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Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics
International Journal of Food Microbiology 98 (2005) 281 – 289 www.elsevier.com/locate/ijfoodmicro
Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics Alejandra Montesia, Raimundo Garcı´a-Albiacha, Marı´a Jose´ Pozueloa, Concepcio´n Pintadob, Isabel Gon˜ic, Rafael Rotgerb,* a
Departamento de Biologı´a Celular, Bioquı´mica y Biologı´a Molecular. Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo-CEU. Urbanizacio´n Monte Prı´ncipe, Boadilla del Monte, 28668 Madrid, Spain b Departamento de Microbiologı´a II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain c Departamento de Nutricio´n y Bromatologı´a I, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain Received 3 March 2004; received in revised form 9 June 2004; accepted 9 June 2004
Abstract The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results. D 2004 Elsevier B.V. All rights reserved. Keywords: Probiotics; Prebiotics; DGGE; Bifidobacterium; Lactobacillus; Rats
1. Introduction
* Corresponding author. Tel.: +34 913 941 755; fax: +34 913 941 745. E-mail address: [email protected] (R. Rotger). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.06.005
The intestinal tract of mammals harbours a complex bacterial ecosystem, which has not yet been fully characterized. Most of its members are obligate anaerobes and many of them (60–80%) have not been cultivated (Suau et al., 1999; Hayashi et al.,
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2003). However, it is now generally accepted that the composition of the human intestinal microbiota has an important role in health and disease (Gionchetti et al., 2000). Constipation, colonic cancer and inflammatory bowel diseases, for example, can be influenced by the microbiota composition and its metabolic activities, but a clear picture of the contribution of the different species in the maintenance of bowel health is not currently available. Over the last few years, the presence of a large number of bifidobacteria has been considered essential to promote intestinal health and to strengthen the local immune response. Lactic acid bacteria, like Lactobacillus, and some Streptococcus species, as Streptococcus thermophilus, are also regarded as beneficial members, whereas some members of other groups, namely Bacteroides, Clostridium and Enterobacteriaceae, would be harmful as a consequence of their metabolic activities or even the pathogenic potentiality of a few species. For example, high risk of colon cancer has been associated with presence of Bacteroides vulgatus and Bacteroides stercoris, and species of Bacteroides and Clostridium could trigger the inflammatory process in human inflammatory bowel diseases (see Guarner and Malagelada, 2003 for a review). As a consequence of these concepts, modification of the human intestinal microbiota has currently become an important objective of dietetics. This goal can be attempted in two ways: either to include in the diet a significant proportion of beneficial bacteria, mainly Bifidobacterium and Lactobacillus species, with the expectation that they will be able to colonize the intestinal tract (probiotics); or to give non-digestible carbohydrates, like fructo-oligosaccharides, which have shown an ability to promote the growth of desirable bacteria (prebiotics) (Kaur et al., 2002; Saarela et al., 2002; Sullivan and Nord, 2002). The complex and time-consuming methods required to isolate and identify the intestinal microbiota members and especially the impossibility of culturing many of them have hindered the study of these populations. However, the development of molecular techniques based on the characterization of 16S rRNA genes has greatly facilitated the work for microbiologists in this respect. Separation of the amplification products generated by PCR by denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE) provides characteristic band profiles
corresponding to the predominant bacterial species (Muyzer, 1999). Sequencing of PCR products reamplified directly from the excised bands permits the identity to be determined (genus or species). Molecular methods have been applied to study the gastrointestinal microbiota of humans (Satokari et al., 2001; Donskey et al., 2003; Nielsen et al., 2003), poultry (Netherwood et al., 1999; Gong et al., 2002) and pigs (Simpson et al., 2000; Leser et al., 2002). It is clear that there is no one single appropriate technique for these studies. For example, quantification of species requires hybridisation techniques (Mangin et al., 2002) or real-time PCR (Matsuki et al., 2004). Obviously, differences can be expected among the results obtained with different techniques and between classical and molecular methods (Wilson and Blitchington, 1996). In this work, we used DGGE to differentiate 16S RNA species amplified by PCR from caecal samples of rats, together with the classical plate-count enumeration of the main bacterial groups. We applied these methods to evaluate the effects on rats of two diet supplements, containing either prebiotics (fructo-oligosaccharides) or probiotics (Bifidobacterium lactis and S. thermophilus).
2. Material and methods 2.1. Animals and diets Thirty male Wistar rats of the same age with a body weight of 195F5 g were supplied by the breeding centre at the Faculty of Pharmacy (Universidad Complutense, Madrid, Spain). After weaning, all animals were fed with a standard maintenance diet (A04, Panlab, Barcelona, Spain). Animals were housed in individual metabolic cages in a room maintained at 22F1 8C, with 12-h light/dark cycles throughout the 28-day experimental period. Rats were divided into three groups and fed with diets containing 70% of a diet adjusted to the animals’ requirements (AIN-93 M purified rodent diet, DYETS, Bethlehem, PA, USA) (Reeves et al., 1993) and 30% of each of the following infant formulas depending on the experimental group: (a)
Control group: Conventional continuation formula (Modar 2, Sandoz, Vienna, Austria).
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(b) (c)
Prebiotic group: Prebiotic formula with 5.7% (w/ w) of fructo-oligosaccharides (Natur 2, Sandoz). Probiotic group: Probiotic formula with B. lactis BL and S. thermophilus (Nativa 2, Nestle´, Zurich, Switzerland).
The nutritional composition of the three infant formulas was similar and the only difference among them was the addition of prebiotics or probiotics. This study was approved by the Department of Nutrition of the Universidad Complutense. Food intake and body weight was controlled every week. After the experimental period, the animals were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight) and their caecum was removed.
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lab) for lactobacilli (Tortuero et al., 1997), supplemented Schaedler (bioMerieux, Marcy-l’Etoile, France) for Bacteroides, and MacConkey (Oxoid) for coliforms (Brigidi et al., 2001). All these plates were incubated anaerobically at 37 8C for 4–5 days, except in the case of the MacConkey plates which were incubated aerobically at 37 8C for 48–72 h. Representative colonies of each selective medium were identified to genus level by standard bacteriological methods such as Gram stain, cellular and colonial morphology and biochemical reactions. Differences in the number of colony forming units (CFU) of the samples were statistically analyzed with Statgraphics plus 5.1 (Statistical Graphics, Rockville, MD, USA) by the Fisher’s Least Significant Differences (LSD) for a confidence level of 90% (Walpole et al., 1992).
2.2. Caecal sample preparation 2.4. Amplification of 16S rDNA The rats were killed under CO2 after the experimental period and caeca were then removed and weighed. For cultivation of bacteria, approximately 1 g of each caecal sample was homogenised in prereduced Wilkins–Chalgren broth (Oxoid, Basingstoke, UK), to a final ratio of 1:10 (w/v); 10% glycerol was added and the samples were frozen at 80 8C. All these procedures were performed in oxygen-free, CO2-saturated atmosphere. For molecular methods, 0.5 g of each caecal specimen was added to 4.5 ml of sterile phosphate-buffered saline– EDTA (PBS–EDTA) and mixed by vortexing the tube for 6 min. One milliliter of each sample was then centrifuged at 13,400 rpm, 4 8C, for 5 min. The pellet was washed twice with PBS–EDTA and finally resuspended in 300 Al of PBS–EDTA, heated at 100 8C for 15 min and immediately frozen at 80 8C. 2.3. Enumeration of cultivable bacteria using plate culture The caecal samples were thawed at room temperature and serial dilutions (from 10 3 to 10 9) were made in pre-reduced Wilkins–Chalgren broth; 0.1 ml of each dilution was spread in duplicate onto the surface of plates which contained the following agar media: Wilkins–Chalgren agar (Oxoid) for enumeration of total anaerobes, supplemented MRS (Hispanlab, Madrid, Spain) for bifidobacteria (Harmsen et al., 1999), SPS (Hispanlab) for clostridia, MRS (Hispan-
The caecal samples were thawed at room temperature and DNA was extracted using a FastDNA spin kit for soil (BIO 101, BuenaVista, CA, USA), in which the cell lysis solution was replaced by CLS-TC (same manufacturer), which is more suitable for animal tissues. The V3 region of the 16S rRNA gene (positions 341–534 in the E. coli gene) was amplified by using primers 518R (5VATTACCGCGGCTGCTGG-3V) and 341F (5VCCTACGGGAGGCAGCAG-3V) with a 5V GC clamp (5V-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGGCACGGGGGG-3V) (Muyzer et al., 1993). PCR was performed in 0.2 ml tubes by using a Gene Amp PCR System 2400 (Perkin Elmer, Boston, MA, USA). Each reaction mixture contained reaction buffer, 1.5 mM MgCl2, deoxynucleosides triphosphate at a concentration of 200 AM, 15 pmol of each primer, 100 ng of caecal DNA and 2 U of Expand High Fidelity (Roche, Basel, Switzerland). The following amplification program was used: 97 8C for 5 min, 30 cycles consisting of 94 8C 1 min, 62 8C 40 s, 72 8C 30 s, and then 72 8C 7 min. Lactobacillus group-specific PCR was performed using the 16S rRNA gene-targeted primers Lac1 (5VAGCAGTAGGGAATCTTCA-3V) and Lac2 (5VATTYCACCGCTACACATG-3V) with a 5V GC clamp (5V-CGCCCGGGGCGCGCCCCGGGCGGCCCGGGGGCACCGGGG-3V) (Walter et al., 2001) that produces a 327 bp amplicon (positions 352–679), using the same amplification program as indicated above.
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Bifidobacterium genus-specific PCR was performed using 16S rRNA gene-targeted primers Bif164F (5VGGGTGGTAATGCCGGATG-3V) and Bif662 (5VCCACCGTTACACCGGGAA-3V) with a 5V GC clamp (5V-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3V) (Satokari et al., 2001), that produce a 523 bp amplicon (positions 164–679), and the following amplification program was used: 95 8C for 5 min, 40 cycles consisting of 94 8C 45 s, 52 8C 50 s, 72 8C 50 s, and then 72 8C for 7 min. 2.5. Denaturing gradient gel electrophoresis (DGGE) DGGE was performed by using a DCodek Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA) with gels of 16100.01 cm. A denaturing gradient of 37–55% was prepared as described elsewhere (Donskey et al., 2003). Electrophoresis was performed at 130 V and 60 8C for about 4:40 h. In the case of the Bifidobacterium-specific amplicons, the denaturing gradient used was of 45– 55% and the electrophoresis was done at 85 V for about 16 h. Gels were stained with a 5 Ag/ml ethidium bromide solution for 20 min, washed with deionised water and viewed by UV trans-illumination. Differences in the number of bands were statistically analyzed with Statgraphics plus 5.1 and the p value was determined for a confidence level of 95%.
2.7. Nucleotide sequence accession numbers The sequences determined in this study have been assigned GenBank accession numbers AY642396– AY642426, AY643067 AY643068, AY649331 and AY649332. 3. Results One rat belonging to the prebiotic group died before the end of the experiment. Examination of pooled caecal content was ruled out since an inter-individual variation could be expected, as it has been reported in humans (Zoetendal et al., 1998). Classical microbiological methods were only applied to three randomly selected rats from each group, due to the complexity of the analysis. Results of the enumeration of cultivable bacteria are presented in Fig. 1, and the inter-individual variation is reflected by the standard deviation bars. Due to the high inter-individual variability, statistically significant differences were evaluated for a confidence level of 90%.
2.6. Sequencing of PCR-amplified products Representative bands were excised from DGGE gels with a sterile scalpel and eluted overnight as described by Ausubel et al. (1993). A 5-Al aliquot was removed and re-amplified by PCR using the original primers without the GC clamp. Resulting PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced with the ABI PRISM bigDye Terminator Cycle sequencing Ready reaction Kit in an automated ABIPRISM 377DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The amplicons were sequenced from the 5V end using the aforementioned 518R and Lac1 primers. Sequences were analyzed with the software Chromas v. 2.23 (Technelysium, Tewantin, Australia), and their similarity with 16S RNA sequences stored in GenBank and EMBL databases was searched with the BLAST software (www.ncbi.nlm.nih.gov/BLAST).
Fig. 1. Enumeration (CFU/g of caecal content) of bacterial groups by plate count from samples of three rats of each experimental group: control (open bars), probiotic (black bars) and prebiotic (grey bars). Logarithmic scale is not the same for each bacterial group, as indicated in the ordinates legend. Lines over each bar indicate standard deviation among individuals and an asterisk indicates a statistically significant difference (90% confidence level).
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Fig. 2. DGGE analysis, in a 37–55% denaturising gradient, of the PCR products of amplification from rat caecal samples using universal primers for eubacterial 16S rRNA gene (518R and 341F; see Materials and methods). A, Control group, except lane bLQ that corresponds to a pure culture of B. lactis isolated from a probiotic. B, Probiotic group. C, Prebiotic group. Numerated arrows in gels B and C point to the bands subjected to re-amplification and sequencing, and correspond to the amplicon IDs of the Table 1. White arrow in gel A indicates the amplification product obtained with the same primers from the sample of B. lactis.
Both diet supplements, probiotic and prebiotic, were similarly effective at reducing bnon-desirableQ bacterial groups, namely Bacteroides and clostridia, and they also provoked a significant reduction in the number of total anaerobes. However, they had opposite effects on the recovery of coliforms: the probiotics induced a significant increase and the prebiotics a larger reduction. None of the diets was able to increase the number of lactobacilli. A higher increment of the bifidobacteria content was detected in the rats fed with prebiotics, whereas the Bifidobacterium-containing probiotics produced a non-significant effect. We enumerated the viable bifidobacteria present in the probiotic formula using the same selective media as for the caecal content, and we found 107 CFU/g. The presence of Streptococcus was not evaluated, since they were not looked for in the caecal content. Molecular analysis of the caecal content by amplification of 16S rRNA genes and DGGE was performed in samples from every individual of each group (Fig. 2). As previously reported in humans (Zoetendal et al., 1998), we found a unique microbiota composition in each rat. The DGGE patterns reflect the predominant bacteria in the caecal samples and we, therefore, expected to detect major changes induced by the different diets. We observed an increase in the number of amplification products in the probiotic and
prebiotic groups and this increment was statistically significant in the case of the probiotic group but not in the prebiotic group (Fig. 3). Some of the bands detected in the probiotic and prebiotic groups were subjected to re-amplification and sequencing (see Fig. 2). Search in the EMBL and GenBank databases allowed us to identify 19 sequences with more than 80% identity (Table 1). Bearing in mind that we have only amplified a 195 base pair fragment, this identification should be considered as only tentative. Most of these 16S rRNA gene
Fig. 3. bBox and whiskersQ representation of the results of the statistical analysis of the number of 16S rRNA gene amplification products obtained from the caecal content of the control group (0), probiotic group (1) and prebiotic group (2). Differences between groups 0 and 1 are statistically significant ( p=0.0012), but not between groups 0 and 2 ( p=0.059) neither between groups 1 and 2 ( p=0.2090), all evaluated for a confidence level of 95%.
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Table 1 Identification by sequencing of the bands amplified with universal primers from caecal samples of the three groups of rats Amplicon IDa
Closest relative, origin and its sequence accession number
% Identityb
B1, C1 B2, B4 C2 B3 C3 B5, C5 C4 C6 C7 B6, C8 B7 C9 B8 C10 C11
Uncultured human fecal bacterium clone 61.25 (AF153867) Uncultured bacterium clone p-1029-a5 from swine intestine (AF371891) Uncultured bacterium clone p-365-a3 from swine intestine (AF371702) Uncultured bacterium clone p-1980-s959-5 from swine intestine (AF371890) Lactobacillus animalis (X61133) Lactobacillus jensenii ATCC 25258 (AF243176) Lactobacillus hamsteri (AJ306298) Uncultured Prevotella sp. isolated from human feces (AB064832) Uncultured bacterium from Gruyere cheese (AJ421821) Uncultured bacterium clone p-2858-6C5 from swine intestine (AF371704) Uncultured bacterium clone p-73-a5 from swine intestine (AF371703) Uncultured bacterium clone p-4205-6Wa5 from swine intestine (AF371520) Uncultured bacterium clone p-2858-6C5 from swine intestine (AF371704) Catenibacterium mitsuokai, isolated from human feces (AB030222) Swine fecal bacterium FPC54 (AF445227)
99, 100 92, 100 99 84 92 88, 99 99 86 100 96, 99 98 100 97 97 100
a b
Refer to Fig. 2; B series correspond to the probiotic group and C series to the prebiotic group. When two figures appear, they refer respectively to the two amplicons listed.
sequences (73%) corresponded to uncultured bacteria. Studies done with human gut samples also revealed a large percentage (76%) of unknown organisms (Suau et al., 1999). The bacteria related to the uncultured human faecal clone 61.25 (bands B1/C1) seem to be predominant in every rat, as deduced from the intensity of the band (Fig. 2). Another two prominent bands present in nearly all the rats (B6/C8 and B7) had sequences related to 16S rRNA gene of bacteria found in the pig gastrointestinal tract. The presence
of these predominant bacteria was not affected by the assayed diets. We found the same closest relative to the sequences of bands B6/C8 and B8, despite of their different migration patterns (Fig. 2). However, their sequences were different, as reflected in their respective % of identity (Table 1), and we checked that the fragments amplified after excision from the gel migrated with the same pattern. We inferred that this coincidence in the BLAST search could reflect the lack in databases of closest sequences to any of them.
Fig. 4. DGGE analysis, in a 45–55% denaturising gradient, of the PCR products of amplification from rat caecal samples using primers specific for Lactobacillus group (Lac1 and Lac2; see Materials and methods). A, Control group. B, Probiotic group. C, Prebiotic group. Numerated arrows point to the bands subjected to re-amplification and sequencing, and correspond to the amplicon IDs of the Table 2. Lanes marked with bLQ show the amplification products obtained with the same primers from pure cultures of L. brevis (L1), L. infantum (L2), L. casei (L3), L. acidophilus (L4) and L. reuteri (L5).
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Among the most prominent bands of the rats fed with prebiotics or probiotics were three fragments corresponding to Lactobacillus spp. Since we did not detect any significant increment in lactobacilli by plate count, we decided to use specific primers to detect the genus Lactobacillus. Results, shown in Fig. 4 and Table 2, indicate that there were no obvious differences in the presence of lactobacilli in the different experimental groups. We did not detect changes in CFU by plate count either (Fig. 1). The amplification products of Bifidobacterium spp. 16S RNA migrate very low in DGGE gels, due to the high C+G content in the DNA of this genus (see the B. lactis control in Fig. 2, lane 7). We observed the presence of bands with this characteristic migration, barely visible in Fig. 1, but the amount of DNA was too small for sequencing purposes. To confirm the presence of Bifidobacterium, we made PCR amplifications from caecal samples with genus-specific primers (see Materials and methods) obtaining positive results from rats belonging to the prebiotic group, but not from any rat of the probiotic group (control rats were not tested). The amplification products corresponding to two randomly selected rats of the prebiotic group
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Fig. 5. DGGE analysis, in a 45–55% denaturising gradient, of the PCR products of amplification with Bifidobacterium genus-specific primers (Bif164F and Bif662; see Materials and methods) from caecal samples of the rats #7 (lane 1) and #8 (lane 2) of the prebiotic group. Sequences of bands A and B corresponded to the 16S RNA of B. animalis (accession number AY151397) with a 99% and a 98% identity, respectively, and band C to the 16S RNA of B. lactis (accession number AB050136) with a 98% identity.
were separated in DGGE and sequenced. We found homology with the 16S rRNA of Bifidobacterium animalis and B. lactis (Fig. 5). B. lactis has been proposed as a subspecies of B. animalis (Ventura and Zink, 2002).
Table 2 Identification of the amplicons generated with Lactobacillus group–specific primers from caecal samples of the three groups of rats Amplicon IDa
Closest relative and its sequence accession number
% Identityb
A1, B1 A2 A3 A4, C2 A5 B2 B3, C3 A6, B6 A7 A8 A9 A10 B4 B5 B7 C1 C4 C5 C6
Lactobacillus intestinalis (AJ306299) Uncultured bacterium clone p-2694-65-a5 (AF371483) Lactobacillus amylolyticus (Y17361) Lactobacillus brevis (AFO90328) Lactobacillus acidophilus johnsonii (M99704) Lactobacillus acidophilus (AJ002515) Lactobacillus crispatus (AJ242969) Lactobacillus murinus (AF157049) Lactobacillus malefermentans (AJ575743) Lactobacillus crispatus (AJ242969) Lactobacillus fermentum (AB125910) Lactobacillus reuteri (AFO90328) Lactobacillus jensenii strain BJ H41-2b (AY339169) Lactobacillus fermentum A12AM (AF329129) Lactobacillus sp. ASF360 (AF157050) Lactobacillus murinus (AF157049) Uncultured swine bacterium clone p-3301-23G (AF371484) Uncultured swine bacterium clone p-165-a2 (AF371482) Lactobacillus gasseri strain BJ H25-8 (AY339178)
99, 95 100 96 96, 92 97 98 100, 96 99, 98 89 98 96 99 94 98 99 98 95 100 85
a b
Refer to Fig. 4; A series corresponds to control group, B series corresponds to the probiotic group and C series to the prebiotic group. See legend to Table 1.
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4. Discussion Both probiotics and prebiotics have been assayed over the last few years with the objective of altering the composition of the intestinal microbiota beneficial to the host. However, the advantages and effects of each strategy are still not clear. In this study we used a rat model, that allowed us to examine the caecal content instead of the feces, because there has been described significant difference in the microbiota composition of these samples (Zoetendal et al., 2002) and the possibility to obtain human colonic samples is more limited (Nielsen et al., 2003). Obviously, there also exist important differences among vertebrates, but the rat model has been already used to evaluate the effects of prebiotics, for example in the prevention of cancer, of ulcerative colitis and in the control of cholesterolemia (Tortuero et al., 1997; Hughes and Rowland, 2001; Fukuda et al., 2002). The diets used in this experiment, supplemented either with prebiotics (fructo-oligosaccharides) or probiotics (B. lactis and S. thermophilus), are designed for use as infant continuation formula. Their effects have been evaluated by two different methods with the aim of getting complementary data. We found both diets to be capable of modifying the bacterial composition of the caecal content in rats and to induce a reduction in clostridia and Bacteroides spp., bacterial groups that include species considered as non-desirable members of the intestinal microbiota (McBain and Macfarlane, 1998). The probiotic formula induced a statistically significant increase in the microbiota diversity as deduced from the DGGE analysis. In other aspects, the prebiotic formula was more effective, namely in the reduction of coliforms and the increasing of Bifidobacterium, which is considered, together with Lactobacillus, to be one of the most beneficial members by their anti-carcinogenic and immunomodulating activities (reviewed by Kaur et al., 2002). PCR amplification with genusspecific primers was unable to detect bifidobacteria in the probiotic group, and since the conditions were the same as those used with a positive result in the prebiotic group, we suspect that the number of bifidobacteria in the faecal samples of these rats was below the sensitivity of the PCR. This result was surprising, bearing in mind that the probiotic formula contained viable B. lactis. This
could be explained by an antagonistic effect exerted by the resident microbiota against the exogenous bifidobacteria, while the growth-promoting fructooligosaccharides of the prebiotic formula can stimulate indigenous strains as well as discourage the growth of non-desirable species. Other authors have reported a significant, but transient, increase of bifidobacteria during treatment with Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium breve in a human trial. However, only 20% of the subjects were colonized by both exogenous B. infantis and B. breve strains (Brigidi et al., 2001). Therefore, these results are not very different from those obtained in this work in the rat model and they emphasize the difficulties that can arise from deliberate colonisation of the intestinal track.
Acknowledgements This work has been supported by a grant from DANONE S.A. and the Universidad Complutense de Madrid (Project 248/01/10148).
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Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics Alejandra Montesia, Raimundo Garcı´a-Albiacha, Marı´a Jose´ Pozueloa, Concepcio´n Pintadob, Isabel Gon˜ic, Rafael Rotgerb,* a
Departamento de Biologı´a Celular, Bioquı´mica y Biologı´a Molecular. Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo-CEU. Urbanizacio´n Monte Prı´ncipe, Boadilla del Monte, 28668 Madrid, Spain b Departamento de Microbiologı´a II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain c Departamento de Nutricio´n y Bromatologı´a I, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain Received 3 March 2004; received in revised form 9 June 2004; accepted 9 June 2004
Abstract The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results. D 2004 Elsevier B.V. All rights reserved. Keywords: Probiotics; Prebiotics; DGGE; Bifidobacterium; Lactobacillus; Rats
1. Introduction
* Corresponding author. Tel.: +34 913 941 755; fax: +34 913 941 745. E-mail address: [email protected] (R. Rotger). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.06.005
The intestinal tract of mammals harbours a complex bacterial ecosystem, which has not yet been fully characterized. Most of its members are obligate anaerobes and many of them (60–80%) have not been cultivated (Suau et al., 1999; Hayashi et al.,
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2003). However, it is now generally accepted that the composition of the human intestinal microbiota has an important role in health and disease (Gionchetti et al., 2000). Constipation, colonic cancer and inflammatory bowel diseases, for example, can be influenced by the microbiota composition and its metabolic activities, but a clear picture of the contribution of the different species in the maintenance of bowel health is not currently available. Over the last few years, the presence of a large number of bifidobacteria has been considered essential to promote intestinal health and to strengthen the local immune response. Lactic acid bacteria, like Lactobacillus, and some Streptococcus species, as Streptococcus thermophilus, are also regarded as beneficial members, whereas some members of other groups, namely Bacteroides, Clostridium and Enterobacteriaceae, would be harmful as a consequence of their metabolic activities or even the pathogenic potentiality of a few species. For example, high risk of colon cancer has been associated with presence of Bacteroides vulgatus and Bacteroides stercoris, and species of Bacteroides and Clostridium could trigger the inflammatory process in human inflammatory bowel diseases (see Guarner and Malagelada, 2003 for a review). As a consequence of these concepts, modification of the human intestinal microbiota has currently become an important objective of dietetics. This goal can be attempted in two ways: either to include in the diet a significant proportion of beneficial bacteria, mainly Bifidobacterium and Lactobacillus species, with the expectation that they will be able to colonize the intestinal tract (probiotics); or to give non-digestible carbohydrates, like fructo-oligosaccharides, which have shown an ability to promote the growth of desirable bacteria (prebiotics) (Kaur et al., 2002; Saarela et al., 2002; Sullivan and Nord, 2002). The complex and time-consuming methods required to isolate and identify the intestinal microbiota members and especially the impossibility of culturing many of them have hindered the study of these populations. However, the development of molecular techniques based on the characterization of 16S rRNA genes has greatly facilitated the work for microbiologists in this respect. Separation of the amplification products generated by PCR by denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE) provides characteristic band profiles
corresponding to the predominant bacterial species (Muyzer, 1999). Sequencing of PCR products reamplified directly from the excised bands permits the identity to be determined (genus or species). Molecular methods have been applied to study the gastrointestinal microbiota of humans (Satokari et al., 2001; Donskey et al., 2003; Nielsen et al., 2003), poultry (Netherwood et al., 1999; Gong et al., 2002) and pigs (Simpson et al., 2000; Leser et al., 2002). It is clear that there is no one single appropriate technique for these studies. For example, quantification of species requires hybridisation techniques (Mangin et al., 2002) or real-time PCR (Matsuki et al., 2004). Obviously, differences can be expected among the results obtained with different techniques and between classical and molecular methods (Wilson and Blitchington, 1996). In this work, we used DGGE to differentiate 16S RNA species amplified by PCR from caecal samples of rats, together with the classical plate-count enumeration of the main bacterial groups. We applied these methods to evaluate the effects on rats of two diet supplements, containing either prebiotics (fructo-oligosaccharides) or probiotics (Bifidobacterium lactis and S. thermophilus).
2. Material and methods 2.1. Animals and diets Thirty male Wistar rats of the same age with a body weight of 195F5 g were supplied by the breeding centre at the Faculty of Pharmacy (Universidad Complutense, Madrid, Spain). After weaning, all animals were fed with a standard maintenance diet (A04, Panlab, Barcelona, Spain). Animals were housed in individual metabolic cages in a room maintained at 22F1 8C, with 12-h light/dark cycles throughout the 28-day experimental period. Rats were divided into three groups and fed with diets containing 70% of a diet adjusted to the animals’ requirements (AIN-93 M purified rodent diet, DYETS, Bethlehem, PA, USA) (Reeves et al., 1993) and 30% of each of the following infant formulas depending on the experimental group: (a)
Control group: Conventional continuation formula (Modar 2, Sandoz, Vienna, Austria).
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(b) (c)
Prebiotic group: Prebiotic formula with 5.7% (w/ w) of fructo-oligosaccharides (Natur 2, Sandoz). Probiotic group: Probiotic formula with B. lactis BL and S. thermophilus (Nativa 2, Nestle´, Zurich, Switzerland).
The nutritional composition of the three infant formulas was similar and the only difference among them was the addition of prebiotics or probiotics. This study was approved by the Department of Nutrition of the Universidad Complutense. Food intake and body weight was controlled every week. After the experimental period, the animals were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight) and their caecum was removed.
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lab) for lactobacilli (Tortuero et al., 1997), supplemented Schaedler (bioMerieux, Marcy-l’Etoile, France) for Bacteroides, and MacConkey (Oxoid) for coliforms (Brigidi et al., 2001). All these plates were incubated anaerobically at 37 8C for 4–5 days, except in the case of the MacConkey plates which were incubated aerobically at 37 8C for 48–72 h. Representative colonies of each selective medium were identified to genus level by standard bacteriological methods such as Gram stain, cellular and colonial morphology and biochemical reactions. Differences in the number of colony forming units (CFU) of the samples were statistically analyzed with Statgraphics plus 5.1 (Statistical Graphics, Rockville, MD, USA) by the Fisher’s Least Significant Differences (LSD) for a confidence level of 90% (Walpole et al., 1992).
2.2. Caecal sample preparation 2.4. Amplification of 16S rDNA The rats were killed under CO2 after the experimental period and caeca were then removed and weighed. For cultivation of bacteria, approximately 1 g of each caecal sample was homogenised in prereduced Wilkins–Chalgren broth (Oxoid, Basingstoke, UK), to a final ratio of 1:10 (w/v); 10% glycerol was added and the samples were frozen at 80 8C. All these procedures were performed in oxygen-free, CO2-saturated atmosphere. For molecular methods, 0.5 g of each caecal specimen was added to 4.5 ml of sterile phosphate-buffered saline– EDTA (PBS–EDTA) and mixed by vortexing the tube for 6 min. One milliliter of each sample was then centrifuged at 13,400 rpm, 4 8C, for 5 min. The pellet was washed twice with PBS–EDTA and finally resuspended in 300 Al of PBS–EDTA, heated at 100 8C for 15 min and immediately frozen at 80 8C. 2.3. Enumeration of cultivable bacteria using plate culture The caecal samples were thawed at room temperature and serial dilutions (from 10 3 to 10 9) were made in pre-reduced Wilkins–Chalgren broth; 0.1 ml of each dilution was spread in duplicate onto the surface of plates which contained the following agar media: Wilkins–Chalgren agar (Oxoid) for enumeration of total anaerobes, supplemented MRS (Hispanlab, Madrid, Spain) for bifidobacteria (Harmsen et al., 1999), SPS (Hispanlab) for clostridia, MRS (Hispan-
The caecal samples were thawed at room temperature and DNA was extracted using a FastDNA spin kit for soil (BIO 101, BuenaVista, CA, USA), in which the cell lysis solution was replaced by CLS-TC (same manufacturer), which is more suitable for animal tissues. The V3 region of the 16S rRNA gene (positions 341–534 in the E. coli gene) was amplified by using primers 518R (5VATTACCGCGGCTGCTGG-3V) and 341F (5VCCTACGGGAGGCAGCAG-3V) with a 5V GC clamp (5V-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGGCACGGGGGG-3V) (Muyzer et al., 1993). PCR was performed in 0.2 ml tubes by using a Gene Amp PCR System 2400 (Perkin Elmer, Boston, MA, USA). Each reaction mixture contained reaction buffer, 1.5 mM MgCl2, deoxynucleosides triphosphate at a concentration of 200 AM, 15 pmol of each primer, 100 ng of caecal DNA and 2 U of Expand High Fidelity (Roche, Basel, Switzerland). The following amplification program was used: 97 8C for 5 min, 30 cycles consisting of 94 8C 1 min, 62 8C 40 s, 72 8C 30 s, and then 72 8C 7 min. Lactobacillus group-specific PCR was performed using the 16S rRNA gene-targeted primers Lac1 (5VAGCAGTAGGGAATCTTCA-3V) and Lac2 (5VATTYCACCGCTACACATG-3V) with a 5V GC clamp (5V-CGCCCGGGGCGCGCCCCGGGCGGCCCGGGGGCACCGGGG-3V) (Walter et al., 2001) that produces a 327 bp amplicon (positions 352–679), using the same amplification program as indicated above.
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Bifidobacterium genus-specific PCR was performed using 16S rRNA gene-targeted primers Bif164F (5VGGGTGGTAATGCCGGATG-3V) and Bif662 (5VCCACCGTTACACCGGGAA-3V) with a 5V GC clamp (5V-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3V) (Satokari et al., 2001), that produce a 523 bp amplicon (positions 164–679), and the following amplification program was used: 95 8C for 5 min, 40 cycles consisting of 94 8C 45 s, 52 8C 50 s, 72 8C 50 s, and then 72 8C for 7 min. 2.5. Denaturing gradient gel electrophoresis (DGGE) DGGE was performed by using a DCodek Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA) with gels of 16100.01 cm. A denaturing gradient of 37–55% was prepared as described elsewhere (Donskey et al., 2003). Electrophoresis was performed at 130 V and 60 8C for about 4:40 h. In the case of the Bifidobacterium-specific amplicons, the denaturing gradient used was of 45– 55% and the electrophoresis was done at 85 V for about 16 h. Gels were stained with a 5 Ag/ml ethidium bromide solution for 20 min, washed with deionised water and viewed by UV trans-illumination. Differences in the number of bands were statistically analyzed with Statgraphics plus 5.1 and the p value was determined for a confidence level of 95%.
2.7. Nucleotide sequence accession numbers The sequences determined in this study have been assigned GenBank accession numbers AY642396– AY642426, AY643067 AY643068, AY649331 and AY649332. 3. Results One rat belonging to the prebiotic group died before the end of the experiment. Examination of pooled caecal content was ruled out since an inter-individual variation could be expected, as it has been reported in humans (Zoetendal et al., 1998). Classical microbiological methods were only applied to three randomly selected rats from each group, due to the complexity of the analysis. Results of the enumeration of cultivable bacteria are presented in Fig. 1, and the inter-individual variation is reflected by the standard deviation bars. Due to the high inter-individual variability, statistically significant differences were evaluated for a confidence level of 90%.
2.6. Sequencing of PCR-amplified products Representative bands were excised from DGGE gels with a sterile scalpel and eluted overnight as described by Ausubel et al. (1993). A 5-Al aliquot was removed and re-amplified by PCR using the original primers without the GC clamp. Resulting PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced with the ABI PRISM bigDye Terminator Cycle sequencing Ready reaction Kit in an automated ABIPRISM 377DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The amplicons were sequenced from the 5V end using the aforementioned 518R and Lac1 primers. Sequences were analyzed with the software Chromas v. 2.23 (Technelysium, Tewantin, Australia), and their similarity with 16S RNA sequences stored in GenBank and EMBL databases was searched with the BLAST software (www.ncbi.nlm.nih.gov/BLAST).
Fig. 1. Enumeration (CFU/g of caecal content) of bacterial groups by plate count from samples of three rats of each experimental group: control (open bars), probiotic (black bars) and prebiotic (grey bars). Logarithmic scale is not the same for each bacterial group, as indicated in the ordinates legend. Lines over each bar indicate standard deviation among individuals and an asterisk indicates a statistically significant difference (90% confidence level).
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Fig. 2. DGGE analysis, in a 37–55% denaturising gradient, of the PCR products of amplification from rat caecal samples using universal primers for eubacterial 16S rRNA gene (518R and 341F; see Materials and methods). A, Control group, except lane bLQ that corresponds to a pure culture of B. lactis isolated from a probiotic. B, Probiotic group. C, Prebiotic group. Numerated arrows in gels B and C point to the bands subjected to re-amplification and sequencing, and correspond to the amplicon IDs of the Table 1. White arrow in gel A indicates the amplification product obtained with the same primers from the sample of B. lactis.
Both diet supplements, probiotic and prebiotic, were similarly effective at reducing bnon-desirableQ bacterial groups, namely Bacteroides and clostridia, and they also provoked a significant reduction in the number of total anaerobes. However, they had opposite effects on the recovery of coliforms: the probiotics induced a significant increase and the prebiotics a larger reduction. None of the diets was able to increase the number of lactobacilli. A higher increment of the bifidobacteria content was detected in the rats fed with prebiotics, whereas the Bifidobacterium-containing probiotics produced a non-significant effect. We enumerated the viable bifidobacteria present in the probiotic formula using the same selective media as for the caecal content, and we found 107 CFU/g. The presence of Streptococcus was not evaluated, since they were not looked for in the caecal content. Molecular analysis of the caecal content by amplification of 16S rRNA genes and DGGE was performed in samples from every individual of each group (Fig. 2). As previously reported in humans (Zoetendal et al., 1998), we found a unique microbiota composition in each rat. The DGGE patterns reflect the predominant bacteria in the caecal samples and we, therefore, expected to detect major changes induced by the different diets. We observed an increase in the number of amplification products in the probiotic and
prebiotic groups and this increment was statistically significant in the case of the probiotic group but not in the prebiotic group (Fig. 3). Some of the bands detected in the probiotic and prebiotic groups were subjected to re-amplification and sequencing (see Fig. 2). Search in the EMBL and GenBank databases allowed us to identify 19 sequences with more than 80% identity (Table 1). Bearing in mind that we have only amplified a 195 base pair fragment, this identification should be considered as only tentative. Most of these 16S rRNA gene
Fig. 3. bBox and whiskersQ representation of the results of the statistical analysis of the number of 16S rRNA gene amplification products obtained from the caecal content of the control group (0), probiotic group (1) and prebiotic group (2). Differences between groups 0 and 1 are statistically significant ( p=0.0012), but not between groups 0 and 2 ( p=0.059) neither between groups 1 and 2 ( p=0.2090), all evaluated for a confidence level of 95%.
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Table 1 Identification by sequencing of the bands amplified with universal primers from caecal samples of the three groups of rats Amplicon IDa
Closest relative, origin and its sequence accession number
% Identityb
B1, C1 B2, B4 C2 B3 C3 B5, C5 C4 C6 C7 B6, C8 B7 C9 B8 C10 C11
Uncultured human fecal bacterium clone 61.25 (AF153867) Uncultured bacterium clone p-1029-a5 from swine intestine (AF371891) Uncultured bacterium clone p-365-a3 from swine intestine (AF371702) Uncultured bacterium clone p-1980-s959-5 from swine intestine (AF371890) Lactobacillus animalis (X61133) Lactobacillus jensenii ATCC 25258 (AF243176) Lactobacillus hamsteri (AJ306298) Uncultured Prevotella sp. isolated from human feces (AB064832) Uncultured bacterium from Gruyere cheese (AJ421821) Uncultured bacterium clone p-2858-6C5 from swine intestine (AF371704) Uncultured bacterium clone p-73-a5 from swine intestine (AF371703) Uncultured bacterium clone p-4205-6Wa5 from swine intestine (AF371520) Uncultured bacterium clone p-2858-6C5 from swine intestine (AF371704) Catenibacterium mitsuokai, isolated from human feces (AB030222) Swine fecal bacterium FPC54 (AF445227)
99, 100 92, 100 99 84 92 88, 99 99 86 100 96, 99 98 100 97 97 100
a b
Refer to Fig. 2; B series correspond to the probiotic group and C series to the prebiotic group. When two figures appear, they refer respectively to the two amplicons listed.
sequences (73%) corresponded to uncultured bacteria. Studies done with human gut samples also revealed a large percentage (76%) of unknown organisms (Suau et al., 1999). The bacteria related to the uncultured human faecal clone 61.25 (bands B1/C1) seem to be predominant in every rat, as deduced from the intensity of the band (Fig. 2). Another two prominent bands present in nearly all the rats (B6/C8 and B7) had sequences related to 16S rRNA gene of bacteria found in the pig gastrointestinal tract. The presence
of these predominant bacteria was not affected by the assayed diets. We found the same closest relative to the sequences of bands B6/C8 and B8, despite of their different migration patterns (Fig. 2). However, their sequences were different, as reflected in their respective % of identity (Table 1), and we checked that the fragments amplified after excision from the gel migrated with the same pattern. We inferred that this coincidence in the BLAST search could reflect the lack in databases of closest sequences to any of them.
Fig. 4. DGGE analysis, in a 45–55% denaturising gradient, of the PCR products of amplification from rat caecal samples using primers specific for Lactobacillus group (Lac1 and Lac2; see Materials and methods). A, Control group. B, Probiotic group. C, Prebiotic group. Numerated arrows point to the bands subjected to re-amplification and sequencing, and correspond to the amplicon IDs of the Table 2. Lanes marked with bLQ show the amplification products obtained with the same primers from pure cultures of L. brevis (L1), L. infantum (L2), L. casei (L3), L. acidophilus (L4) and L. reuteri (L5).
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Among the most prominent bands of the rats fed with prebiotics or probiotics were three fragments corresponding to Lactobacillus spp. Since we did not detect any significant increment in lactobacilli by plate count, we decided to use specific primers to detect the genus Lactobacillus. Results, shown in Fig. 4 and Table 2, indicate that there were no obvious differences in the presence of lactobacilli in the different experimental groups. We did not detect changes in CFU by plate count either (Fig. 1). The amplification products of Bifidobacterium spp. 16S RNA migrate very low in DGGE gels, due to the high C+G content in the DNA of this genus (see the B. lactis control in Fig. 2, lane 7). We observed the presence of bands with this characteristic migration, barely visible in Fig. 1, but the amount of DNA was too small for sequencing purposes. To confirm the presence of Bifidobacterium, we made PCR amplifications from caecal samples with genus-specific primers (see Materials and methods) obtaining positive results from rats belonging to the prebiotic group, but not from any rat of the probiotic group (control rats were not tested). The amplification products corresponding to two randomly selected rats of the prebiotic group
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Fig. 5. DGGE analysis, in a 45–55% denaturising gradient, of the PCR products of amplification with Bifidobacterium genus-specific primers (Bif164F and Bif662; see Materials and methods) from caecal samples of the rats #7 (lane 1) and #8 (lane 2) of the prebiotic group. Sequences of bands A and B corresponded to the 16S RNA of B. animalis (accession number AY151397) with a 99% and a 98% identity, respectively, and band C to the 16S RNA of B. lactis (accession number AB050136) with a 98% identity.
were separated in DGGE and sequenced. We found homology with the 16S rRNA of Bifidobacterium animalis and B. lactis (Fig. 5). B. lactis has been proposed as a subspecies of B. animalis (Ventura and Zink, 2002).
Table 2 Identification of the amplicons generated with Lactobacillus group–specific primers from caecal samples of the three groups of rats Amplicon IDa
Closest relative and its sequence accession number
% Identityb
A1, B1 A2 A3 A4, C2 A5 B2 B3, C3 A6, B6 A7 A8 A9 A10 B4 B5 B7 C1 C4 C5 C6
Lactobacillus intestinalis (AJ306299) Uncultured bacterium clone p-2694-65-a5 (AF371483) Lactobacillus amylolyticus (Y17361) Lactobacillus brevis (AFO90328) Lactobacillus acidophilus johnsonii (M99704) Lactobacillus acidophilus (AJ002515) Lactobacillus crispatus (AJ242969) Lactobacillus murinus (AF157049) Lactobacillus malefermentans (AJ575743) Lactobacillus crispatus (AJ242969) Lactobacillus fermentum (AB125910) Lactobacillus reuteri (AFO90328) Lactobacillus jensenii strain BJ H41-2b (AY339169) Lactobacillus fermentum A12AM (AF329129) Lactobacillus sp. ASF360 (AF157050) Lactobacillus murinus (AF157049) Uncultured swine bacterium clone p-3301-23G (AF371484) Uncultured swine bacterium clone p-165-a2 (AF371482) Lactobacillus gasseri strain BJ H25-8 (AY339178)
99, 95 100 96 96, 92 97 98 100, 96 99, 98 89 98 96 99 94 98 99 98 95 100 85
a b
Refer to Fig. 4; A series corresponds to control group, B series corresponds to the probiotic group and C series to the prebiotic group. See legend to Table 1.
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4. Discussion Both probiotics and prebiotics have been assayed over the last few years with the objective of altering the composition of the intestinal microbiota beneficial to the host. However, the advantages and effects of each strategy are still not clear. In this study we used a rat model, that allowed us to examine the caecal content instead of the feces, because there has been described significant difference in the microbiota composition of these samples (Zoetendal et al., 2002) and the possibility to obtain human colonic samples is more limited (Nielsen et al., 2003). Obviously, there also exist important differences among vertebrates, but the rat model has been already used to evaluate the effects of prebiotics, for example in the prevention of cancer, of ulcerative colitis and in the control of cholesterolemia (Tortuero et al., 1997; Hughes and Rowland, 2001; Fukuda et al., 2002). The diets used in this experiment, supplemented either with prebiotics (fructo-oligosaccharides) or probiotics (B. lactis and S. thermophilus), are designed for use as infant continuation formula. Their effects have been evaluated by two different methods with the aim of getting complementary data. We found both diets to be capable of modifying the bacterial composition of the caecal content in rats and to induce a reduction in clostridia and Bacteroides spp., bacterial groups that include species considered as non-desirable members of the intestinal microbiota (McBain and Macfarlane, 1998). The probiotic formula induced a statistically significant increase in the microbiota diversity as deduced from the DGGE analysis. In other aspects, the prebiotic formula was more effective, namely in the reduction of coliforms and the increasing of Bifidobacterium, which is considered, together with Lactobacillus, to be one of the most beneficial members by their anti-carcinogenic and immunomodulating activities (reviewed by Kaur et al., 2002). PCR amplification with genusspecific primers was unable to detect bifidobacteria in the probiotic group, and since the conditions were the same as those used with a positive result in the prebiotic group, we suspect that the number of bifidobacteria in the faecal samples of these rats was below the sensitivity of the PCR. This result was surprising, bearing in mind that the probiotic formula contained viable B. lactis. This
could be explained by an antagonistic effect exerted by the resident microbiota against the exogenous bifidobacteria, while the growth-promoting fructooligosaccharides of the prebiotic formula can stimulate indigenous strains as well as discourage the growth of non-desirable species. Other authors have reported a significant, but transient, increase of bifidobacteria during treatment with Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium breve in a human trial. However, only 20% of the subjects were colonized by both exogenous B. infantis and B. breve strains (Brigidi et al., 2001). Therefore, these results are not very different from those obtained in this work in the rat model and they emphasize the difficulties that can arise from deliberate colonisation of the intestinal track.
Acknowledgements This work has been supported by a grant from DANONE S.A. and the Universidad Complutense de Madrid (Project 248/01/10148).
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