The secret life of a probiotic Bacillus

What happens to probiotic bacteria as they travel from the feed through the digestive tract? Should live cells be used or spores? The activity of probiotic preparations in the digestive tracts of pigs and chickens has been explored for one species of Bacillus.
By Ortwin Simon, Anke Jadamus and Wilfred Vahjen
activity we find that they belong to different microbial groups (yeasts, bacteria, moulds) and to different genera with different metabolic characteristics and different natural habitats (Table 1). The microbial preparations currently authorised as feed additives contain different strains of the yeast Saccharomyces cerevisiae, spores of the bacterial genus Bacillus (whose natural habitat is soil) and vegetative cells of acidproducing intestinal bacteria (strains of Enterococcus faecium and Pediococcus spp.). These differences alone demonstrate their varying ability to maintain vital functions in the gut. Furthermore, the beneficial activities generally postulated for probiotics cannot apply equally to all microorganisms. Some general actions of probiotics are described as follows: - Aggregation with pathogenic organisms; - Attachment to receptors on the gut wallin competition with pathogenic organisms; - Formation of inhibitory metabolic products (bacteriocins, reuterin); - Competition with undesirable microbes for specific nutrients; - Lowering of pH through acid formation; - Modulation of the intestinal epithelium modified transport kinetics; - Modification of the breakdown of bile

eed additives have different core areas antibiotics exhibit very different modes of of activity. Preservatives, antioxidants action. While antibiotics act within hours or various technical treatments exert and directly upon contact, probiotics their effects primarily within the feedstuffs require several days to indirectly induce a themselves. Yet antioxidants, vitamins and shift in the dynamic microbial equilibrium. trace elements can also act in intermediary It was shown that even non-starch polysacmetabolism and affect product quality by charide-hydrolysing feed enzymes influence accumulation within the animal. Another the composition of the bacterial population group of feed additives, such as antibiotics, in the small intestine of poultry (Vahjen et al., 1998). Thus, various feed additives modimicro-organisms, coccidiostats and enzymes fy the microbial flora of the digestive tract, act primarily in the digestive tract. Organic but the underlying mechanisms of action acids and their salts are also known to exert must differ widely. physiological effects in the proximal sections of the digestive tract (Bolduan et al., A heterogeneous group 1988). With the exception of enzymes, it is Even when we consider only those micropresumed that their primary action occurs organisms described as having probiotic by modification of the microbial population in the digestive tract, albeit via different routes. For example, coccidiostats mainTable 1 - Probiotic micro-organisms applied alone or in combination in animal feeding ly inhibit the development of protozoa. Organic acids Microorganisms Spore Acid O2-behaviour Habitat affect the pH, especially in formation formation the stomach and possibly in Yeasts the proximal small intestine Saccharomyces cerevisiae (E5) Aerobic/anaerobic Fermenting fruits, food of monogastric animals and Candida pintolopesii may indirectly influence Fungi microbial colonisation by Aspergillus oryzae + Aerobic/anaerobic Soil altering the microbial enviBacteria ronment. Bacillus spp. (E3) + Aerobic/facult.anaerob. soil, dust The use of micro-organClostridium butyricum + Anaerobic intestine, soil, waters Bifidobacterium spp. + intestine, mucosa isms as feed additives (popuEnterococcus faecium* (E9) + Aerobic/facult.anaerob. intestine, mucosa larly known as probiotics) is Lactobacillus spp. (E3) + intestine, milk, dairy products (plants) currently widely promoted as L. reuteri* + an alternative to antibiotic Pediococcus spp. (E1) + intestine growth promoters (Zani et Streptococcus spp. (E1) + intestine, (milk, mucosa) al., 1988; Iben and Leibetseder, 1989). However, * Bacteriocin formation this proposition must be E = representatives of these microbial groups are authorised provisionally or permanently as feed additives in the viewed with considerable European Union (Official Journal of the European Communities L299/1 ff.) reservations. Probiotics and

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FEED MIX Volume 10 Number 6 2002

Figure 1 - Germination of B. toyoi in the digestive tract of broilers

Figure 2 - Germination of B. toyoi in the lumen of the digestive tract of piglets

Total microbial activity ( ) and spore content ( after different passage times 1E+06 16 h CFU/mgTiO2 1E+05 1E+04 1E+03 Duod.1 Duod.2 Ileum1 Ileum2 Stomach Caecum Caecum Colon Colon Jej.1 Jej.2 Jej.3 1E+02 )

Total microbial activity ( ) and spore content ( in the digestive tract of broilers 1E+6 30 min 1E+5 1E+4 1E+3 Crop Stomach Jejunum Ileum

CFU/mg TiO2 in the diet

CFU/mg TiO2 in the diet

Caecum

1E+6 60 min 1E+5 1E+4 1E+3

CFU/mg TiO2 in the diet

1E+06 24 h CFU/mgTiO2 Crop Stomach Jejunum Ileum Caecum 1E+05 1E+04 1E+03 Stomach Jej.1 Jej.2 Jej.3

CFU/mg TiO2 in the diet

1E+6 120 min 1E+5 1E+4 1E+3 Crop Stomach Jejunum

CFU/mg TiO2 in the diet

Ileum1

Duod.1

Ileum

Caecum

Figure 3 - Sporulation of germinated B. toyoi cells in the digestive tract of broilers

Total microbial activity ( 2h CFU/mg TiO2 1E+7 1E+6 1E+5 1E+4 1E+3 1E+2 1E+1 1E+0 ) and spore content ( ) after ingestion of germinated cells

- Permanent spores (resistant to high temperatures and pressures and extreme pH ranges) - Natural resistance to antibiotics Several studies were carried out, regarding the mode of action of this probiotic, which is used as a spore preparation.

Behaviour in chicks and piglets

As probiotic bacteria usually exert their effect via their metabolic activity it was necessary to examine the germination behaviour of Bacillus spores in the digestive tract. This was of particular interest in chicks, because germination was not certain in view of the high digesta passage rate (four hours for the distal small intestine; 6.5 hours for the entire gut, according to Shires et al., 1987). The experimental design was identical for both species: after starving the animals for a short period, feed containing B. toyoi spores was offered in a single meal. The feed contained TiO2 as indicator substance. The animals were sacrificed at defined times after feeding, the digestive tract ligated in segments and digesta samples removed for the determination of total and spore colony forming units (CFU) of B. toyoi. Tests in broilers followed the same procedure, except that sample collection times after administration of the probiotic were considerably shorter than for pigs (30 to 120 min). Dilutions of the digesta were plated out on agar media containing antibiotics. Differentiation of total and spore counts was performed by heating the samples to 80C for 15 minutes. This treatment kills vegetative cells, but not spores. Sporulation of vegetative cells in the digestive tract was studied by the same proce-

Diet

Crop

Stomach Jejunum

Ileum

Caecum

4h CFU/mg TiO2

1E+7 1E+6 1E+5 1E+4 1E+3 1E+2 1E+1 1E+0

Diet

Crop

Stomach Jejunum

Ileum

Caecum

acids or deconjugation of bile acids. Such diversity in effects implies that the routes by which different probiotic microorganisms influence the dynamic balance of the intestinal microflora will also differ. General statements like probiotics promote a state of eubiosis or ...stabilise the gut flora do not clarify their specific modes of action. In order to gain an understanding of the mode of action of different probiotics, these statements must be explained and substantiated for each probiotic strain.

Bacillus cereus var. toyoi (B. toyoi)

The following main characteristics of B. toyoi suggest that suitable conditions for the performance of metabolic functions by this micro-organism exist in the digestive tract: - Isolated from soil samples in Japan - Facultative anaerobe - Non-toxin-forming Bacillus strain - Usable substrates are: starch, glucose, gelatine, propionate - Temperature range for growth: 25-40C - Optimum pH 7.2

Duod.2

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Ileum2

1E+02

Absorption [690 nm] after 24 h

dure, except that a liquid culture of only vegetative cells instead of spores was orally administered by intubation. The results in broilers (Figure 1) indicate that spores of B. toyoi germinate very rapidly in the digestive tract. Within 30 minutes after consuming the spore-containing diet, over 90 % of the probiotic spores were present in the crop as vegetative cells. All but 1% of spores had germinated before reaching the proventriculus, so the small intestinal digesta contained the probiotic primarily in the form of vegetative cells. The spore content relative to total probiotic counts was of similar magnitude in the digesta of the stomach and small intestine of piglets at 16 and 24 hours post administration (Figure 2). In the distal sections of the digestive tract, a trend was observed towards higher proportions of spores, suggesting that the vegetative probiotic cells sporulate again in the digestive tract. This was demonstrated in experiments with both piglets and broilers. The results of studies in broilers are presented in Figure 3. Two and four hours after oral administration of vegetative B. toyoi cells, up to 100 % of this organism could also be detected as spores in the various sections of the digestive tract. These studies suggest that B. toyoi germinates, grows and sporulates again during its passage through the digestive tract, which in turn would indicate a high persistence rate of the bacterium. This assumption was tested in broilers using an experimental diet containing B. toyoi and an indicator (2 x 105 CFU/mg TiO2). One week after changing to a B. toyoi-free, but TiO2-containing diet, the contents of segments of the digestive tract were examined for total counts of B. toyoi and the concentration of the marker. While the concentration of the indicator in relation to the total probiotic counts had declined by one to two powers of magnitude in the proximal sections of the digestive tract, the digesta of the ileum and caecum still contained 4.5-5.0 x 105 CFU/mg TiO2, approximately equivalent to the concentration of the probiotic in the original diet. The spore content was about 30% of the total probiotic CFU. Investigations have shown that the bacterium also has a high persistence rate in pigs. In sows, the concentration of B. toyoi in the faeces declined from 105 to 103 - 104 CFU/g of dry faecal matter after two weeks and was still detectable in the faeces after three weeks.

Figure 4 - Growth pattern of B. toyoi in the lumen of the digestive tract of piglets
Total microbial activity (
CFU/g wet matter CFU/g wet matter CFU/g wet matter CFU/g wet matter 1E+7 1E+5 1E+3 1E+1 21 days, 1 week supplementary feed 1E+7 1E+5 1E+3 1E+1 28 days, day of weaning 1E+7 1E+5 1E+3 1E+1 32 days, piglet starter 1E+7 1E+5 1E+3 1E+1 Stomach Duod.1 Duod.2 Jej.1 Jej.2 Jej.3 Ileum1 Ileum2 Caecum Colon Limit of detection of the method Limit of detection of the method

) and spore content (

) at different ages

13 days, suckling piglets

Figure 5 - Growth potential of enterobacteriacae in mucosal samples from the digestive tract of piglets

Absorption [690 nm] after 24 h

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Day 13 * *

** 1.4 * * 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.4 1.2 1.0 *** ** 0.8 0.6 0.4 0.2

Day 21 * ** ***

**

1.4 1.2 1.0 0.8

Day 28

Day 32

Establishment of B. toyoi in piglets

As explained above, effective probiotic function requires an adaptation period for the strain, because it indirectly modifies the intestinal microflora. If the objective of probiotic supplementation for piglets is the prevention of weaning problems, starting the medication at weaning would be inefficient. It was therefore proposed to bring piglets into contact with the probiotic as early as possible and to monitor the estab-

**

0.6 * 0.4 0.2 0.0 Duod.1 Duod.2 Jej.

0.0 Ileum Caecum Colon Duod.1 Duod.2 Jej.

(Simon et al. 1999)

Ileum Caecum Colon

Control B. toyoi

*Significant, p<0.05, **p<0.01, ***p<0.001

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Figure 6 - Growth potential of enterococci in mucosal samples from the digestive tract of piglets
Absorption [690 nm] after 24 h Absorption [690 nm] after 24 h

Diet complicates matters

Knowledge of how dietary factors influence microbial populations in the digestive tract is very limited at present. This is reflected by the fact that although some 400 to 500 micro-organisms are believed to colonise the digestive tract, a characterisation of these populations can only be carried out with a fraction of bacteria that grow on selective media. Important work on this issue has been carried out by Professor Gedek of the University of Munich in Germany, who studied the effect of selected probiotics on the main and secondary flora (Gedek et al., 1993). According to Professor Gedek, microbial populations in the intestine consist of the main flora (representatives of the genera Lactobacillus, Bifidobacterium, Bacteroides and Eubacterium), the secondary flora (E. coli and Enterococcus) and the residual flora (comprising primarily pathogenic species such as Staphylococcus, Proteus, Pseudomonas etc.). The desired ratio of main:secondary:residual flora is considered to be 90:1:0.01, a state referred to as eubiosis. Hence probiotics are designed to promote the main flora while inhibiting the remaining microbial groups. Samples for microbiological studies may consist of digesta from the intestinal lumen or of tissues from mucosal layers, which will contain mainly wall-associated microorganisms. Figures 5 and 6 show the in vitro growth potential of enterobacteria and enterococci (representatives of the secondary flora) from samples of control and probiotic-treated animals. It can be seen that the greatest changes occurred in the first two sampling days. However, at that time the probiotic was still poorly established in the digestive tract and it can be presumed that these alterations are attributable to the sows microbial population as modified by the probiotic. In addition to these microbial groups, the growth of all anaerobic and lactic acid bacteria was tested. However, no differences were observed between the treatments, so a more advanced interpretation of these findings is probably inappropriate. Knowledge about specific bacterial metabolic activities in the digestive tract is needed before their

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

Day 13

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

Day 21

***

0.35 0.30 0.25 0.20 0.15 0.10 0.05

Day 28

0.35 0.30 0.25 0.20 0.15 0.10 0.05

Day 32

* * Ileum Caecum Colon

Control B. toyoi

0.00 Duod.1 Duod.2 Jej.

0.00 Ileum Caecum Colon Duod.1 Duod.2 Jej.

(Simon et al. 1999)

*Significant, p<0.05, **p<0.01, ***p<0.001

lishment of B. toyoi during the first few weeks after birth and the potential effects on other groups of microbes. In the treated group the sows were given 109 CFU B. toyoi /kg diet as early as 14 days ante partum. The same concentration was added to the supplementary feed of the piglets during the suckling period and to the starter ration after weaning. The animals in the control group received the same diet without the probiotic. Only litters with >10 suckling piglets were included in the studies. Supplementary feed was offered from 14 days of age and the piglets were weaned at 28 days. Samples were collected from three piglets of each group on days 13 (before the first ingestion of supplementary feed), 21 (one week after introducing the supplementary feed), 28 (at weaning) and 32 (four days post weaning). The piglets were then sacrificed and the intestinal segments to be examined were ligated and placed in an anaerobic chamber. Sampling was performed under anaerobic conditions. The in vitro growth potential of gut wall-associated bacterial groups was determined by washing segments of intestinal tissue, scraping off the mucosal layer and incubating dilutions in selective liquid media in microtitre plates under anaerobic conditions. The establishment of B. toyoi in the digestive tract of the piglets is shown in Figure 4. B. toyoi was detectable in piglets as early as 13 days of age, especially in the distal sections of the digestive tract, although these piglets had not yet started to consume sup-

plementary feed. It can be presumed that the probiotic is ingested with small quantities of faeces from the sow. The fact that the probiotic was detected first in distal sections of the digestive tract may be due to low cell numbers, a rapid digesta passage and an unfavourable environment for the probiotic micro-organism in young piglets. Establishment of a bacterial population in the digestive tract can only occur in one of two situations: either there are factors facilitating its attachment to the gut wall or the organism is able to colonise the lumen against the flow of the digesta due to a high rate of cell division (Fox, 1988). Once piglets begin to consume supplementary feed, the small intestine becomes progressively colonised. At the time of weaning a balanced probiotic flora is present in all sections of the digestive tract.

Conclusions
The key findings of the studies described here may be summarised as follows: * Bacillus toyoi spores germinate very rapidly in the intestinal environment and may pass through several germination and sporulation cycles within the digestive tract. * Oral administration of spores of Bacillus toyoi results in colonisation of the entire digestive tract of broilers and piglets. * Bacillus toyoi persists for several days to weeks in the intestine after administration. * The composition of microbial populations in the digestive tract of suckling piglets is influenced by feeding Bacillus toyoi to the sow shortly before farrowing. * The most striking effect of Bacillus toyoi in young piglets was the suppression of the growth of enterobacteria and in part enterococci. * Future studies on the mode of action of probiotics must address the characterisation of bacterial physiological activities in the digestive tract.

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Figure 7 - Activity of taurocholic saltdeconjugating microbial enzymes in digesta supernatant from the jejunum of piglets
Area of lysis zone [mm2]

60 50 40 30 20 10 0

a a a a

Control B. toyoi

Suckling 1 week Weaning 4 days piglets supple- date post mentary weaning feed a,b= significant, Student's t-test, p<0.05

consequences for the host metabolism can be estimated. Moreover, these methods say very little about the residual flora, which also comprises microbes that are harmful to the host animal. Future studies on the mode of action of probiotics requires the application of new methodologies. This includes the use of the polymerase chain reaction (PCR), which enables scientists to demonstrate the incidence of genes of specific pathogenic organisms even at very low concentrations. Furthermore, activity of bacterial ribosomes can be determined with gene probes. Methodological work along these lines is currently being carried out in our working group. Physiological activities of microbial populations can also be characterised by the use of specific enzyme activities expressed solely by bacteria (Figure 7). In piglets of the treated group the activity of taurocholic salt-deconjugating enzymes in the jejunal digesta was significantly lower than in the control animals throughout the entire suckling period, indicating a shift of bacterial activities and thus composition of active bacterial populations in the intestine. In theory this might have an effect on fat absorption in the small intestine, but this hypothesis has not yet been tested experimentally.

A more detailed account of the experimental method is available on request.

References Bolduan et al. (1988). J. Anim. Physiol. Anim. Nutr. 59, 72-78 Fox (1988). Vet. Med. 83, 806-830 Gedek and Kirchgessner (1993). Arch. Tierernhr. 44, 215-226 Iben and Leibetseder (1989). Wien. Tierrztl. Mschr. 76, 363-366 Shires et al. (1987). Poult. Sci. 66, 289 - 298. Vahjen et al. (1998). J. Agric. Sci. 130, 489-500 Zani et al. (1988). J. Appl. Microbiol. 84, 68-71

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