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Applied and Environmental Microbiology, September 2006, p. 6271-6276, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00477-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Increase in Terminal Restriction Fragments of Bacteroidetes-Derived 16S rRNA Genes after Administration of Short-Chain Fructooligosaccharides

Yusuke Nakanishi,^1, Koichiro Murashima,^2*, Hiroki Ohara,² Takahisa Suzuki,³ Hidenori Hayashi,⁴ Mitsuo Sakamoto,⁴ Tomoyuki Fukasawa,² Hidetoshi Kubota,² Akira Hosono,¹ Toshiaki Kono,² Shuichi Kaminogawa,¹ and Yoshimi Benno⁴

Department of Food Science and Technology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510,¹ Food and Health R&D Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1 Chiyoda, Sakado, Saimata 350-0289,² Pharmaceutical Research Department, Meiji Seika Kaisha, Ltd., 760 Morooka, Kohoku-Ku, Yokohama, Kanagawa 222-8567 and,³ Microbe Division, Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan⁴

Received 27 February 2006/ Accepted 22 June 2006

Top Abstract Introduction Materials and Methods Results and Discussion References

 
It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Short-chain fructooligosaccharide (scFOS) is one of the popular prebiotics (4). The scFOS is a mixture of oligosaccharides consisting of a glucose (G) unit linked to fructose (F) units (n = 2 to 4) (6). The scFOS arrives at the gastrointestinal tract without digestion and is used as a carbon source by intestinal bacteria (14). Consequently, the composition of intestinal microbiota is changed by scFOS administration (5).

It is known that scFOS has several physiological effects on humans and animals. For example, in murine models, scFOS shows several immunomodulating functions (13). Recently, we showed that scFOS administration enhanced the secretion of murine intestinal immunoglobulin A (8), which is expected to inhibit infection by pathogens. The change in murine intestinal microbiota due to scFOS administration is suspected to be a trigger of immunomodulating functions (8), although the mechanisms are still unclear. Comparison of the timing and dose response between changes in murine intestinal microbiota and immunomodulating functions should give us important information on the mechanisms of scFOS immunomodulating functions. For such purposes, it is necessary to determine the markers of change in murine intestinal microbiota after scFOS administration.

Effects of scFOS administration on murine intestinal microbiota have not been well characterized, although the effects on human intestinal microbiota have been studied intensively (17, 20). In the case of humans, fecal bacteria are cultured on selective media to determine the effects of scFOS administration on intestinal microbiota. This technique showed that an increase in the number of bifidobacteria had a major effect on human intestinal microbiota after scFOS administration. Based on these results, the CFU of bifidobacteria are utilized as a marker of the positive change in human intestinal microbiota after scFOS administration. In contrast, no consistent effects of scFOS administration on intestinal microbiota have been detected by the culturing method in the murine model (8). Therefore, no markers of change in murine intestinal microbiota after scFOS administration have been identified so far. Salzman et al. estimated that at least 60% of murine intestinal bacteria are still uncultured (18). In this context, the culturing method might not be suitable to determine changes in murine intestinal microbiota.

Molecular techniques are becoming popular to comprehensively determine intestinal microbiota (21). Most of the molecular techniques used to identify bacteria are based on the determination of 16S rRNA gene amplicons by PCR with universal primers. After PCR, the composition of the amplicons is determined by fingerprint analysis, such as terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis, and temperature gradient gel electrophoresis. These molecular techniques allow access to uncultured bacteria, since the DNA template for PCR is extracted directly from samples without bacterial cultivation. Therefore, these molecular techniques are powerful methods to detect the effects of scFOS administration on murine intestinal microbiota in which most bacteria are still uncultured.

In this study, we determined the effects of scFOS administration on the T-RFLP profiles of murine intestinal microbiota and sought markers for the positive change in murine intestinal microbiota as a result of scFOS administration.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Animals.
Female 6-week-old BALB/c mice (n = 7 for each diet group; Clea Japan, Tokyo, Japan) were housed at 23 to 25°C and 50 to 60% relative humidity with a 12-h light-dark cycle. The mice were fed a pelleted mouse flat diet (Oriental Yeast, Tokyo, Japan) for 1 week before commencement of the experimental diet. All experiments were performed in accordance with the Nihon University guidelines for laboratory animal care.

Diets.
Mice were fed one of four experimental diets (scFOS diet, kestose [GF2] diet, nystose [GF3] diet, or control diet) for 5 weeks. The control diet consisted of 200 g of casein, 532 g of cornstarch, 70 g of corn oil, 10 g of vitamin mixture, 35 g of mineral mixture, 50 g of cellulose, and 100 g of sucrose. For the scFOS diet, GF2 diet, or GF3 diet, 75 g of the sucrose in the control diet was substituted with 75 g of scFOS, GF2, or GF3. All components, with the exception of scFOS, GF2, and GF3, were obtained from Oriental Yeast (Tokyo, Japan). Vitamin and mineral mixtures were prepared according to the AIN-93 formulation (16).The scFOS consisted of 34% 1-kestose (GF2), 53% nystose (GF3), and 10% fructofuranosyl nystose (GF4) (14) that was supplied by Meiji Seika Kaisha, Ltd. (Tokyo, Japan). The GF2 and GF3 were also supplied by Meiji Seika Kaisha, Ltd.

Sampling of fecal and cecal samples.
Fecal samples were collected before commencement of the experimental diet (0 weeks) and at 5 weeks after changing the mouse flat diet to the experimental diet (5 weeks). Four to five fecal pellets were collected from each mouse. Also, cecal contents were collected from mice in the GF2-fed group, the GF3-fed group, and the control group at 5 weeks. Each collected fecal or cecal sample was stored at –80°C until DNA was extracted.

Extraction of DNA from fecal and cecal samples.
DNA was extracted from 50 mg of each collected fecal or cecal sample with the UltraClean Soil DNA isolation kit (Mo Bio Labs, Solana Beach, CA) according to the method described by Clement and Kitts (2), with some modifications. To efficiently degrade bacterial cell walls, 50 µl of cell lysing solution (60 mg/ml of lysozyme, 1 mg/ml of N-acetylmuramidase SG [Seikagaku Kogyo Ltd., Tokyo, Japan] in TE buffer) was added to the bead solution from the kit. Subsequently, 50 mg of each fecal or cecal sample was added to the bead solution and incubated at 37°C for 30 min. After incubation, DNA was extracted according to the manufacturer's instructions. The DNA yields from fecal and cecal samples were 228 ± 65 ng and 222 ± 38 ng, respectively.

T-RFLP analysis.
To amplify partial 16S rRNA gene fragments, PCR was done utilizing DNA from the fecal sample and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (11). The 27F primer was labeled with 6'-carboxyfluorescein at the 5' end. Amplification reactions were performed in a total volume of 50 µl containing 5 µl of DNA, 1.25 U of Takara Ex Taq (Takara shuzo, Tokyo, Japan), 5 µl of Ex Taq buffer, 8 µl of deoxynucleoside triphosphate mixture (2.5 mM each), and 25 pmol of each primer. The PCR program was 95°C for 3 min, then 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 90 s, followed by 72°C for 10 min. PCR products were purified with the MinElute PCR purification kit (QIAGEN, Valencia, CA) and then digested with MspI. Lengths of terminal restriction fragments (T-RFs) in digested PCR products were analyzed by the ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA) in Genescan mode, with the GS-500 ROX and GS-1000 ROX internal standards as described previously (10). The obtained T-RFLP profiles were analyzed by BioNumerics software (Applied Maths, Ver 3.0, Austin, TX) as described previously (10). To remove background and small peaks, T-RFs whose relative areas were less than 3.0% of total area were deleted by setting the "Min. profiling," which is a criteria to remove background, at 3.0% on the "auto search band" command of the software. Bands ranging from 50 to 600 bp were used to construct average T-RFLP profiles.

Construction of average T-RFLP profiles.
To standardize all of the obtained T-RFLP profiles, we calculated the relative area of each T-RF against the summation of total area of all obtained T-RFs in each profile. The total area of each T-RFLP profile was set at 100%; therefore, the summation of all relative areas in each profile was 100%. The average relative area of each T-RF in the scFOS-fed group or the control group was then calculated, and the averaged T-RFLP profiles of both feeding groups were constructed. The statistical differences of the relative T-RF areas were determined by paired two-sided Student's t test. A P value of <0.05 was considered significant.

Sequence and phylogenetic analysis of the 16S rRNA gene.
To amplify partial 16S rRNA gene fragments, PCR was performed utilizing one of the fecal DNAs of the scFOS-fed group or the control group as a template and the universal primers 27F and 1492R as described above. The amplified PCR fragments were directly ligated into pCR2.1 (Invitrogen, San Diego, CA) by TA cloning techniques (7) and then transformed into One Shot INVaF' competent cells (Invitrogen) to construct 16S rRNA gene libraries. Clones isolated from the libraries were used as templates for sequence analysis. In the scFOS-fed and control groups, 77 and 67 clones were isolated, respectively. The partial sequences of the 16S rRNA gene (Escherichia coli positions 27 to 519) in all of the isolated clones were determined by the dideoxy chain termination reaction with the universal primer 27F or 519R (5'-ACCGCGGCYGCTGGC-3') (11). The 16S rRNA gene sequences were submitted to the BLAST search program of the National Center for Biotechnology Information (1) to find similar sequences. Also, the 16S rRNA gene sequences were aligned with the CLUSTALW (19) with reference sequences obtained from the RDP Select Sequence function (12) and GenBank. To check secondary structures of the 16S rRNA gene sequences and correct the alignment, Se-Al (15) was used. A phylogenetic tree was constructed by the neighbor-joining method (3).

Real-time PCR with primers specific for Bacteroidetes.
The 16S rRNA partial gene sequences determined as described above were aligned with CLUSTAL-W (19). Two regions where the sequences of the class Bacteroidetes are well-conserved but different from other sequences were found. Based on these two sequence regions, we designed two primers specific for the class Bacteroidetes: MIBF, 5'-GGCGACCGGCGCACGGG-3' (forward primer); MIBR, 5'-GRCCTTCCTCTCAGAACCC-3' (reverse primer). The amplification reactions were carried out in a total volume of 50 µl, which consisted of 1 µl of fecal DNA, 25 µl of SYBR green PCR master mix (Applied Biosystems), and 24 µl of deionized water. The fecal DNA was diluted to 1/10 concentration with deionized water. The PCR program was 95°C for 10 min, then 30 cycles of 95°C for 20 s, and 65°C for 60 s with the real-time ABI PRISM 7000 PCR machine (Applied Biosystems). To determine the specificity of the primer set MIBF and MIBR, the amplification rate of 2 pg of the 16S rRNA gene plasmids described above were determined. The amounts of 16S rRNA genes of the class Bacteroidetes were shown as a relative amount against the standard fecal DNA. The standard fecal DNA was extracted from a fecal sample from a mouse of the scFOS-fed group at 5 weeks. The standard curve was calculated with the data from standard fecal DNA, which was diluted to 1/1, 1/10, 1/100, or 1/1,000 concentration with deionized water. The statistical differences between amounts of 16S rRNA genes were determined by the two-sided Student t test. A P value of <0.05 was considered significant.

Nucleotide sequence accession numbers.
The partial 16S rRNA gene sequences from the unique clones were deposited in the DNA Data Bank of Japan (DDBJ). The accession numbers are shown in Fig. 1.

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FIG. 1. Phylogenetic tree showing the relationships of obtained 16S rRNA partial genes in murine fecal microbiota with published sequences. The obtained 16S rRNA partial genes are displayed as accession numbers. The theoretical lengths of the T-RFs are also shown. The 16S rRNA partial genes with red underlining indicate candidate genes assigned to marker T-RFs at 83, 88, 93, and 95 bp. The genes with blue underlining and the gene with green underlining indicate candidate genes for marker T-RF at 215 bp and 475 bp, respectively.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Search for increasing T-RFs after scFOS administration as markers of positive change in intestinal microbiota.
To find increasing T-RFs after scFOS administration, we constructed the average T-RFLP profiles of 16S rRNA genes from murine intestinal microbiota in the scFOS-fed groups and compared this profile with that of the control group. The average T-RFLP profiles of both groups at 5 weeks are shown in Fig. 2. Six T-RFs (83, 88, 93, 95, 215, and 475 bp) in the T-RFLP profiles were significantly larger in the scFOS-fed group than in the control group. Therefore, these T-RFs were selected as marker T-RFs for positive changes in intestinal microbiota after scFOS administration.

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FIG. 2. Average T-RFLP profiles of the scFOS-fed group and control group at 5 weeks. Error bars indicate standard deviations of the mean relative areas. In the x axis of the figure, T-RF lengths are described. Asterisks next to the T-RF lengths indicate significance of difference of relative T-RF areas. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

We attempted to assign these marker T-RFs to phylogenetic groups with information on the theoretical lengths of T-RFs derived from 16S rRNA of murine fecal microbiota and the phylogenetic positions of these 16S rRNA genes. The accession numbers and theoretical T-RF lengths of the obtained 16S rRNA partial genes are shown in Fig. 1. Also shown in Fig. 1 are the phylogenetic positions of these obtained 16S rRNA partial genes and those of known bacteria. Khan et al. indicated that the lengths of observed T-RFs were sometimes different from their theoretical lengths by 1 to 8 bp (9). In consideration of Khan et al.'s observation, the 16S rRNA partial genes, whose theoretical lengths were within ±8 bp of the lengths of the marker T-RFs, were selected as candidate 16S rRNA partial genes assigned to the marker T-RFs. We then attempted to assign the marker T-RFs to phylogenetic groups based on the phylogenetic position of the candidate 16S rRNA partial genes.

For marker T-RFs at 83, 88, 93, and 95 bp, we sought obtained 16S rRNA partial genes whose theoretical lengths were between 75 and 103 bp. Figure 1 shows that all 16S rRNA partial genes whose theoretical lengths were between 75 and 103 bp were similar to Bacteroides spp. or to the MIB group. The MIB group was found recently by Salzman et al. as a separate and still unrecognized branch of the Bacteroides group in the murine intestine (18). Therefore, we concluded that these marker T-RFs were derived from Bacteroidetes. For the marker T-RF at 215 bp, we sought the obtained 16S rRNA partial genes whose theoretical lengths were between 207 and 222 bp. Figure 1 shows that such 16S rRNA partial genes were scattered on the phylogenetic tree. For the marker T-RF at 475 bp, we sought the obtained 16S rRNA partial genes whose theoretical lengths were between 487 and 483 bp. Figure 1 shows that such 16S rRNA was only plasmid AB126304, which was also similar to some Clostridium spp.

Increase in T-RFs derived from Bacteroidetes after scFOS administration.
As described above, four of six marker T-RFs were derived from Bacteroidetes. Another two T-RFs (at 84 and 86 bp), also derived from Bacteroidetes, were larger in the scFOS-fed group, although the differences were not significant. The total average relative areas of these six T-RFs derived from Bacteroidetes of the scFOS-fed group and the control group were 25.0% ± 12.5% and 4.1% ± 3.2% (Fig. 3), respectively. The difference between the total relative areas of the scFOS-fed group and the control group was significant (P < 0.01). These results indicated that the T-RFs derived from Bacteroidetes were consistently larger in the scFOS-fed group than in the control group; therefore, these T-RFs were considered good markers for murine intestinal microbiota change after scFOS administration. Figure 3 also shows that the total relative areas from Bacteroidetes of the scFOS-fed group were significantly larger than those at 0 weeks. On the other hand, the difference in total relative areas from Bacteroidetes between the control group and mouse at 0 weeks was not significant. These results suggest that scFOS administration significantly increased the total area of T-RFs from Bacteroidetes.

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FIG. 3. Average total relative areas of T-RFs derived from Bacteroidetes. Average total relative areas of T-RFs derived from 16S rRNA genes of Bacteroidetes are shown. Error bars indicate standard deviations of the mean relative areas. Asterisks indicate significances of difference of relative T-RF areas. *, P < 0.05; **, P < 0.01; ***, P < 0.001. "NS" indicates that the difference was not significant.

The 16S rRNA gene-based methods, including T-RFLP analysis, have inherent problems related to the quantification of bacterial populations (21). The amplification speeds of 16S rRNA genes in different bacteria genomes vary because of different operon number and PCR bias (21). Therefore, the quantity of PCR amplicons does not necessarily reflect the actual numbers of bacteria. Thus, the quantitative differences in the relative areas of T-RFs between the scFOS-fed group and the control group do not necessarily reflect quantitative differences in their actual bacterial populations.

In this context, we preliminarily determined the relative amount of 16S rRNA genes of Bacteroidetes using real-time PCR with Bacteroidetes-specific primers to confirm that fecal samples of the scFOS-fed group had more Bacteroidetes bacteria than those from the control group. To confirm that the primers were specific for Bacteroidetes, we amplified the obtained 16S rRNA partial genes in plasmids with this primer set. All 16S rRNA partial genes which were similar to those of Bacteroidetes, with the exception of AB126311, were amplified. On the other hand, the other plasmids (AB126313, AB126301, AB126299, AB126300, AB126303, AB126302, AB126318, AB126316, and AB126302) were not amplified. Based on these results, the primers were judged to be specific to the Bacteroidetes bacteria found in mouse intestine.

Bacteroidetes bacteria in murine intestine have not been cultured so far (18). Therefore, DNA extracted from a known number of Bacteroidetes was not obtained. Thus, we quantified only a relative number of Bacteroidetes in mouse intestine. Real time PCR of fecal samples showed that the average amount of 16S rRNA genes derived from Bacteroidetes in the scFOS-fed group was 123 times more than that of the control group (P < 0.01). Therefore, as suggested by T-RFLP analysis, the fecal samples from the scFOS-fed group likely possessed more Bacteroidetes bacteria than the samples from the control group.

Effects of kestose and nystose administration on T-RFs derived from Bacteroidetes.
The scFOS used in this study contained two major components, kestose (GF2) and nystose (GF3) (6). To compare the efficiency of the increase in T-RFs derived from Bacteroidetes between mice fed GF2 and GF3, BALB/c mice were fed a GF2 diet, a GF3 diet, or a control diet for 5 weeks, and then we determined the T-RFLP profile of the fecal DNA of each mouse. The total relative areas of the T-RFs derived from the 16S rRNA genes of Bacteroidetes are shown in Fig. 3. The results show that the total relative areas of T-RFs derived from Bacteroidetes were significantly larger in the GF3-fed group than in the GF2-fed group and the control group. These results suggest that GF3 is a more efficient component than GF2 to increase T-RFs derived from Bacteroidetes in fecal microbiota.

We also determined the T-RFLP profiles of cecal microbiota of mice fed a GF2 or a GF3 diet for 5 weeks. The total relative areas of the T-RFs derived from the 16S rRNA genes of Bacteroidetes are shown in Fig. 3. The results show that the total relative areas of T-RFs derived from Bacteroidetes were significantly larger in the GF3-fed group than in the GF2-fed group and the control group. These results suggest that GF3 is a more efficient component than GF2 to increase the T-RFs derived from Bacteroidetes in cecal microbiota as well as in fecal microbiota.

To our knowledge, this is the first report to describe markers of change in murine intestinal microbiota after scFOS administration. As described above, the T-RFLP is less suitable for quantification of an absolute number of intestinal bacteria than other methods, such as culturing, fluorescent in situ hybridization, and real-time PCR with internal standards. Therefore, the use of T-RFs as markers for diet evaluation could be limited. However, T-RF analysis could be a practical approach, as most mouse intestinal bacteria, including Bacteroidetes, are still uncultured. Also, T-RFLP is a high-throughput method. This feature should be preferable for determining changes in intestinal microbiota due to scFOS administration, since approximately 50 fecal samples need to be analyzed in even a small animal study. Therefore, we believe the results obtained here should contribute to our understanding of the mechanisms of physiological functions induced by scFOS administration in a murine model.

 
We thank Roy H. Doi (UC—Davis) for comments on the manuscript.

 
* Corresponding author. Mailing address: Food and Health R&D Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1 Chiyoda, Sakado-shi, Saitama 350-0289, Japan. Phone: 81 492847592. Fax: 81 492847598. E-mail: koichiro_murashima{at}meiji.co.jp.

These authors contributed equally to this work.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, September 2006, p. 6271-6276, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00477-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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