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On the fate of ingested Bacillus spores
Res. Microbiol. 151 (2000) 361–368 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923250800001595/FLA
On the fate of ingested Bacillus spores Maria R. Spinosaa, Tiziana Braccinia, Ezio Riccab, Maurilio De Feliceb, Lorenzo Morellic, Gianni Pozzia, Marco R. Oggionia* a
Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Università di Siena, Via Laterina 8, 53100 Siena, Italy b Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli Federico II, Napoli, Italy c Istituto di Microbiologia, Università Cattolica S. C., Piacenza, Italy Received 22 November 1999; accepted 24 February 2000
Abstract — Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine. © 2000 Éditions scientifiques et médicales Elsevier SAS Bacillus / probiotic / spore / bile / Bacillus subtilis / lymph node / germination / transit marker
1. Introduction The use of bacteria for equilibrating the intestinal microflora and the optimization of intestinal functions has received attention for human and veterinary applications. Among the bacteria used as probiotics, bacteria belonging to the genus Bacillus represent a peculiar situation. Unlike other bacteria, B. subtilis [31], B. cereus [2, 3, 18, 31], B. clausii [8, 13] (Spinosa et al., Microbial Ecology in Health and Disease 2000, in press) and B. coagulans [1] are given orally as spores, not as vegetative forms. The beneficial effect of other probiotics, such as lactobacilli, has been ascribed to metabolic products or proteins of actively multiplying cells in the anaerobic environment of the intestine (reviewed by Salminen and colleagues) [28, 29]. No such metabolites or proteins have been identified in
* Correspondence and reprints [email protected]
Bacillus, even if they are suspected, for example in the case of Bacillus natto [17]. An immunestimulating activity has been reported [11, 20], but the mechanism of stimulation is not fully explained. So far, the only known direct effect of Bacillus in the intestinal environment (of mosquito larvae) is due to a protoxin (crystal protein) produced by B. thuringiensis and B. sphaericus during sporulation [16]. Since the mammalian intestine is an anaerobic environment and Bacillus spp. are preferentially aerobic [15, 21], germination and outgrowth of spores in the intestine seem difficult to envisage. Moreover, the capacity to survive the lytic action of bile salts, one of the criteria used to select potentially probiotic strains [4, 6], is highly uncommon in nonenteric microorganisms [5]. To analyse which biological form of Bacillus could be responsible for the probiotic effect(s), in the work presented here we fed mice with two nonpathogenic Bacillus species and tested
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for the presence of spores and vegetative cells in the murine intestine.
2. Materials and methods 2.1. Bacterial strains and growth conditions
The B. subtilis strain MO1099 (amyE::erm) [9] was used to isolate a spontaneous rifampicinresistant mutant ER229. Spores of the erythromycin- and rifampicin-resistant strain were prepared by growing cells for 36 h in Difco sporulation medium (DSM; Difco Laboratories), and treated with lysozyme to eliminate vegetative cells as previously described [22]. Bacillus clausii [23, 34] spores were from an Italian pharmaceutical preparation for human use which has been licensed for oral treatment of intestinal disorders (Enterogerminat; Sanofi Winthrop, Milan, Italy). This strain was previously allotted to the B. subtilis [8] species and later to B. alcalophius [13]. Here it is reported as B. clausii [34] due to new 16S rRNA sequences (GenBank Accession No. AF172603). Bacteria from animal samples were plated on tryptic soy agar (TSA; Difco Laboratories) at 37 °C in aerobic conditions. Antibiotics for selection of strains were rifampicin (50 µg/mL) or erythromycin (5 µg/µL). 2.2. Animals
Seven-week-old female BALB/c mice, obtained from Charles River (Italy), were used for experiments and housed in our animal facility for the duration of the experiment. 2.3. Experimental set-up
Two distinct experiments were performed: one using B. subtilis and the other one B. clausii spores. In both experiments, nine BALB/c mice were inoculated, while three control mice did not receive any inoculum. Prior to inoculation and killing, animals were left without food for 16 h. Animals were killed at 4, 24 or 72 h after inoculum and samples from intestinal sites and lymphoid organs and blood were taken.
2.4. Inoculum
Spores were centrifuged and resuspended at room temperature in distilled water at a concentration of 109 spores/200 µL. The precise number of spores injected intragastrically was calculated by microscopical count with a Burker chamber. Intragastric inoculum of 1 × 109 spores was performed using an animal feeding needle. 2.5. Samples for microbiological analysis
Blood samples were obtained from anaesthetized mice prior to killing from the cheek (100–200 µL) which had been disinfected by ethanol and swabbed for control of superficial contamination. Samples from lymphoid organs were the mesenteric lymph nodes and the spleen. Lymph nodes were fragmented by needle disruption or mild sonication (1 mL of saline), while the spleen contents were ‘washed out ‘ with a syringe (1 mL). Samples from the intestinal tract were from the ileum or colon and a faecal sample was obtained from the terminal rectum. About 2-cm fragments of the ileum and colon were washed with 1 mL of saline. Care was taken to collect colon samples free of faecal content. The faecal sample was homogenized with a sterile toothpick and resuspended in 1 mL of saline. All samples were immediately divided into two aliquots. One of these was subjected to heat inactivation at 80 °C for 20–25 min to kill vegetative cells, while the other was left at room temperature. Samples obtained from lymph nodes, spleen and blood were plated on nonselective tryptic soy agar plates (TSA, Difco Laboratories) to monitor contamination of environmental or intestinal microorganisms. Intestinal samples were plated on rifampicin-containing TSA plates to counterselect enteric flora, at dilutions ranging from 101 to 106. Intestinal samples were also plated on MacConkey agar (Biotec, Grosseto, Italy) to monitor possible changes in the enteric flora counterselected in the plates above. An inactivated food sample and an inactivated faecal sample of a control mouse were plated on TSA medium (Difco Laboratories) to control environmental spore contamination.
M.R. Spinosa et al. / Res. Microbiol. 151 (2000) 361–368 2.6. MIC determination for bile salts
Minimum inhibitory concentration (MIC) of the conjugated bile salt taurodeoxycholic acid sodium salt (TDOC 25 mM = 1.2% w/v) and the nonconjugated salt deoxycholic acid (DOC 25 mM = 1% w/v) was determined on 96-well microtitre plates. Bacteria grown overnight on plates were resuspended in liquid medium at a concentration of 105 CFU/mL and aliquoted into the microtitre plate (100 µL/well). TDOC and DOC were distributed by two-fold dilutions in a range 51.2–0.05 mM. Growth was registered after 24 h of incubation. Media were Muller Hinton broth for enterobacteriaceae, Pseudomonas and Bacillus, TSB (tryptic soy broth; Difco Laboratories) for streptococci and staphylococci and supplemented Muller Hinton Broth (SR158E, Oxoid) for Haemophilus, Neisseria and Branhamella. The following strains were used: B. subtilis (ER229, see above), B. cereus (Bactisubtilt; AF172711), B. clausii (Enterogerminat; AF142576; AF172603), Streptococcus gordonii (Challis), Enterococcus faecalis (OG1 SS), Streptococcus pneumoniae (R6), Moraxella catarrhalis (BRA022 22-77, clinical isolate), Enterococcus faecium (ENTO85 23-56, clinical isolate), Escherichia coli (ATCC25922), Klebsiella planticola (ATCC 33531), Klebsiella pneumoniae (KL0023, clinical isolate), Pseudomonas maltophilia (clinical isolate), Proteus mirabilis (PRO14 22-60, clinical isolate), Staphylococcus aureus (ATCC29923), Neisseria lactamica (NE007bis 22-42, clinical isolate) and Haemophilus influenzae (HAE089 23-27, clinical isolate). All clinical strains (identified by conventional biotyping) were human isolates from the collection of the Microbiology Section, Department of Molecular Biology, University of Siena. 2.7. Inhibition of B. subtilis germination by bile salts
B. subtilis spores were prepared and purified as described by Nicholson and Setlow [22]. Purified spores were heat-activated (10 min at 80 °C) and diluted in 10 mM Tris-HCl pH 8.4 to give a final OD580 of approximately 0.3 (2 × 107 spores/mL). D-glucose (1 mM), D-fructose
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(1 mM) and KCl (10 mM), each dissolved in 10 mM Tris-HCl pH8.4, were added to the suspension and equilibrated at 37 °C for 15 min. Germination was initiated by the addition of 10 mM L-alanine. Optical density (580 nm) was measured at 5-min intervals until a constant reading was reached. The efficiency of germination was expressed as percentage decrease in OD with respect to initial OD of the sample. Experiments, performed in duplicate, were carried out in the presence of 0.2 and 0.4 mM DOC and 0.2, 0.4 and 0.8 mM TDOC.
3. Results 3.1. Biological state of B. subtilis and B. clausii in mice after intragastric inoculation of spores
To verify whether vegetative forms of bacilli are present in the intestinal environment after inoculation of spores, two groups of mice were inoculated intragastrically with a single dose of 109 spores of B. subtilis and the probiotic B. clausii, respectively. After sampling, the specimens were immediately divided into two aliquots one of which was incubated at 80 °C for 20 min. This heat treatment was applied in order to eliminate all possible vegetative cells from one of the two samples. In this way, one of the two samples (heat-treated) would permit the counting of the spores only, while the other would yield, upon plating, the count resulting from the sum of spores and vegetative cells. A 1-min 80 °C treatment was found to reduce the viable count of vegetative cells to < 0.0001% (B. subtilis) and < 0.001% (B. clausii), while it did not affect spore viability (data not shown). 3.2. Distribution of B. subtilis spores in the intestinal tract
At 4 h after inoculation Bacillus spores were found at all intestinal sites (figures 1 and 2). Spore counts in faeces and other intestinal sites diminished with an exponential rate over the duration of the experiment (figure 3), and were reduced beyond the detection limit (5 × 102/g of feces) in additional mice followed up to 15 days
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Figure 1. Presence of intragastrically inoculated Bacillus species in faeces of mice. The data refer to two distinct experiments. Upper panel: with probiotic B. clausii spores; lower panel: with spores of B. subtilis ER229. Three mice were killed at 4, 24 and 72 h (left to right), respectively, after a single intragastric inoculum of 109 spores. Bacillus spore counts after heat inactivation of one part of the sample are shown by black bars. Counts from direct plating of the samples are reported by white bars (cumulative counts of spores and vegetative cells).
(data not shown). After day 3, the spore count of the inoculated antibiotic-resistant Bacillus strains was about ten-fold lower than total spore count (environmental/food-derived spores). The presence of spores in food and faeces (5 × 103 and 1 × 103 CFU/g, respectively) (data not shown) is in agreement with previous reports [1, 10, 32]. 3.3. Transit tolerance in the intestinal tract
Bile salts present in the intestine are toxic to many bacterial species and resistance to these salts is one of the main parameters for determining transit tolerance of a bacterium [4–6]. The MIC of two bile salts was determined for B. subtilis, the probiotic B. clausii and B. cereus strains (Bactisubtilt). Both the conjugated bile salt taurodeoxycholate (TDOC) and the unconjugated deoxycholate (DOC) were chosen for
MIC determination. As shown in table I all three Bacillus stains had exceptionally low MICs to TDOC when compared to other human commensal bacteria. Comparable profiles were also obtained with DOC, but while MIC for TDOC correlated with intestinal survival of enteric flora (E. coli, Proteus, Klebsiella, enterococci), DOC did not (low MIC for E. coli). The 100- to 1 000-fold lower resistance of Bacillus to TDOC, with respect to intestinal bacteria, probably explains the absence of spore outgrowth in the intestinal environment. The effect of both bile salts on spore germination was measured and revealed a slight inhibition of germination (about 20% at 20–25 min) (data not shown). This decrease in the germination efficiency of B. subtilis spores was observed at all concentrations of bile salts tested
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Figure 2. Presence of Bacillus spores (B. subtilis in panels A and B; B. clausii in panels C and D) in ileum and colon of mice killed at 4, 24 and 72 h, respectively, after a single intragastric inoculum of 109 spores. Data reported are the mean of the two samples (inactivated and noninactivated). Error bars refer to standard deviation calculated on samples of three mice killed at each time point (mice 1–3, 4–6 and 7–9, respectively, for both experiments).
(one dilution above and below the MIC on vegetative cells) (data not shown). 3.4. Bacterial translocation
Translocation of bacteria to lymphoid organs [12, 33] and the presence in blood was con-
trolled for all animals at death. No significant presence of bacteria or spores could be detected in blood samples. Bacilli were detected in mesenteric lymph nodes and spleen of one mouse inoculated with B. clausii (mouse 3, figure 1). In both mesenteric lymph nodes and the spleen of
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Figure 3. Bacillus counts in murine faeces over time. As in figure 1, the data refer to two distinct experiments, one with probiotic B. clausii spores (open rectangles), the other with spores of B. subtilis ER229 (filled circles).
mouse 3 the cumulative count of vegetative cells and spores was about ten-fold higher than the count of spores alone (1.4 × 104 versus 1.6 × 103 CFU/g and 2.0 × 104 versus 3.6 × 103 CFU/g in lymph nodes and spleen, respectively).
4. Discussion The present work was intended to determine which biological form of Bacillus, vegetative
cells or spores, is present in intestinal samples. The interest in this question derives from an intrinsic conflict concerning the use as probiotics of microorganisms that are not part of the normal intestinal flora. The conflict is generated on one hand by the general belief (deriving from work on other bacteria) that probiotics act upon the host organism through metabolic products or cell components of actively multiplying vegetative cells [28, 29], and, on the other, by the knowledge that the Bacillus is not part of the normal intestinal microflora of man. In spite of the daily intake with food (wheat, milk and their derived products) [7, 27], Bacillus species do not colonize the intestine of man, and it has been previously reported, at least for one species, “that carriage of B. cereus in stools is transient and its presence at any one time reflects solely its intake with foods” [31]. In this work we used a probiotic B. clausii strain and a derivative of B. subtilis MO1099 in a murine animal model. Our data, in agreement with previous reports on thermophilic bacilli generally used as gut transit markers [19, 25], show that elimination of spores from the intestinal tract of mice is a logarithmic phenomenon (figure 3). In case of any establishment, even at low level, the curve in figure 3 should level up (see also figure 2.1 in ref. [25]), but no such event takes place within the detection limit of
Table I. Minimal inhibitory concentration of bile salts. Species B. subtilis B. cereus (Bactisubtilt) B. clausii (Enterogerminat) S. gordonii S. pneumoniae E. faecalis E. faecium S. aureus H. influenzae N. lactamica E. coli K. planticola K. pneumoniae M. catarrhalis P. maltophilia P. mirabilis
MIC of TDOC µg/mL (mM)
MIC of DOC µg/mL (mM)
195 (0.4) 98 (0.2) ≤ 24 (≤ 0.05) 12 500 (25.6) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) 3 125 (6.4) ≥ 25 000 (≥ 51.2) ≤ 24 (≤ 0.05) 195 (0.4) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2)
78 (0.2) 78 (0.2) 78 (0.2) ≤ 20 (≤ 0.05) ≤ 20 (≤ 0.05) 625 (1.6) 625 (1.6) ≥ 20 000 (≥ 51.2) ≥ 20 000 (≥ 51.2) ≥ 20 000 (≥ 51.2) ≤ 20 (≤ 0.05) ≥ 20 000 (≥ 51.2) ≤ 20 (≤ 0.05) 39 (0.1) 312 (0.8) ≥ 20 000 (≥ 51.2)
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our experiments (103 CFU/g) and the time span analysed. In addition, throughout the sampling no evidence of a significant quantity of vegetative cells could be detected by differential plating, either in the ileum, colon or faeces. The 100- to 1 000-fold lower MICs of the bile salt TDOC on Bacillus (B. subtilis and the probiotics B. clausii and B. cereus), when compared to normal commensals of the human intestine (enterobacteria, enterococci), may well be one of the main factors inhibiting the intestinal growth of these species. Although we cannot rule out the possibility that a small fraction of spores germinate (less than 1%) and may give rise to a limited population of vegetative cells which undergo few or no duplications, our data indicate that Bacillus spores inoculated intragastrically transit through the murine intestine and are excreted in the faeces as spores. A different situation was encountered when analysing samples of lymphoid organs. In one mouse, both mesenteric lymph nodes and spleen were positive for B. clausii and, most interestingly, about 90% of the viable cells of these samples were vegetative cells. Although this was the case for a single mouse, one of the three mice having over 108 spores of B. clausii per gram of faeces, it suggests that outside the intestine Bacillus species are able to germinate and grow. It is therefore tempting to speculate that the intestinal environment (i.e. bile salts) is able to block germination/outgrowth of spores, but that these bacterial forms are capable of returning to the vegetative life when translocated outside the gastrointestinal tract. This is in concordance with a variety of clinical reports ([10, 24, 26, 30] and references therein), which document a significant increase in blood cultures positive for ‘nonpathogenic’ Bacillus species in immunocompromised hosts (cancer, transplant patients). In this context, it should be noted that B. subtilis has been shown to survive to some extent in macrophages [3] while the pathogenic B. anthracis does determine respiratory anthrax (haemorragic lymphadenitis) after germination in macrophages [14]. Further studies on germination in the mammalian host of ‘nonpathogenic’ bacilli may be needed to deter-
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mine the importance of inoculum, survival rate, and survival time or other factors affecting spore germination and outgrowth outside the intestinal environment. In conclusion, this report indicates that if a probiotic effect of bacilli does occur, this may be due to spores or to some not yet defined extraintestinal life cycle rather than to metabolically active cells, as shown for lactobacilli and other members of the intestinal microflora.
Acknowledgments The generous support of the Commission of the European Union (BIOTECH Programme, contract n. BIO4-98-0144) is gratefully acknowledged.
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[13] Green D.H., Wakely P.R., Page A., Barnes A., Baccigalupi L., Ricca E., Cutting S., Characterization of two Bacillus probiotics, Appl. Environ. Microbiol. 65 (1999) 4288–4291. [14] Guidi-Rontani C., Weber-Levy M., Labruyere E., Mock M., Germination of Bacillus anthracis spores within alveolar macrophages, Mol. Microbiol. 31 (1999) 9–17. [15] Hoffman T., Troup P., Szabo A., Hungerer C., Jahn D., The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system, FEMS Microbiol. Lett. 131 (1995) 219–225. [16] Hofte H., Whiteley H.R., Insecticidal crystal proteins of Bacillus thuringiensis, Microbiol. Rev. 53 (1989) 242–245. [17] Hosoi T., Amenati A., Kiuchi K., Kaminogawa S., Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components, Can. J. Microbiol. 45 (1999) 59–66. [18] Kirchgessner M., Roth F.X., Eidelsburger U., Gedek B., The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets, 1. Effect on the growth parameters and gastrointestinal environment, Arch. Tierernahr. 44 (1993) 111–121. [19] Mathers J.K., Smith H., Carter S., Dose-response effects of raw potato starch on small-intestinal escape, large-bowel fermentation and gut transit time in the rat, Br. J. Nutr. 78 (1997) 1015–1029. [20] Muscettola M., Grasso G., Blach-Olszewska Z., Migliaccio P., Borghesi-Nicoletti C., Giarratana M., Gallo V.C., Effects of Bacillus subtilis spores on interferon production, Pharmacol. Res. 26 (1992) 176–177. [21] Nakano M.N., Dailly Y.P., Zuber P., Clark D.P., Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth, J. Bacteriol. 179 (1997) 6749–6755. [22] Nicholson W.L., Setlow P., Sporulation, germination and outgrowth, in: Harwood C., Cutting S. (Eds.), Molecular Biological Methods for Bacillus, John Wiley and Sons, Chichester, 1990, pp. 391–451. [23] Nielsen P., Fritze D., Priest F.G., Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species, Microbiology 141 (1995) 1745–1761.
[24] Oggioni M.R., Galieni P., Bigazzi C., Pozzi G., Valensin P.E., Recurrent septicaemia in immunocompromised patient due to probiotic strains of Bacillus subtilis, J. Clin. Microbiol. 36 (1998) 325–326. [25] Raibaud P., Bacterial interactions in the gut, in: Fuller R. (Ed.), Probiotics: The Scientific Basis, Chapman and Hall, London, 1992, pp. 9–28. [26] Richard V., Van der Auwera P., Snoeck R., Daneau D., Meunier F., Nosocomial bacteremia caused by Bacillus species, Eur. J. Clin. Microbiol. 7 (1988) 783–785. [27] Rosenkvist H., Hansen A., Contamination profiles and characterisation of Bacillus species in wheat bread and raw materials for bread production, Int. J. Food Microbiol. 26 (1995) 353–363. [28] Salminen S., Salminen E., Clinical use of probiotics for stabilising the gut mucosal barrier: successful strains and future challenges, Antonie van Leeuwenhoek 2–4 (1996) 347–358. [29] Salminen S., von Wright A., Morelli L., Marteau P., Brassart D., De Vos W.M., Fonden R., Saxelin M., Collins K., Mogensen G., Birkeland S.E., Mattila Sandholm T., Demonstration of safety of probiotics – a review, Int. J. Food Microbiol. 44 (1998) 93–106. [30] Santini F., Borghetti V., Amalfitano G., Mazzucco A., A Bacillus licheniformis prosthetic aortic valve endocarditis, J. Clin. Microbiol. 33 (1995) 3070–3073. [31] Sorokulova I.B., Kirik D.L., Pinchuk I.V., Probiotics against Campylobacter pathogens, J. Travel Med. 4 (1997) 167–170. [32] Turnbull P.C., Kramer J.M., Intestinal carriage of Bacillus cereus: faecal isolation studies in three population groups, J. Hyg. (London) 95 (1985) 629–638. [33] Waitzberg D.L., Mester M., Significance of bacterial traslocation in nutrition, Arq. Gastroenterol. 35 (1998) 110–115. [34] Validation of the publication of new names and new combinations previously effectively published outside the IJSB. No. 55, Int. J. Syst. Bacteriol. 45 (1995) 879–880.
On the fate of ingested Bacillus spores Maria R. Spinosaa, Tiziana Braccinia, Ezio Riccab, Maurilio De Feliceb, Lorenzo Morellic, Gianni Pozzia, Marco R. Oggionia* a
Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Università di Siena, Via Laterina 8, 53100 Siena, Italy b Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli Federico II, Napoli, Italy c Istituto di Microbiologia, Università Cattolica S. C., Piacenza, Italy Received 22 November 1999; accepted 24 February 2000
Abstract — Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine. © 2000 Éditions scientifiques et médicales Elsevier SAS Bacillus / probiotic / spore / bile / Bacillus subtilis / lymph node / germination / transit marker
1. Introduction The use of bacteria for equilibrating the intestinal microflora and the optimization of intestinal functions has received attention for human and veterinary applications. Among the bacteria used as probiotics, bacteria belonging to the genus Bacillus represent a peculiar situation. Unlike other bacteria, B. subtilis [31], B. cereus [2, 3, 18, 31], B. clausii [8, 13] (Spinosa et al., Microbial Ecology in Health and Disease 2000, in press) and B. coagulans [1] are given orally as spores, not as vegetative forms. The beneficial effect of other probiotics, such as lactobacilli, has been ascribed to metabolic products or proteins of actively multiplying cells in the anaerobic environment of the intestine (reviewed by Salminen and colleagues) [28, 29]. No such metabolites or proteins have been identified in
* Correspondence and reprints [email protected]
Bacillus, even if they are suspected, for example in the case of Bacillus natto [17]. An immunestimulating activity has been reported [11, 20], but the mechanism of stimulation is not fully explained. So far, the only known direct effect of Bacillus in the intestinal environment (of mosquito larvae) is due to a protoxin (crystal protein) produced by B. thuringiensis and B. sphaericus during sporulation [16]. Since the mammalian intestine is an anaerobic environment and Bacillus spp. are preferentially aerobic [15, 21], germination and outgrowth of spores in the intestine seem difficult to envisage. Moreover, the capacity to survive the lytic action of bile salts, one of the criteria used to select potentially probiotic strains [4, 6], is highly uncommon in nonenteric microorganisms [5]. To analyse which biological form of Bacillus could be responsible for the probiotic effect(s), in the work presented here we fed mice with two nonpathogenic Bacillus species and tested
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for the presence of spores and vegetative cells in the murine intestine.
2. Materials and methods 2.1. Bacterial strains and growth conditions
The B. subtilis strain MO1099 (amyE::erm) [9] was used to isolate a spontaneous rifampicinresistant mutant ER229. Spores of the erythromycin- and rifampicin-resistant strain were prepared by growing cells for 36 h in Difco sporulation medium (DSM; Difco Laboratories), and treated with lysozyme to eliminate vegetative cells as previously described [22]. Bacillus clausii [23, 34] spores were from an Italian pharmaceutical preparation for human use which has been licensed for oral treatment of intestinal disorders (Enterogerminat; Sanofi Winthrop, Milan, Italy). This strain was previously allotted to the B. subtilis [8] species and later to B. alcalophius [13]. Here it is reported as B. clausii [34] due to new 16S rRNA sequences (GenBank Accession No. AF172603). Bacteria from animal samples were plated on tryptic soy agar (TSA; Difco Laboratories) at 37 °C in aerobic conditions. Antibiotics for selection of strains were rifampicin (50 µg/mL) or erythromycin (5 µg/µL). 2.2. Animals
Seven-week-old female BALB/c mice, obtained from Charles River (Italy), were used for experiments and housed in our animal facility for the duration of the experiment. 2.3. Experimental set-up
Two distinct experiments were performed: one using B. subtilis and the other one B. clausii spores. In both experiments, nine BALB/c mice were inoculated, while three control mice did not receive any inoculum. Prior to inoculation and killing, animals were left without food for 16 h. Animals were killed at 4, 24 or 72 h after inoculum and samples from intestinal sites and lymphoid organs and blood were taken.
2.4. Inoculum
Spores were centrifuged and resuspended at room temperature in distilled water at a concentration of 109 spores/200 µL. The precise number of spores injected intragastrically was calculated by microscopical count with a Burker chamber. Intragastric inoculum of 1 × 109 spores was performed using an animal feeding needle. 2.5. Samples for microbiological analysis
Blood samples were obtained from anaesthetized mice prior to killing from the cheek (100–200 µL) which had been disinfected by ethanol and swabbed for control of superficial contamination. Samples from lymphoid organs were the mesenteric lymph nodes and the spleen. Lymph nodes were fragmented by needle disruption or mild sonication (1 mL of saline), while the spleen contents were ‘washed out ‘ with a syringe (1 mL). Samples from the intestinal tract were from the ileum or colon and a faecal sample was obtained from the terminal rectum. About 2-cm fragments of the ileum and colon were washed with 1 mL of saline. Care was taken to collect colon samples free of faecal content. The faecal sample was homogenized with a sterile toothpick and resuspended in 1 mL of saline. All samples were immediately divided into two aliquots. One of these was subjected to heat inactivation at 80 °C for 20–25 min to kill vegetative cells, while the other was left at room temperature. Samples obtained from lymph nodes, spleen and blood were plated on nonselective tryptic soy agar plates (TSA, Difco Laboratories) to monitor contamination of environmental or intestinal microorganisms. Intestinal samples were plated on rifampicin-containing TSA plates to counterselect enteric flora, at dilutions ranging from 101 to 106. Intestinal samples were also plated on MacConkey agar (Biotec, Grosseto, Italy) to monitor possible changes in the enteric flora counterselected in the plates above. An inactivated food sample and an inactivated faecal sample of a control mouse were plated on TSA medium (Difco Laboratories) to control environmental spore contamination.
M.R. Spinosa et al. / Res. Microbiol. 151 (2000) 361–368 2.6. MIC determination for bile salts
Minimum inhibitory concentration (MIC) of the conjugated bile salt taurodeoxycholic acid sodium salt (TDOC 25 mM = 1.2% w/v) and the nonconjugated salt deoxycholic acid (DOC 25 mM = 1% w/v) was determined on 96-well microtitre plates. Bacteria grown overnight on plates were resuspended in liquid medium at a concentration of 105 CFU/mL and aliquoted into the microtitre plate (100 µL/well). TDOC and DOC were distributed by two-fold dilutions in a range 51.2–0.05 mM. Growth was registered after 24 h of incubation. Media were Muller Hinton broth for enterobacteriaceae, Pseudomonas and Bacillus, TSB (tryptic soy broth; Difco Laboratories) for streptococci and staphylococci and supplemented Muller Hinton Broth (SR158E, Oxoid) for Haemophilus, Neisseria and Branhamella. The following strains were used: B. subtilis (ER229, see above), B. cereus (Bactisubtilt; AF172711), B. clausii (Enterogerminat; AF142576; AF172603), Streptococcus gordonii (Challis), Enterococcus faecalis (OG1 SS), Streptococcus pneumoniae (R6), Moraxella catarrhalis (BRA022 22-77, clinical isolate), Enterococcus faecium (ENTO85 23-56, clinical isolate), Escherichia coli (ATCC25922), Klebsiella planticola (ATCC 33531), Klebsiella pneumoniae (KL0023, clinical isolate), Pseudomonas maltophilia (clinical isolate), Proteus mirabilis (PRO14 22-60, clinical isolate), Staphylococcus aureus (ATCC29923), Neisseria lactamica (NE007bis 22-42, clinical isolate) and Haemophilus influenzae (HAE089 23-27, clinical isolate). All clinical strains (identified by conventional biotyping) were human isolates from the collection of the Microbiology Section, Department of Molecular Biology, University of Siena. 2.7. Inhibition of B. subtilis germination by bile salts
B. subtilis spores were prepared and purified as described by Nicholson and Setlow [22]. Purified spores were heat-activated (10 min at 80 °C) and diluted in 10 mM Tris-HCl pH 8.4 to give a final OD580 of approximately 0.3 (2 × 107 spores/mL). D-glucose (1 mM), D-fructose
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(1 mM) and KCl (10 mM), each dissolved in 10 mM Tris-HCl pH8.4, were added to the suspension and equilibrated at 37 °C for 15 min. Germination was initiated by the addition of 10 mM L-alanine. Optical density (580 nm) was measured at 5-min intervals until a constant reading was reached. The efficiency of germination was expressed as percentage decrease in OD with respect to initial OD of the sample. Experiments, performed in duplicate, were carried out in the presence of 0.2 and 0.4 mM DOC and 0.2, 0.4 and 0.8 mM TDOC.
3. Results 3.1. Biological state of B. subtilis and B. clausii in mice after intragastric inoculation of spores
To verify whether vegetative forms of bacilli are present in the intestinal environment after inoculation of spores, two groups of mice were inoculated intragastrically with a single dose of 109 spores of B. subtilis and the probiotic B. clausii, respectively. After sampling, the specimens were immediately divided into two aliquots one of which was incubated at 80 °C for 20 min. This heat treatment was applied in order to eliminate all possible vegetative cells from one of the two samples. In this way, one of the two samples (heat-treated) would permit the counting of the spores only, while the other would yield, upon plating, the count resulting from the sum of spores and vegetative cells. A 1-min 80 °C treatment was found to reduce the viable count of vegetative cells to < 0.0001% (B. subtilis) and < 0.001% (B. clausii), while it did not affect spore viability (data not shown). 3.2. Distribution of B. subtilis spores in the intestinal tract
At 4 h after inoculation Bacillus spores were found at all intestinal sites (figures 1 and 2). Spore counts in faeces and other intestinal sites diminished with an exponential rate over the duration of the experiment (figure 3), and were reduced beyond the detection limit (5 × 102/g of feces) in additional mice followed up to 15 days
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Figure 1. Presence of intragastrically inoculated Bacillus species in faeces of mice. The data refer to two distinct experiments. Upper panel: with probiotic B. clausii spores; lower panel: with spores of B. subtilis ER229. Three mice were killed at 4, 24 and 72 h (left to right), respectively, after a single intragastric inoculum of 109 spores. Bacillus spore counts after heat inactivation of one part of the sample are shown by black bars. Counts from direct plating of the samples are reported by white bars (cumulative counts of spores and vegetative cells).
(data not shown). After day 3, the spore count of the inoculated antibiotic-resistant Bacillus strains was about ten-fold lower than total spore count (environmental/food-derived spores). The presence of spores in food and faeces (5 × 103 and 1 × 103 CFU/g, respectively) (data not shown) is in agreement with previous reports [1, 10, 32]. 3.3. Transit tolerance in the intestinal tract
Bile salts present in the intestine are toxic to many bacterial species and resistance to these salts is one of the main parameters for determining transit tolerance of a bacterium [4–6]. The MIC of two bile salts was determined for B. subtilis, the probiotic B. clausii and B. cereus strains (Bactisubtilt). Both the conjugated bile salt taurodeoxycholate (TDOC) and the unconjugated deoxycholate (DOC) were chosen for
MIC determination. As shown in table I all three Bacillus stains had exceptionally low MICs to TDOC when compared to other human commensal bacteria. Comparable profiles were also obtained with DOC, but while MIC for TDOC correlated with intestinal survival of enteric flora (E. coli, Proteus, Klebsiella, enterococci), DOC did not (low MIC for E. coli). The 100- to 1 000-fold lower resistance of Bacillus to TDOC, with respect to intestinal bacteria, probably explains the absence of spore outgrowth in the intestinal environment. The effect of both bile salts on spore germination was measured and revealed a slight inhibition of germination (about 20% at 20–25 min) (data not shown). This decrease in the germination efficiency of B. subtilis spores was observed at all concentrations of bile salts tested
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Figure 2. Presence of Bacillus spores (B. subtilis in panels A and B; B. clausii in panels C and D) in ileum and colon of mice killed at 4, 24 and 72 h, respectively, after a single intragastric inoculum of 109 spores. Data reported are the mean of the two samples (inactivated and noninactivated). Error bars refer to standard deviation calculated on samples of three mice killed at each time point (mice 1–3, 4–6 and 7–9, respectively, for both experiments).
(one dilution above and below the MIC on vegetative cells) (data not shown). 3.4. Bacterial translocation
Translocation of bacteria to lymphoid organs [12, 33] and the presence in blood was con-
trolled for all animals at death. No significant presence of bacteria or spores could be detected in blood samples. Bacilli were detected in mesenteric lymph nodes and spleen of one mouse inoculated with B. clausii (mouse 3, figure 1). In both mesenteric lymph nodes and the spleen of
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Figure 3. Bacillus counts in murine faeces over time. As in figure 1, the data refer to two distinct experiments, one with probiotic B. clausii spores (open rectangles), the other with spores of B. subtilis ER229 (filled circles).
mouse 3 the cumulative count of vegetative cells and spores was about ten-fold higher than the count of spores alone (1.4 × 104 versus 1.6 × 103 CFU/g and 2.0 × 104 versus 3.6 × 103 CFU/g in lymph nodes and spleen, respectively).
4. Discussion The present work was intended to determine which biological form of Bacillus, vegetative
cells or spores, is present in intestinal samples. The interest in this question derives from an intrinsic conflict concerning the use as probiotics of microorganisms that are not part of the normal intestinal flora. The conflict is generated on one hand by the general belief (deriving from work on other bacteria) that probiotics act upon the host organism through metabolic products or cell components of actively multiplying vegetative cells [28, 29], and, on the other, by the knowledge that the Bacillus is not part of the normal intestinal microflora of man. In spite of the daily intake with food (wheat, milk and their derived products) [7, 27], Bacillus species do not colonize the intestine of man, and it has been previously reported, at least for one species, “that carriage of B. cereus in stools is transient and its presence at any one time reflects solely its intake with foods” [31]. In this work we used a probiotic B. clausii strain and a derivative of B. subtilis MO1099 in a murine animal model. Our data, in agreement with previous reports on thermophilic bacilli generally used as gut transit markers [19, 25], show that elimination of spores from the intestinal tract of mice is a logarithmic phenomenon (figure 3). In case of any establishment, even at low level, the curve in figure 3 should level up (see also figure 2.1 in ref. [25]), but no such event takes place within the detection limit of
Table I. Minimal inhibitory concentration of bile salts. Species B. subtilis B. cereus (Bactisubtilt) B. clausii (Enterogerminat) S. gordonii S. pneumoniae E. faecalis E. faecium S. aureus H. influenzae N. lactamica E. coli K. planticola K. pneumoniae M. catarrhalis P. maltophilia P. mirabilis
MIC of TDOC µg/mL (mM)
MIC of DOC µg/mL (mM)
195 (0.4) 98 (0.2) ≤ 24 (≤ 0.05) 12 500 (25.6) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) 3 125 (6.4) ≥ 25 000 (≥ 51.2) ≤ 24 (≤ 0.05) 195 (0.4) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2) ≥ 25 000 (≥ 51.2)
78 (0.2) 78 (0.2) 78 (0.2) ≤ 20 (≤ 0.05) ≤ 20 (≤ 0.05) 625 (1.6) 625 (1.6) ≥ 20 000 (≥ 51.2) ≥ 20 000 (≥ 51.2) ≥ 20 000 (≥ 51.2) ≤ 20 (≤ 0.05) ≥ 20 000 (≥ 51.2) ≤ 20 (≤ 0.05) 39 (0.1) 312 (0.8) ≥ 20 000 (≥ 51.2)
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our experiments (103 CFU/g) and the time span analysed. In addition, throughout the sampling no evidence of a significant quantity of vegetative cells could be detected by differential plating, either in the ileum, colon or faeces. The 100- to 1 000-fold lower MICs of the bile salt TDOC on Bacillus (B. subtilis and the probiotics B. clausii and B. cereus), when compared to normal commensals of the human intestine (enterobacteria, enterococci), may well be one of the main factors inhibiting the intestinal growth of these species. Although we cannot rule out the possibility that a small fraction of spores germinate (less than 1%) and may give rise to a limited population of vegetative cells which undergo few or no duplications, our data indicate that Bacillus spores inoculated intragastrically transit through the murine intestine and are excreted in the faeces as spores. A different situation was encountered when analysing samples of lymphoid organs. In one mouse, both mesenteric lymph nodes and spleen were positive for B. clausii and, most interestingly, about 90% of the viable cells of these samples were vegetative cells. Although this was the case for a single mouse, one of the three mice having over 108 spores of B. clausii per gram of faeces, it suggests that outside the intestine Bacillus species are able to germinate and grow. It is therefore tempting to speculate that the intestinal environment (i.e. bile salts) is able to block germination/outgrowth of spores, but that these bacterial forms are capable of returning to the vegetative life when translocated outside the gastrointestinal tract. This is in concordance with a variety of clinical reports ([10, 24, 26, 30] and references therein), which document a significant increase in blood cultures positive for ‘nonpathogenic’ Bacillus species in immunocompromised hosts (cancer, transplant patients). In this context, it should be noted that B. subtilis has been shown to survive to some extent in macrophages [3] while the pathogenic B. anthracis does determine respiratory anthrax (haemorragic lymphadenitis) after germination in macrophages [14]. Further studies on germination in the mammalian host of ‘nonpathogenic’ bacilli may be needed to deter-
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mine the importance of inoculum, survival rate, and survival time or other factors affecting spore germination and outgrowth outside the intestinal environment. In conclusion, this report indicates that if a probiotic effect of bacilli does occur, this may be due to spores or to some not yet defined extraintestinal life cycle rather than to metabolically active cells, as shown for lactobacilli and other members of the intestinal microflora.
Acknowledgments The generous support of the Commission of the European Union (BIOTECH Programme, contract n. BIO4-98-0144) is gratefully acknowledged.
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