Pyrosequencing Analysis Reveals Changes in Intestinal Microbiota of Healthy Adults Who Received a Daily Dose of Immunomodulatory Probiotic Strains

The colon microbiota plays a crucial role in human gastrointestinal health. Current attempts to manipulate the colon microbiota composition are aimed at finding remedies for various diseases. We have recently described the immunomodulatory effects of three probiotic strains (Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034, and Bifidobacterium breve CNCM I-4035). The goal of the present study was to analyze the compositions of the fecal microbiota of healthy adults who received one of these strains using high-throughput 16S ribosomal RNA gene sequencing. Bacteroides was the most abundant genus in the groups that received L. rhamnosus CNCM I-4036 or L. paracasei CNCM I-4034. The Shannon indices were significantly increased in these two groups. Our results also revealed a significant increase in the Lactobacillus genus after the intervention with L. rhamnosus CNCM I-4036. The initially different colon microbiota became homogeneous in the subjects who received L. rhamnosus CNCM I-4036. While some orders that were initially present disappeared after the administration of L. rhamnosus CNCM I-4036, other orders, such as Sphingobacteriales, Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes, were detected after the intervention. In summary, our results show that the intake of these three bacterial strains induced changes in the colon microbiota.

of probiotic administration on intestinal colonization, which might contribute to the current understanding of the immunomodulatory effects of these three strains [15].

Ethical Statement
The probiotic strains Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 were obtained from the feces of breast-fed newborns, as previously described (SETOPROB study) [15][16][17]. These strains were assayed for enzymatic activity and carbohydrate utilization, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur [18]. This study followed the guidelines of the Declaration of Helsinki, and all procedures involving human subjects were approved by the ethics committees of the University of Granada, Murcia and Valencia. Written informed consent was obtained from the subjects after a careful explanation of the nature of the study. Additionally, this trial was registered at www.clinicaltrials.gov as NCT01479543.

Subjects and Experimental Design
Twenty-five healthy volunteers underwent a 15-day washout period (t1), after which they were randomly and blindly divided into five groups and received either a placebo, a capsule containing 9 × 10 9 CFUs of one of the three strains, or a capsule containing 9 × 10 9 CFUs of a mixture of Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 each day for 30 days (t2). The intervention period was followed by a second washout of another 15 days (t3) for the five different groups. The patients did not consume any fermented milk products, or products containing either probiotic bacteria or prebiotics for the entire duration of the study [15]. There were no additional dietary restrictions.

Fecal Samples, DNA Extraction and 454 Pyrosequencing
Fecal samples were collected from each volunteer at t1, t2, and t3 under anaerobic conditions by using the Anaerogen Compact System (Oxoid, Basingstoke, UK). The system consists of a plastic pouch and a paper gas generating sachet. The paper sachet contained ascorbic acid and activated carbon, which reacts with air. Oxygen is rapidly absorbed and carbon dioxide is produced. The fecal samples were placed inside the plastic pouch together with the sachet, and the pouch was immediately sealed. Samples were kept under anaerobic conditions for 4 h, and then transferred to −80°C until analysis.
Fecal samples were homogenized in a Stomacher-400 blender. DNA was extracted using a QIAamp DNA Stool Mini Kit (QIAGEN, Barcelona, Spain) as directed by the manufacturer, with the exception that samples were mixed with the lysis buffer and incubated at a temperature of 95°C instead of 70°C to ensure lysis of both Gram-positive and negative bacteria. Quantification was conducted with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, DE, USA). Extracted DNA samples from each participant for each collection period were sent to the Department of Microbiology, University Hospital San Cecilio (Granada, Spain). The amplification of a 600-bp sequence in the variable region V1-V3 of the 16S rRNA gene was performed using barcoded primers. PCR was performed in a total volume of 15 µL for each sample containing the universal 27F and Bif16S-F forward primers (10 μmol/L) at a 9:1 ratio, respectively, and the barcoded universal reverse primer 534R (10 µmol/L) in addition to dNTP mix (10 mmol/L), FastStart 10× buffer with 18 mmol/L of MgCl2, FastStart HiFi polymerase (5 U in 1 mL), and 2 µL of genomic DNA. The dNTP mix, FastStart 10× buffer with MgCl2, and FastStart HiFi polymerase were included in a FastStart High Fidelity PCR System, dNTP Pack (Roche Applied Science). The PCR conditions were as follows: 95 °C for 2 min, 30 cycles of 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 5 min, and final step at 4 °C. After PCR, amplicons were further purified using AMPure XP beads (Beckman-Coulter) to remove smaller fragments. DNA concentration and quality were measured using a Quant-iT™ PicoGreen ® dsDNA Assay Kit. Finally, the PCR amplicons were combined in equimolar ratios to create a DNA pool (10 9 DNA molecules) that was used for clonal amplification (emPCR) and pyrosequencing according to the manufacturer's instructions.
Pyrosequencing of the PCR amplicons was performed using a Roche/454 GS Titanium technology platform (Branford, CT, USA). After the sequencing was completed, all reads were scored for quality, and any poor quality and short reads were removed.

Taxonomic Analysis
Sequences were selected to estimate the total bacterial diversity of the DNA samples in a comparable manner and were trimmed to remove barcodes, primers, chimeras, plasmids, mitochondrial DNA and any non-16S bacterial reads and sequences <150 bp. MG-RAST (metagenomics analysis server) [19] with the Ribosomal Database Project (RDP) were used for analyses of all sequences. The input processing steps in MG-RAST included demultiplexing, quality filtering, length filtering, dereplication, and removal of model organism sequences. The preprocessing options and details of data provided were: FASTQ sequences were filtered using a dynamic trimming. Fifteen was the specific lowest Phred quality score that was counted as a high-quality base and the sequences containing 5 bases below the value score 15 were trimmed.
The raw microbiologic data were reported as relative abundances at the taxonomic levels of phylum, family, and genus. The Shannon index, which is based on species richness (the number of species present) and species abundance (the number of individuals per species), was calculated using all three times t1, t2 and t3. Differences among times were compared by using the Mann-Whitney U-test.

Statistical Analysis
Because the data of relative abundance were not normally distributed, median and ranges were used to express those results. The differences in the relative abundances of the families and genera for each sample were computed using the Mann-Whitney U-test. The clustering of the colon microbiota within the L. rhamnosus CNCM I-4036 group was calculated using a principal component analysis (PCoA). In the PCoA plot the data were analyzed with RDP. The data were normalized to values between 0 and 1 and Euclidean distance was measured. A PERMANOVA analysis was done using the PRIMER 7 software. Finally, a tree diagram was created with the MG-RAST software, which allowed for comparisons of the datasets at the level/rank of order.

Subject Information and Sequencing Coverage
We randomly selected 25 healthy adult volunteers and placed five into each group; however, two individuals were excluded due to insufficient fecal samples. Accordingly, the number of subjects was decreased to twenty-three (14 males and 9 females) after the DNA extractions and pyrosequencing analysis. We evaluated five healthy volunteers in the placebo, Lactobacillus rhamnosus CNCM I-4036, and Lactobacillus paracasei CNCM I-4034 groups and four healthy volunteers in the mixture and Bifidobacterium breve CNCM I-4035 groups. A total of 343,063 sequences were generated using 454 pyrosequencing, and the number of sequences per sample varied from 1725 to 10,569 (median of 4782) ( Table 1). Our analysis of the intestinal microbiota revealed that the volunteers enrolled in this study possessed colon microbiota compositions at t1 which did not associate with any intestinal disorder. At the phylum level (Figure 1), the prevalences of the four major phyla (Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria) varied considerably among the samples collected at different time points. All four phyla were present, varying from less than 2% to more than 50% in abundance.

Figure 1.
Compositions of fecal microbiota in 23 healthy adults at the phylum level. The subjects were divided into five groups and received either a placebo, a capsule containing 9 × 10 9 CFUs of one of the three strains, or a capsule containing 9 × 10 9 CFUs of a mixture of B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 each day for 30 days. Each column represents 1 healthy adult, as described in Table 1.
At the genus level (Table 2), Bacteroides was the most abundant genus in all of the study groups, and the levels of Faecalibacterium, Alistipes, Prevotella, Clostridium, Eubacterium, Ruminococcus, Phascolarctobacterium, Parabacteroides, Veillonella, Butyrivibrio, Akkermansia, Bifidobacterium, Lactobacillus, and Bacillus were found to vary at the different time points of the study, as extensively described elsewhere [20].

Impact of Lactobacillus rhamnosus CNCM I-4036 on the Colon Microbiota Compositions of Healthy Volunteers
We next focused on L. rhamnosus CNCM I-4036 because it was the only probiotic strain that modified the colon microbiota (Table 2). To explore the manner by which the intestinal microbiota responds to L. rhamnosus CNCM I-4036 intake, we performed PCoA (Figure 2) to assess the fecal samples prior to the administration of the strain (first washout), after 30 days of its consumption (intervention) and after another washout (second washout). An apparent clustering pattern was identified for most of the subjects during the different L. rhamnosus CNCM I-4036 phases. A large amount of data points shifted from the external sides of the score plot before the L. rhamnosus CNCM I-4036 intake phase (first washout, black circles) to the center of the graph after the treatment was stopped (intervention, white squares) and at 15 days after the intervention (second washout, grey triangles), as shown in Figure 2. A PERMANOVA analysis showed statistically significant differences in t1 vs. t2, and t1 vs. t3 (P < 0.05). The tree diagram ( Figure 3A-C) revealed differences in the three time points. The first washout ( Figure 3A) presented a characteristic composition at the order level. The orders Gemmatimonadales, Rhodobacterales, and Chroococcales disappeared after the intervention and also after the second washout. The order Nautilales also vanished after the intervention but was detected again after the second washout (t3).  Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes orders were detected in the fecal samples ( Figure 3B). All of these orders remained in the samples after the second washout.

Discussion
In a recent multicenter, randomized, double-blind, placebo-controlled trial (SETOPROB), we have described that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 administration modifies the bacterial populations in fecal samples obtained from volunteers, as evidenced by real-time PCR and fluorescence in situ hybridization (FISH). Some of these changes were transient, whereas others were stable [15]. These two methodological evaluations only measured 10 bacterial groups; thus, their findings were not representative of the true colon microbiota composition following probiotic administration. Advancements in gene-sequencing technologies, as well as the increased availability of powerful bioinformatics tools, have enabled novel insights into the composition of the human colon microbiota and the effects of microbial communities on human physiology and disease. Studies using these technologies have indicated that dysbiosis (i.e., an abnormal microbiota composition) and a decreased complexity of the gut microbial ecosystem are common features in patients with Crohn's disease or ulcerative colitis [21]. The pyrosequencing analysis performed in the present study allowed for the determination of both the entire phylogenetic spectrum and the flexibility to analyze populations at different taxonomic levels.
Although some studies have suggested that bacterial taxa in the gut are continuously distributed, recent studies have identified three robust categories, termed enterotypes, on the basis of the abundances of key bacterial genera in adults [22][23][24]. These are the Bacteroides-, the Prevotella-and the Ruminococcus-dominated enterotypes, profiles which may, in part, be determined by long-term nutritional habits [20,24]. Our results with regard to the four major phyla (Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria) were similar to those obtained by Zhang et al. [25] and Ferrario et al. [8] in adult volunteers who received probiotics and to the findings of Azad et al. [26] in healthy infants.
Bacteroides was the most abundant genus in all of the study groups, but only those individuals who received L. rhamnosus CNCM I-4036 or L. paracasei CNCM I-4034 showed significant changes in their fecal compositions. Additionally, the Shannon indices were significantly increased at the end of the intervention in these two groups.
In the SETOPROB study, we found that healthy adults who received these two Lactobacillus strains showed decreases in pro-inflammatory cytokines together with increases in anti-inflammatory cytokines [15]. In addition, cell culture studies performed by Bermudez-Brito et al. [27,28] have shown that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 inhibit the production of pro-inflammatory cytokines and chemokines by human intestinal dendritic cells challenged with pathogenic bacteria, and that these effects seem to be mediated through the decreased expression of toll-like receptor (TLR)-1, TLR-5 and TLR-9 [27,28].
We considered the impact of L. rhamnosus CNCM I-4036 on the colon microbiota of healthy volunteers because previous data have suggested that the administration of this strain to volunteers for 30 days results in its intestinal colonization [15]. Our results revealed a significant increase in the Lactobacillus genus after the 30-day intervention with this strain, and its level remained elevated after the second washout. These important modifications in the colon microbiota detected with highthroughput 16S ribosomal RNA gene sequencing techniques were not observed in the previous FISH analysis [15].
Additionally, we discovered an apparent clustering pattern after the administration of L. rhamnosus CNCM I-4036. The data points shifted from the external sides of the score plot to the center of the graph, as shown in Figure 2. The initial colon microbiota compositions, represented by black circles, were altered in all of the healthy adults who received L. rhamnosus CNCM I-4036, becoming similar to one another after the intervention period (white squares). These alterations in the colon microbiota remained in the absence of probiotic administration (grey triangles) (Figure 2). We are aware of the limitations of the PERMANOVA analysis that we used to ascertain whether these changes were statistically significant: (1) the number of factors must not be greater than nine; (2) the design must have replication within cells (i.e., n ≥ 2); (3) the sample size must be equal within each cell (i.e., balanced designs only); and (4) all cells in the design must be filled: no cells (combinations of factors given) can be missing.
Furthermore, the tree diagram revealed differences at the three study time points at the order level. Some orders that were initially present in the colon microbiota disappeared after the administration of L. rhamnosus CNCM I-4036, whereas other orders, such as Sphingobacteriales, Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes, were detected. These analyses confirm the impact of L. rhamnosus CNCM I-4036 on the colon microbiota. Similarly, Zhang et al. [25] found that the consumption of Lactobacillus casei significantly alters the composition of intestinal microbiota and colon microbiota diversity, and Ferrario et al. [8] have demonstrated that the intake of L. paracasei DG increases the Blautia/Coprococcus ratio, which can potentially confer a health benefit to the host [29,30].
Gender differences in microbial patterns induced by the intake of the probiotic strains cannot be excluded due to the small number of subjects. We are aware of the limitations of the small sample size of our study. However, although small, this size was similar to that described elsewhere [8,25,26]. Future trials enrolling a greater number of healthy adults would strengthen these preliminary results.

Conclusions
In summary, results obtained demonstrate that the intake of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 induced changes in the colon microbiota. Overall, our results warrant further studies to ascertain whether the modification of the microbiota composition might explain the immunomodulatory effects previously described for these probiotic strains.