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.The gram-positive soil microorganism Bacillus subtilis has been studied extensively, primarily as a model with which to study cell differentiation and for exploitation in the biotechnology industry. While some Bacillus species are pathogenic (e.g., B. anthracis and some B. cereus strains), B. subtilis has, at most, been associated with opportunistic infections of immunocompromised patients (6,11,17). For these reasons, it has received relatively little clinical interest. Bacillus spores, though, are currently available as probiotics and as competitive exclusion agents (CE agents). Probiotics are live bacterial supplements which can enhance the normal intestinal flora, while CE agents are bacteria which can suppress infection and may contain undefined mixtures of more than one bacterial species (7,8,19). Ingestion of significant quantities of spores is thought to restore the normal microbial flora following extensive antibiotic usage or illness (13). How this occurs is unclear but could include competitive exclusion of pathogens, whether by immunostimulation or competition for adhesion sites. Spore probiotics are primarily used by humans as an over-the-counter supplement for oral bacteriotherapy and bacterioprophylaxis of mild gastrointestinal disorders, many of which lead to diarrhea (13). In the livestock and poultry industries, probiotics containing Bacillus spores are used extensively; an example is Biogrow (Provita Eurotech Ltd., County Tyrone, Northern Ireland), which contains a mixture of B. subtilis and B. licheniformis spores. With the recent ban on the use of antibiotics as growth promoters in Denmark, the use of probiotics or CE agents as antibiotic alternatives seems likely to increase.The validity of spores as probiotics or CE agents was recently demonstrated by showing that oral inoculation of 1-dayold chicks with 2.5 ϫ 10 8 B. subtilis spores suppressed all aspects of infection when chicks were challenged with Escherichia coli 078:K80 (12). One dogma regarding the use of spores as probiotics or CE agents is their mode of action, which presumably must be substantially different from that of the other, better known bacterial supplements, such as Lactobacillus spp....

Bacillus subtilis Spores Germinate in the Chicken Gastrointestinal Tract

“…We decided to use a molecular approach, as preliminary work revealed that the process of serial dilution and plating onto medium supplemented with chloramphenicol was an unreliable method for accurately determining total viable counts of B. subtilis SC2362 (i.e., spores and vegetative cells) in samples of gut tissue. Other researchers have reported similar problems (14,15), the issues being (i) inefficient and nonsynchronous spore germination in the absence of a heat treatment/activation step, which leads to an underestimation of total viable units, and (ii) growth of background contaminants which obscure colonies of B. subtilis on agar plates. Ultimately, our decision to use RT-PCR was based on successful work by researchers elsewhere (6,25).…”

The effect of probiotic BioPlus 2B on feed efficiency and metabolic parameters in swine

“…Hoa et al (2001) revealed that the number of eliminated Bacillus subtilis bacteria was higher than that in the inoculum, i.e. one may assume that germination and multiplication of probiotic spores occurred.…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…Definition of specific events relevant in the triggering of humoral and cellular immune responses can contribute to the development of more rational and efficient vaccine strategies with enhanced immunogenicity. Recent evidences indicate that germination of B. subtilis spores located into phagosomes is a key step on the activation of immune responses since it permit antigen presenting cells to sample, process and display peptides to lymphocytes (Hoa et al 2001). Similar events seem to occur with the spores of the more immunogenic B. anthracis vaccine strains, which germinate and transiently multiply inside macrophages (Cohen et al 2000).…”

“…Expression of listeriolysin has allowed phagosome escape and transient intracellular multiplication of recombinant B. subtilis (Bielecki et al 1990, Hoa et al 2001. Mutations allowing early germination of B. subtilis spores are known but the role during the transit into the mammal host is unknown (Setlow 1994).…”