Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Bacillus subtilis SF106 and Bacillus clausii SF174 spores reduce the inflammation and modulate the gut microbiota in a colitis model.

Chronic intestinal inflammation is associated with strong alterations of the microbial composition of the gut. Probiotic treatments and microbiota-targeting approaches have been considered to reduce the inflammation, improve both gut barrier function as well as overall gastrointestinal health. Here, a murine model of experimental colitis was used to assess the beneficial health effects of Bacillus subtilis SF106 and Bacillus clausii (recently renamed Shouchella clausii) SF174, two spore-forming strains previously characterised in vitro as potential probiotics. Experimental colitis was induced in BALB/c mice by the oral administration of dextran sodium sulphate (DSS) and groups of animals treated with spores of either strain. Spores of both strains reduced the DSS-induced inflammation with spores of B. clausii SF174 more effective than B. subtilis SF106. Spores of both strains remodelled the mouse gut microbiota favouring the presence of beneficial microbes such as members of the Bacteroidetes and Akkermansia genera.

Carotenoids found in Bacillus.

AIMS: To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation. MATERIALS AND RESULTS: Sampling of heat-resistant bacterial counts from soil, sea water and the human gastrointestinal tract. Phylogenetic profiling using analysis of 16S rRNA sequences to define species. Pigment profiling using high-performance liquid chromatography-photo diode array analysis. CONCLUSIONS: The most commonly found pigments were yellow, orange and pink. Isolates were nearly always members of the Bacillus genus and in most cases were related with known species such as Bacillus marisflavi, Bacillus indicus, Bacillus firmus, Bacillus altitudinis and Bacillus safensis. Three types of carotenoids were found with absorption maxima at 455, 467 and 492 nm, corresponding to the visible colours yellow, orange and pink, respectively. Although the presence of other carotenoids cannot be ruled out, these three predominant carotenoids appear to account for the pigments obtained in most pigmented bacilli, and our analysis reveals the existence of a C30 biosynthetic pathway. Interestingly, we report the presence of a water-soluble pigment that may also be a carotenoid. The function of carotenoids is photoprotection, and carotenoid-containing spores exhibited significantly higher levels of resistance to UV radiation than non-carotenoid-containing Bacillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that pigmented bacilli are ubiquitous and contain new carotenoid biosynthetic pathways that may have industrial importance.

Safety and probiotic evaluation of two Bacillus strains producing antioxidant compounds.

Bacillus species are becoming increasingly relevant for use as probiotics or feed additives where their heat stability can ensure survival in the food matrix or enable long-term storage at ambient temperature. Some Bacillus species are pigmented and in this study, we have examined two strains, one Bacillus pumilus (pigmented red) and the other Bacillus megaterium (pigmented yellow) for their safety for potential use in humans as dietary supplements. In addition, we have set out to determine if they might confer any potential health benefits. Both strains produce C30 carotenoids while the B. pumilus strain also produced large quantities of riboflavin equivalent to genetically modified Bacillus strains and most probably contributing to this strain's pigmentation. Riboflavin's and carotenoids are antioxidants, and we have evaluated the ability of vegetative cells and/or spores to influence populations of Faecalibacterium prausnitzii in the colon of mice. While both strains increased levels of F. prausnitzii, spores of the B. pumilus strain produced a significant increase in F. prausnitzii levels. If found to be reproducible in humans such an effect might, potentially, confer health benefits particularly for those suffering from inflammatory bowel disease.

The safety of Bacillus subtilis and Bacillus indicus as food probiotics.

AIMS: To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. METHODS AND RESULTS: Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions. CONCLUSIONS: Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the use of B. subtilis and B. indicus strains as food supplements.

Beneficial effects of carotenoid-producing cells of Bacillus indicus HU16 in a rat model of diet-induced metabolic syndrome.

A well-established rat model of diet-induced metabolic syndrome was used to evaluate the effects of the oral administration of spores or cells of HU16, a carotenoid-producing strain of Bacillus indicus. Symptoms of metabolic syndrome were induced in 90-days old, male Sprague-Dawley rats maintained for eight weeks on a high-fat diet, as previously reported. Parallel groups of animals under the same diet regimen also received a daily dose of 1×1010 cells or spores of B. indicus HU16. Cells of strain HU16 were able to reduce symptoms of metabolic syndrome, plasma markers of inflammation and oxidative markers in plasma and liver to levels similar to those observed in rats under a standard diet. HU16 cells did not affect obesity markers or the accumulation of triglycerides in the liver of treated animals. Denaturing gradient gel electrophoresis analysis showed that the oral administration of HU16 cells did not significantly affect the gut microbiota of high fat-fed rats, suggesting that the observed beneficial effects are not due to a reshaping of the gut microbiota but rather to metabolites produced by HU16 cells.

Structural and functional studies on an FtsH inhibitor from Bacillus subtilis.

The small 3 kDa SpoVM protein is essential for development of the spore in Bacillus subtilis. Genetic and biochemical experiments have shown that the function of SpoVM is to inhibit the proteolytic activity of FtsH during sporulation. We have used a combination of genetic and biophysical techniques to characterise the role of this small polypeptide. SpoVM was found to be widespread in Bacillus as well as in two Clostridia species, suggesting that SpoVM provides a common mechanism for inactivating the FtsH protease during spore differentiation. Using site-specific mutagenesis, we have identified C-terminal residues of SpoVM essential for biological activity. Analysis of SpoVM's structure showed that it is able to assume an alpha-helical conformation in the presence of a lipid interface which may be important in interacting with FtsH.

Identification of a new sigmaB-controlled gene, csbX, in Bacillus subtilis.

We have identified a new member, csbX, of the general stress regulon controlled by sigmaB in Bacillus subtilis. As with other members of the sigmaB regulon csbX is expressed during the stationary phase of cell growth and inactivation of this gene produces no obvious phenotype during cell growth or early development. csbX lies uspstream from the sporulation gene bofC which is co-transcribed during stationary phase.

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.

Evaluation of Bacillus subtilis strains as probiotics and their potential as a food ingredient.

Spores of Bacillus subtilis including one strain used commercially were evaluated for their potential value as a probiotic and as potential food additives. Two isolates of B. subtilis examined here were HU58, a human isolate and PXN21, a strain used in an existing commercial product. Compared to a domesticated laboratory strain of B. subtilis both isolates carried traits that could prove advantageous in the human gastro-intestinal tract. This included full resistance to gastric fluids, rapid sporulation and the formation of robust biofilms. We also showed that PXN21 spores when administered weekly to mice conferred non-specific cellular immune responses, indicative signs of the stimulation of innate immunity. Spores mixed in wholemeal biscuits were found to survive baking at 235 °C for 8 minutes with only a 1-log reduction in viability. That spores can survive the baking process offers the possibility of using spores as probiotic supplements in a range of novel food products.

Cloning and dependence pattern of the sporulation operon spoVH.

The spoVH locus, involved in the sporulation of Bacillus subtilis, was cloned in derivatives of the temperate bacteriophage luminal diameter 105. Two recombinant phages were obtained which contained 4.2 kilobases of chromosomal DNA. Both phages only partially complemented a mutation in the spoVH operon, spoVH516. Nevertheless, analysis of the cloned locus with integrational plasmids showed that the complete operon had been cloned. A spoVH'-lacZ transcriptional fusion was constructed, and this indicated that the spoVH operon was expressed 2.25 h after the start of sporulation. The distribution of beta-galactosidase in sporulating cells containing a spoVH'-lacZ fusion showed that spoVH was expressed in the spore compartment; lac fusion experiments were also used to study spoVH expression in the presence of other sporulation mutations. Expression of spoVH was prevented by mutations in any of the stage 0 or stage II loci and also by mutations in spoIIIA, spoIIIB, and spoIIIE. A similar pattern of dependence was found previously for the expression of spoVA, which is also expressed in the spore compartment.

Expression of the Bacillus subtilis spoIVB gene is under dual sigma F/sigma G control.

The expression of the Bacillus subtilis spoIVB gene, which encodes a developmental cell-cell signalling molecule, has been characterized. In some conditions, this gene can be transcribed by RNA polymerase associated with either sigma F or sigma G, in contrast to previous studies implying exclusive control by sigma G. However, during sporulation, only sigma G directs significant levels of spoIVB expression.

Membrane topology of the Bacillus subtilis pro-sigma(K) processing complex.

Activation of the final sporulation-specific transcription factor, sigma(K), is regulated by a signal emanating from the forespore which interacts with the pro-sigma(K) processing complex, comprising SpoIVFA, BofA, and the pro-sigma(K) processing protease, SpoIVFB. Mature sigma(K) then directs late gene expression in the parental compartment of the developing sporangial cell. The nature of this complex and how it is activated to process pro-sigma(K) are not understood. All three proteins are predicted to be integral membrane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of spoIVFA and spoIVFB with the complementary reporters phoA and lacZ and analyzing activity in Escherichia coli. SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in the membrane. SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB. Although the bofB8 allele, which uncouples the sigma(K) checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-sigma(K).

Branched pattern of regulatory interactions between late sporulation genes in Bacillus subtilis.

We measured the synthesis of dipicolinic acid (DPA) during sporulation in spo mutants of Bacillus subtilis by a sensitive biological assay based on cross-feeding of a spoVF mutant strain and also chemically. Many spo mutations, including several that block sporulation at stage III, did not prevent synthesis of DPA but instead prevented its incorporation into the spore. In general, strains with mutations in loci that are expressed in the spore compartment synthesized DPA, whereas strains with mutations in most of the loci that are expressed in the mother-cell compartment did not. Transcription of the gerE gene, as measured by DNA-RNA hybridization, followed a dependence pattern very similar to that of DPA synthesis. However, the dependence patterns of the two operons show that at about stage IV of sporulation there is a branch in the sequence of operon expression in the mother cell. One branch leads through spoVC to synthesis of DPA synthetase, and the other leads through spoVD to expression of gerE.

The PDZ domain of the SpoIVB serine peptidase facilitates multiple functions.

During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the sigma(K) regulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor sigma(K) in the outer mother cell chamber of the sporulating cell. This forms the basis of the sigma(K) checkpoint and ensures accurate sigma(K)-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for trans proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-sigma(K) processing, and (iv) signaling of the second function of SpoIVB.

Proteolysis of SpolVB is a critical determinant in signalling of Pro-sigmaK processing in Bacillus subtilis.

SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K). We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.

Characterization of two Bacillus probiotics.

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.

Fate and dissemination of Bacillus subtilis spores in a murine model.

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.