desirable for a probiotic. In addition, spores are thought to be able to germinate in the intestine under certain conditions (see Chapter 7). Ingestion of intact B. subtilis cells and natto probably increase the Lactobacillus spp. and Bifidobacterium spp. in the intestine. B. subtilis cells induce cytokine responses of human intestinal epithelial-like Caco-2 cells with less cytotoxicity than nonpathogenic Escherichia coli, pathogenic Salmonella enteritidis, and Pseudomonas aeruginosa. In addition, a serine protease, subtilisin, produced by B. subtilis degrades soybean allergens and shows fibrinolytic activity. Ingestion of vitamin K2 (menaquinone-7) helps coagulant activity and prevent osteoporosis. Natto contains phytoestrogens (isoflavones) that originate in soybeans and they seem to have preventive effects on breast and prostate cancer, osteoporosis, menopausal symptoms, and heart disease.

Chapter 13
Spore Probiotics as Animal Feed Supplements
Stephen T. Cartman and Roberto M. La Ragione

Concern over the emergence of bacterial pathogens with multiple antibiotic resistance has led the European Commission to restrict the use of antibiotic feed supplements on European farms, with a complete ban proposed for 2006. Therefore, alternative approaches are required to maximise livestock performance and reduce morbidity and mortality rates through controlling infectious enteric disease. Probiotics are a promising alternative and currently there are six bacterial spore-based products available for veterinary application. However, only two of these are approved for use within the European Union. The remaining products are deemed to be unsafe due to the bacterial strains concerned having either donor potential in antibiotic resistance gene transfer, or potential for producing toxins. Recent studies have demonstrated benefits to both animal health and performance following supplementation of feed with bacterial spores. Furthermore, the intrinsic resistance of bacterial spores to adverse environmental conditions means they are ideally suited to long term storage and distribution as a commercial product. However, as is the case for probiosis in general, evidence explaining spore probiotic functional mechanisms of action is extremely limited at present. Ongoing research aims to identify these as well as desirable traits of effective probiotic bacteria so that spore-based feed supplements may be optimised for veterinary use and become an effective alternative to antibiotic feed supplements.

Chapter 14
The Potential for Use of Bacillus spp. in Sports Turf Management
Alan C. Gange and Karen J. Hagley

Sports turf is a long-term crop, heavily reliant on water, fertilizers and pesticides to maintain it in perfect condition. Recent studies have shown that microbial populations in turf soils, including numbers of Bacillus spp., are low compared with natural grasslands. The main reason for this is carbon limitation, but compaction, pesticides and fertilizers may also be responsible. It has been argued that Bacillus spp. have great potential for use in turf management, because many of the problems (poor root growth, pest and disease attack and weed control) could be ameliorated by these bacteria. There is now research on all of these topics and the promise of Bacillus as plant protectants has been shown by their incorporation into new biopesticides. However, a cause for concern is that a number of bacterial products are available for use on turf and, while the theory behind their use is clear, their efficacy and mode of action is largely untested.

Chapter 15
Antimicrobial Activity of Bacillus Probiotics
Maria C. Urdaci and I. Pinchuk

The production of antimicrobials is one of the mechanisms by which probiotic bacteria are believed to exert their beneficial effect on the host, presumably by inhibiting the growth and colonization of the gastrointestinal tract by pathogenic bacteria. This chapter summarises what is now known about the production of bacteriocins, bacteriocin-like inhibitory substances (BLIS) and antibiotics in Bacillus species. An attempt is also made to demonstrate the potential that these antimicrobial substances may have in specific commercial formulations.

Chapter 16
Gut Sporeformers
Teresa M. Barbosa, Cláudia R. Serra and Adriano O. Henriques

Some intestinal symbionts use modified forms of sporulation for propagation and survival between gastrointestinal tracts, or to produce viviparous multiple offspring. Others, such as Bacillus cereus and presumably its close relatives show preferential filamentous growth in the invertebrate intestine. Moreover, spores of B. subtilis germinate and the vegetative cells appear to be capable of sporulation in the gut of mice. However, with the exception of Clostridium, which is a common and well-described component of the gut anaerobic microflora, relatively little is known about the incidence, nature, and properties of sporeformers in gut ecosystems. Without intending to be exhaustive, we try in this chapter to review some of the present knowledge, including the information that we recently obtained from a study aimed towards the isolation of sporeforming bacteria from the gastrointestinal tract of poultry and other animals.

Section III. Spores as Vaccine Vehicles and in Therapeutics

Chapter 17
Display of Molecules on the Spore Surface
Rachele Isticato, Giuseppina Cangiano, Maurilio De Felice and Ezio Ricca

Surface display systems for the expression of heterologous molecules on the surface of viruses and cells has proven to be invaluable for a wide range of applications such as the development of bioadsorbents and biocatalysts, the identification of new antibiotics and antigens and the delivery of vaccines and drugs. The recent development of strategies to expose bioactive molecules on the surface of Bacillus subtilis spores, in which efficient display is coupled with spore robustness, has driven attention to the spore-based system as an attractive new tool to deliver antigens and enzymes of industrial or pharmaceutical relevance. In this chapter we first outline methods for presenting heterologous proteins on the surface of bacterial cells and then review the known examples of surface presentation on bacterial spores.

Chapter 18
Spores as Oral Vaccines
Simon M. Cutting

Recently, the first use of bacterial spores as oral vaccine vehicles has been reported showing that mice orally immunised with Bacillus subtilis spores expressing a tetanus antigen could be protected against lethal challenge with tetanus toxin. Unlike many 2nd Generation vaccine systems currently under development none offer the heat stability of bacterial spores or the flexibility for genetic manipulation. The current use of Bacillus spores as probiotics for both humans and animals may facilitate their eventual licensing as oral vaccines. Although this field is only now emerging initial proof of principle studies are outlined here as well as the potential advantages and disadvantages of the spore vaccine approach.

Chapter 19
Recombinant Clostridial Spores in Tumour Therapy
Oliver J. Pennington, Lieve Van Mellaert, Jan Theys, Sofie Barbé, Philippe Lambin, Jozef Anné and Nigel P. Minton

Effective tumour therapies are reliant on the selective delivery of anti-cancer agents at high therapeutic doses. In recent years, gene therapy using viral vectors as the delivery vehicle has received considerable attention. However, such vectors exhibit a lack of tumour specificity, promote poor levels of transgene expression and are inefficiently distributed throughout the tumour mass. In contrast, the ability of intravenously injected clostridial spores to infiltrate and thence selectively germinate in the hypoxic regions of solid tumours appears a totally natural phenomenon. The organism requires no fundamental alterations, and its ability to target solid tumours is exquisitely specific. As a consequence, clostridial spores have now been used to deliver various therapeutic proteins to the tumours of selected animal models. In the case of prodrug converting enzymes, delivery has been shown, in certain models, to result in complete tumour regression following the systemic administration of prodrug.

Appendices

Appendix I
Commercial Probiotic Products Containing Bacillus Spores
Simon M. Cutting

No abstract provided

Appendix II
EU Regulations on Bacillary Probiotics For Animal Feeds
Elinor McCartney

The use of live micro-organisms (probiotics) in animal feeds has come under legislative pressure in the EU during the last decade, resulting in increased costs of obtaining marketing authorisation. For example, current estimates to license a probiotic for broilers and piglets, two common target animal categories, are around €1,400,000, a considerable cost for an industry where sales volumes and margins are significantly lower than for feed or pharmaceutical products. Of particular concern to regulators has been the risk of transference of antibiotic resistance in probiotic bacteria to other bacteria in the environment and the food chain. Another perceived risk concerning the use of Bacillus species as animal probiotics, linked to the robustness and longevity of bacillary spores, is the potential for toxin production and enteropathogenicity in some strains. The intense scrutiny of animal probiotics in relation to possible transfer of antibiotic resistance is in marked contrast to regulatory attitudes to live bacteria used in human pharmaceuticals or in food or beverages. The recently established EFSA (European Food Safety Authority) is charged, among other tasks, with harmonising food and feed law in the EU. This may result in tougher legislation for live micro-organisms used in human food, beverages and as pharmaceuticals.

Appendix III
The Fungicidal Activity of Spore Forming Bacteria
Rudolf R. Azizbekyan

Today, especially in less developed countries, food deficits are being observed. One of the primary reasons behind the global food deficit are fungal diseases of commercially important plants which amount to annual losses to agriculture of between 20 to 30%. Current methods for control of plant diseases is by the use of chemical pesticides, which are harmful to non target organisms and pollute the environment. For this reason, in the last decade increasing attention has been given to biological methods of plant protection. One major attribute of Bacillus species is their ability to synthesize biologically active compounds, including insecticidal toxins and antifungal antibiotics. B. thuringiensis-based insecticides have been used for more than forty years for the control of the harmful insects (e.g., Lepidoptera, Coleoptera and Diptera). The advantages of the large-scale application of biological insecticides is the relative simplicity and cost-effectiveness of their manufacture. Bacillus-based fungicides have not yet received widespread attention nor application. Nevertheless, an increasing number of Bacillus strains with anti-fungal activity are being reported. In most cases, Bacillus species synthesize a number of fungicidal factors that enhance their antagonistic effect on plant pathogens and have been shown to reduce the probability of infection during storage and some examples are given in this chapter. Biological fungicides do not pollute the environment and produce no measurable effect on the microenvironment. In the near future we predict an increased interest in Bacillus-based biofungicides and their application in plant protection.

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