Characteristics of the study population

A total of 20 overweight, male Taiwanese adults were recruited. All participants were omnivorous. Their age ranged from 28 to 53 years, with an average of 40.5 years (SD = 7.03); and their body mass index (BMI) ranged from 25.7 to 34.2 (kg/m2), with an average BMI of 28.9 (SD = 2.6). All participants did not have systemic diseases such as anemia, hypertension, diabetes mellitus, chronic kidney disease or abnormal liver function. The clinical data and biochemical data were shown in Table 1. Participants provided fecal samples and blood samples at three time points: prior to bowel prep, 7 days after colonoscopy, and 28 days after colonoscopy. For convenience, the three collection time points were denoted as SB, D7, and D28, respectively.

Table 1 Characteristics of 20 male samples and biochemical data (Mean values with their standard deviation) Full size table

Prevotella-ratio separated into two groups

We analyzed the microbiota composition in fecal samples using 16S rRNA gene amplicon sequencing. The number of genera detected in the three time collections of fecal samples was 131. Of these, 16 genera were detected in every fecal sample collected, regardless of the collection time point (Fig. 2 and Additional file 2: Table S2). According to the results of an average relative abundance in all fecal samples, the most abundant genera were Bacteroides (31.7% of all assigned reads) and Prevotella (23.8%). The next were Phascolarctobacterium (5.5%), Faecalibacterium (4.9%), and Megamonas (4.1%). Figure 3 depicts fecal microbiota composition at the genus level within individuals. It also reveals that Bacteroides and Prevotella abundance in the SB fecal sample is essentially restored 28 days after colonoscopy.

Fig. 2 Venn diagram of the number of genera in the three fecal sample groups. SB, D7, and D28 mean the three sample collection time points: prior to bowel preparation; 7 days after colonoscopy; and 28 days after colonoscopy, respectively. The number of genera detected in all three fecal groups is 131 Full size image

Fig. 3 Microbiota composition of each fecal sample over the three collection times. The fecal microbiota composition profiles at genus level are revealed by 16S rRNA amplicon sequencing. Each color represents one bacterial genus. The two most abundant genera within most individuals were Bacteroides and Prevotella Full size image

By evaluating the ratio of Prevotella to the sum of Prevotella and Bacteroides within individuals, the SB fecal samples was separated into two groups. One group had a Prevotella-ratio lower than 0.11, denoted as Type 1; and the other had a Provetella-ratio higher than 0.45, denoted as Type 2 (Table 2). The participants will be separated into two groups depending on their SB fecal type. Based on the two group, the results of the analysis will be explained from two aspects: first, comparing the differences of fecal microbiome between types 1 and 2, at each of the three collection times; and second, over the three collection times, comparing the differences of fecal microbiome of each type separately.

Table 2 Prevotella ratios of the sum of Bacteroides and Prevotella Full size table

A degree of microbial diversity change in fecal community between the two groups

In this study, the richness of microbial diversity means the number of genera detected within each individual. In the SB fecal samples, the interquartile range of richness was wider in the Type 1 group than in the Type 2 group, but the difference in richness was not statistically significant (Additional file 3: Figure S1A). However, in the D28 fecal samples, the interquartile range of richness was wider in the Type 2 group than the Type 1 group, and the richness was significantly different between the two types (P = 0.025, Additional file 3: Figure S1A). As for the Shannon diversity index, there was no statistical difference between the two types at each of the three collection times, but in the SB fecal samples, the interquartile range of Shannon diversity index was wider in the Type 1 group than in the Type 2 group (Additional file 3: Figure S1B and Additional file 4: Table S3). Furthermore, the correlation coefficient between richness and Shannon diversity index of each type became closer from SB to D28 (Additional File 3: Figure S1C).

Next, in the Type 1 group, both the richness and Shannon diversity index did not show significant differences between any of the collection time points, but the interquartile range of richness decreased over the three collection times (Fig. 4). Unlike in the Type 1 group, the richness of the Type 2 group significantly differed between SB and D28 (P < 0.032, Fig. 4A); and the Shannon diversity index significantly differed between SB and D7 (P = 0.018), but not between SB and D28 (Fig. 4B). In addition, the interquartile range of richness in the Type 2 group increased over the three collection times (Fig. 4B).

Fig. 4 The change of microbial diversity for each type. (a) Boxplot of richness. In the Type 1 group, the richness does not show significant differences between any of the collection time points, but the spread of richness decreases over the three collection times. In the Type 2 group, the richness significantly differed between SB and D28 (P = 0.032). (b) Boxplot of Shannon diversity index. In the Type 1 group, the Shannon diversity index does not show significant differences between any of the collection time points. In the Type 2 group, the Shannon diversity index significantly differs between SB and D7 (P = 0.018) Full size image

According to the results of the weighted UniFrac distance, the 60 fecal samples were separated into two clusters. The Type 1 fecal samples formed one cluster in which the most abundant genus was Bacteroides; and the other group was formed from the Type 2 fecal samples in which the most abundant genus was either Prevotella, Megomonas, or Klebsiella (Fig. 5A and Additional file 5: Fig. S2). Whereas, regardless of the type, the difference of microbial diversity within individuals based on the weighted UniFrac distance did not change remarkably over the three collection times (Fig. 5B).

Fig. 5 Principal coordinate analysis (PCoA) based on weighted UniFrac distance. Each point represents one fecal sample. (a) Red color means the Type 1 group, and blue color means the Type 2 group. The 60 fecal samples were separated into two clusters. (b) The three colors represent the three collection sample times. Gray means prior to bowel preparation, denotes as SB; orange means 7 days after colonoscopy, denoted as D7; and blue means 28 days after colonoscopy, denoted as D28. The difference of microbial diversity within individuals did not change remarkably over the three collection times Full size image

Significant differences in microbiota between the two groups

Among the genera which were detected in at least 50% of the all fecal samples, no genera differed significantly in the Type 1 fecal samples from SB, through D7, to D28. Only four genera: Bacteroides, Prevotella, Oscillospira, Holdemania changed significantly in the Type 2 fecal samples from SB to D7 (P < 0.05). In addition, at any sample collection time, Bacteroides and Prevotella differed markedly between the Type 1 and Type 2 groups (P < 0.05). The following genera also changed significantly between the Type 1 and Type 2 groups at differing sample collection times: two genera, Akkermansia and Paraprevotella, at SB; five genera, Akkermansia, Paraprevotella, Actinomyces, Oscillospira, and Fusobacterium at D7; and 3 genera Paraprevotella, Streptococcus, and Enterococcus at D28.

We also found that at the phylum level, the percentage of three phyla, Bacteroidetes, Firmicutes, and Proteobacteria, of each type changed slightly over the three collection times (Fig. 6A). At SB and D7 collection times, the correlations between Bacteroidetes and Firmicutes were negative (Spearman ρ = − 0.53 and − 0.45, respectively; P < 0.05) (Fig. 6B). At D28, the correlation between Bacteroidetes and Firmicutes was weakly negative without statistical significance (Spearman ρ = − 0.25; P = 0.27) (Fig. 6B). Furthermore, in the Type 1 group, the correlations were 0.00, − 0.40, and 0.28 at SB, D7, and D28, respectively; and in the Type 2 group, the correlations were − 0.35, − 0.58, and − 0.65 at SB, D7, and D28, respectively (Fig. 6C).

Fig. 6 Microbial distribution at phylum level. (a) Phylum-level microbial composition of fecal community. Two phyla, Bacteroidetes and Firmicutes, are dominant within most individuals. (b) Scatter plots between Bacteroidetes and Firmicutes for each type at the three collection time points. The correlations between Bacteroidetes and Firmicutes at SB and D7 were negative (Spearman ρ = − 0.53 and − 0.45, respectively; P < 0.05). (c) The correlation between Bacteroidetes and Firmicutes in the Type 1 samples changed markedly from SB to D28 compared with the Type 2 samples Full size image

A degree of microbial correlation change after bowel preparation

The correlation networks for 61 genera, which were detected in at least 50% of the fecal samples at a particular collection time, are presented in the supplementary figures (Additional file 6: Figure S3-S5). The strength of correlation was evaluated using Spearman’s correlation coefficient. To further observe the changes of the correlation over the three collection times, the 1830 correlation coefficients were separated into three subintervals [− 1, − 0.5], (− 0.5, 0.5), and [0.5,1]. An obvious phenomena was that there were fewer pairs of microbiota which remained weakly correlated in the Type 1 group than in the Type 2 group (Additional file 7: Figure S6).

We also found that the correlation of 25 genera, which were detected in at least 90% of all 60 fecal samples, had different patterns. From six heat maps of the correlation networks, it is obvious that the pattern of these microbial correlations within the Type 2 group was remarkably different at D7 compared with the other two collection times, whereas these microbial correlations within the Type 1 group seemed not to change significantly (Fig. 7 and Additional file 8: Table S4-S9). It would appear that participants whose fecal type was the Type 2 were temporarily susceptible to bowel preparation.

Fig. 7 Six heat maps of correlation networks. Six heat maps of 25 genera which were detected in at least 90% of all 60 fecal samples. The pattern of theses microbial correlations within the Type 2 group is remarkably different at D7 compared with the other two collection time. Whereas these microbial correlations within the Type 1 group seemed not to change significantly Full size image

Variation of predictive functional modules in microbial community between the two types

Functional potential profiling of microbial communities was evaluated using PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) via the KEGG database. At level 3 of the KEGG pathway database, 220 functional modules presented in the SB, D7, and D28 samples. In the SB fecal sample, 26 functional modules were significantly different between the Type 1 group and the Type 2 group (P < 0.05), of which mineral absorption and arachidonic acid metabolism differed remarkably (P = 1.19 × 10− 5 and P = 8.34 × 10− 5, respectively). (Additional file 9: Table S10).

In the Type 1 group, of 220 functional modules, only one module (Bacterial invasion of epithelia cells) changed significantly from SB to D28 (Additional file 10: Table S11). Whereas, in the Type 2 group, 174 functional modules differed significantly between SB and D7 (P < 0.05); and 187 between SB and D28 (P < 0.05), of which lipopolysaccharide biosynthesis changed remarkably (P = 0.00018, Additional file 11: Table S12). Figure 8 depicts 12 functional modules which differed significantly between the Type 1 and Type 2 groups for each of the three collection times. Using principal component analysis, the 60 fecal samples were mostly separated into two clusters corresponding to the type (Fig. 9A). We also found that the SB and D7 fecal samples were roughly separated into two parts: the first part consisted of 80% of the SB fecal samples; the second part consisted of 70% of the D7 fecal samples. As for the D28 fecal samples, 55% of them were closer to the predominantly SB part and 45% were in the predominantly D7 part (Fig. 9B).

Fig. 8 Boxplots of 12 predictive functional modules. The 12 predictive functional modules differed significantly either between the Type 1 and Type 2 groups, or over the three collection times (P < 0.05). The full module names are abbreviated as follows (1) Mineral = Mineral absorption; (2) Arachidonic = Arachidonic acid metabolism; (3) Carbohydrate = Carbohydrate digestion and absorption; (4) LPS = Lipopolysaccharide biosynthesis; (5) Protein = Protein digestion and absorption; (6) Membrane = Membrane and intracellular structural molecules; (7) Phenylalanine = Phenylalanine metabolism; (8) Cellular = Cellular antigens; (9) Citrate = Citrate cycle and TCA cycle; (10) Amino = Amino acid metabolism; (11) Phenylpropanoid = Phenylpropanoid biosynthesis; (12) Aminobensoate = Aminobensoate degradation Full size image

Fig. 9 Principal component analysis of 12 predictive functional modules using PICRUSt in level 3 KEGG database. Each sample is represented by a colored point. (a) Red means type 1 samples, and blue means type 2 samples. The 60 fecal samples are mainly separated into two clusters. (b) Three colors denote the three different sample collection times. Gray means prior to bowel preparation, denoted as SB; orange means 7 days after colonoscopy, denoted as D7; and blue means 28 days after colonoscopy, denoted as D28. The SB and D7 fecal samples are roughly separated into two parts: the first part consists of 80% of the SB samples; the second part consists of 70% of the D7 samples. As for the D28 samples, 55% of them are closer to the predominantly SB part and 45% are in the predominantly D7 part Full size image

Association of each type with inflammation cytokine and blood tests

In our blood samples, the averages of these inflammation cytokine, including C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), were within normal range, but the distribution of the test results differed in the participants of the two types. At all three time points, the participants in the Type 2 group generally had higher median for CRP, IL-6, and TNF-alpha than ones in the Type 1 group (Additional file 12: Figure S7 and Additional file 13: Table S13). In addition, the variance of CRP between the Type 1 and Type 2 groups was statistically significant at each sample collection time (P < 0.05, Additional file 13: Table S13). For each type, by comparing individually CRP, IL-6 and TNF-alpha, there is no statistically significant over the three collection times.

We also found that the variances of liver functions (serum glutamic oxaloacetic transaminase, also known as SGOT; and serum glutamic pyruvic transaminase, also known as SGPT), and the variance of basophils between the two types was significantly different at D7 and at D28 (P < 0.05, Additional file 14: Table S14). When comparing each type individually over the three collection times, only the variance of albumin differed significantly in the Type 2 group. Whereas the variance of the other blood tests between the two types generally did not change remarkably (Additional file 14: Table S15). The descriptive statistics of the blood test results present in Additional file 15: Table S16.

Figure 10 depicts the correlations between Bacteroides abundance and each of TG, HDL, and BMI. There were three noteworthy phenomena. First, at each of the sample collection time, Bacteroides negatively correlated with TG in the Type 2 group, whereas Bacteroides positively correlated with TG in the Type 1 group. Furthermore, at SB, Bacteroides positively correlated with HDL in the Type 2 group (ρ = 0.66, P < 0.1) but very weakly negatively correlated with HDL in the Type 1 group (ρ = − 0.08) without statistical significance. Finally, the correlation between Bacteroides and BMI differed for each type at SB and D7. It was positive in the Type 2 group at SB and D7. In contrast, it was negative in the Type 1 group at SB but positive at D7.