Simon M. Cutting

Characterization of Bacillus Probiotics Available for Human Use

ABSTRACT Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

Bacillus Probiotics: Spore Germination in the Gastrointestinal Tract

ABSTRACT Spores of Bacillus species are being used commercially as probiotics and competitive exclusion agents. Unlike the more commonly used Lactobacillus-type probiotics, spores are dormant life forms. To address how spore probiotics might function we have investigated whether spores can germinate in the gastrointestinal tract by using a murine model. Using a genetically engineered chimeric gene, ftsH-lacZ, which is strongly expressed only in vegetative cells, we have developed a sensitive competitive reverse transcription-PCR assay which has enabled detection of as few as 102 vegetative bacteria in the mouse gut. Using this method we have administered doses of ftsH-lacZ spores to groups of mice and shown that spores can germinate in significant numbers in the jejunum and ileum. The levels of detection we obtained suggest that spores may colonize the small intestine, albeit briefly.

The Intestinal Life Cycle of Bacillus subtilis and Close Relatives

Bacillus subtilis is considered a soil organism for which endospore formation provides a means to ensure long-term survival in the environment. We have addressed here the question of what happens to a spore when ingested. Spores displaying on their surface a heterologous antigen, tetanus toxin fragment C (TTFC), were shown to generate anti-TTFC responses not to the antigen contained in the primary oral inoculum but to those displayed on spores that had germinated and then resporulated. We then used reverse transcription-PCR to determine expression of vegetative genes and sporulation-specific genes in the mouse gut following oral dosing with spores. Significant levels of germination and sporulation were documented. Using natural isolates of B. subtilis that could form biofilms, we showed that these strains could persist in the mouse gut for significantly longer than the laboratory strain. Moreover, these isolates could grow and sporulate anaerobically and exhibited a novel phenomenon of being able to form spores in almost half the time required for the laboratory isolate. This suggests that spores are not transient passengers of the gastrointestinal tract but have adapted to carry out their entire life cycle within this environment. This is the first report showing an intestinal life cycle of B. subtilis and suggests that other Bacillus species could also be members of the gut microflora.

Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders.

ABSTRACT Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.

Bacterial Spores as Vaccine Vehicles

ABSTRACT For the first time, bacterial spores have been evaluated as vaccine vehicles. Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) antigen were used for oral and intranasal immunization and were shown to generate mucosal and systemic responses in a murine model. TTFC-specific immunoglobulin G titers in serum (determined by enzyme-linked immunosorbent assay) reached significant levels 33 days after oral dosing, while responses against the spore coat proteins were relatively low. Tetanus antitoxin levels were sufficient to protect against an otherwise lethal challenge of tetanus toxin (20 50% lethal doses). The robustness and long-term storage properties of bacterial spores, coupled with simplified genetic manipulation and cost-effective manufacturing, make them particularly attractive vehicles for oral and intranasal vaccination.

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.

Bacillus subtilis isolated from the human gastrointestinal tract

As part of an ongoing study to determine the true habitat of Bacillus species, we report here the isolation and characterisation of Bacillus subtilis from the human gastrointestinal tract (GIT). Strains were obtained from ileum biopsies as well as from faecal samples and their biotypes defined. 16S rRNA analysis revealed that most isolates of B. subtilis were highly conserved, in contrast to RAPD-PCR fingerprinting that showed greater diversity with 23 distinct RAPD types. The majority of B. subtilis strains examined possessed features that could be advantageous to survival within the GIT. This included the ability to form biofilms, to sporulate anaerobically and secretion of antimicrobials. At least one isolate was shown to form spores that carried an exosporium, a loosely attached outer layer to the mature endospore, this being the first report of B. subtilis spores carrying an exosporium. This study reinforces a growing view that B. subtilis and probably other species have adapted to life within the GIT and should be considered gut commensals rather than solely soil microorganisms.

Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

ABSTRACT Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.

Bacillus subtilis spores competitively exclude Escherichia coli O78:K80 in poultry.

Newly hatched specific pathogen-free chicks were dosed with a suspension of Bacillus subtilis spores prior to challenge with Escherichia coli O78:K80, a known virulent strain associated with avian colibacillosis, 24h later. A single oral inoculum of 2.5x10(8) spores was sufficient to suppress all aspects of E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over 2log(10) whilst colonisation of the intestine, as measured by direct caecal count, was reduced over 3log(10). Shedding of E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the duration of the experiment, 35 days. B. subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores.

Regulatory studies on the promoter for a gene governing synthesis and assembly of the spore coat in Bacillus subtilis

gerE is a regulatory gene of Bacillus subtilis that governs the synthesis and assembly of the spore coat and is required for the production of spores that are lysozyme-resistant and germination-proficient. We report the identification of the promoter for gerE and studies on the regulation of its expression. We show that gerE is switched on at the fourth hour of sporulation (stage-V) and that this expression is restricted to the mother-cell chamber of the sporangium. Dependency studies in which the level of gerE expression was measured in 36 different developmental mutants indicate that efficient expression of gerE requires the products of almost all spo0-IV genes tested as well as certain spoV genes. On the basis of its time of induction, compartmentalization of expression and pattern of dependence on other spo genes, gerE is inferred to be regulated co-ordinately with the previously studied spore coat protein gene cotA. gerE and cotA may be members of a developmental regulon of genes whose products are involved in the assembly of the spore coat.