Selection of samples with similar symptoms for sequencing
On the basis of similar symptoms, we selected 26 samples (13 each) out of 1000 screened samples for shotgun metagenomic sequencing of HC and BC fecal microbiota.
Microbial profiling of HS and BC fecal samples was carried out using MEGAN6 software . The microbial profiles of dominated phyla, families, and genera of HS and BC fecal samples are shown in Figures 3 and 4, respectively.
Microbial profiling at the phylum level
In total, 5 phyla were identified in HS fecal samples namely Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Tenericutes. Conversely, 4 phyla were identified in BC fecal samples namely Proteobacteria, Firmicutes, Bacteroidetes, and Tenericutes. The fecal microbial profile of HS samples showed the presence of Actinobacteria whereas this specific phylum was not found in BC fecal samples. Among all these phyla, Proteobacteria was the most dominated phylum in both HS and BC fecal samples accounted for 45.6% and 38.9%, respectively. In HS samples, the second most abundant phylum was Bacteroidetes (36.6%), followed by Firmicutes (12.5%), Actinobacteria (5%), and Tenericutes (0.3%). In BC fecal samples, the second most abundant phylum was Firmicutes (36.4%), followed by Bacteroidetes (15.8%) and Tenericutes (8.9%).
Microbial profiling at the family level
In both HS and BC fecal samples, 10 microbial families were found to be the most abundant. Families dominated in HS fecal microbiota were Enterobacteriaceae, Bacteroidaceae, Ruminococcaceae, Clostridiaceae, Bifidobacteriaceae, Enterococcaceae, Selenomonadaceae, Sphingobacteriaceae, Streptococcaceae and Aeromonadaceae. In contrast to the HS fecal microbiota, families dominated in BC fecal samples were Enterobacteriaceae, Bacteroidaceae, Clostridiaceae, Lactobacillaceae, Pseudomonadaceae, Acholeplasmataceae, Staphylococcaceae, Lachnospiraceae, Ruminococcaceae, and Caulobacteraceae. Families specifically found only in HS fecal microbiota were Bifidobacteriaceae, Enterococcaceae, Selenomonadaceae, Sphingobacteriaceae, Streptococcaceae, and Aeromonadaceae whereas Lactobacillaceae, Pseudomonadaceae, Acholeplasmataceae, Staphylococcaceae, Lachnospiraceae, and Caulobacteraceae dominated only in BC fecal microbiota. Among all these families, Enterobacteriaceae was the most abundant in both HS and BC fecal samples, which accounted for 40% and 24.1% respectively. Similarly, family Bacteroidaceae was the second most abundant family in both HS and BC fecal samples, which accounted for 32% and 16.4% respectively. The third most abundant family in HS fecal microbiota was Ruminococcaceae (8.8%) followed by Clostridiaceae (5.1%), Bifidobacteriaceae (4.4%), Enterococcaceae (3.3%), Selenomonadaceae (1.8%), Sphingobacteriaceae (1.3%), Streptococcaceae (0.9%) and Aeromonadaceae (0.7%). Conversely, the third most abundant family in BC fecal samples was Clostridiaceae (11.7%) followed by Lactobacillaceae (11.4%), Pseudomonadaceae (10.9%), Acholeplasmataceae (8%), Staphylococcaceae(7.3%), Lachnospiraceae (5.2%), Ruminococcaceae (4.7%) and Caulobacteraceae (0.3%). Overall, 6 families were found unique in both HS and BC fecal samples.
Microbial profiling at the genus level
Microbial profiling revealed 10 abundant genera in both HS and BC fecal samples. Those included Bacteroides, Escherichia, Enterobacter, Salmonella, Faecalibacterium, Ruminococcus, Alistipes, Bifidobacterium, Enterococcus, Coprococcus, Pseudomonas, Lactobacillus, Faecalibacterium, Phytoplasma and Staphylococcus. Among these, genus Bifidobacterium, Enterococcus and Coprococcus dominated specifically only in HS samples while Pseudomonas, Lactobacillus, Faecalibacterium, Phytoplasma, and Staphylococcus dominated only in BC fecal samples. The most abundant genera in HS samples were Bacteroides (22.9%) followed by Escherichia (20.1%), Enterobacter (9.7%), Salmonella (9.1%), Faecalibacterium (7.1%), Ruminococcus (6.1%), Alistipes (4.8%), Bifidobacterium (3.1%), Enterococcus (2.9%) and Coprococcus (2.7%). In contrast, the most abundant genera found in BC fecal microbiota were Escherichia (15.9%) followed by Alistipes (13.5%), Bacteroides (10.9%), Pseudomonas (10.9%), Lactobacillus (10%), Faecalibacterium (10.3%), Phytoplasma (8%), Ruminococcus (7.4%), Staphylococcus (6.9%) and Salmonella (6.4%).
Microbial profiling at the species level
The various abundant and common species found in HS and BC fecal samples are shown in Figures 5, 6 and 7 respectively. In total, 37 species were found in HS fecal microbiota while 58 species were found in BC fecal samples. Among the various species, E.coli, Salmonella enterica, Klebsiella oxytoca, Klebsiella aerogenes, Klebsiella pneumoniae, Raoultella ornithinolytica, Bacteroides fragilis, Bacteroides thetaiotaomicron, Butyrate-producing bacterium SS3/4, Ruminococcus bromii, Ruminococcus champanellensis, Ruminococcus torques, Faecalibacterium prausnitzii, Alistipes shahii, Arsenophonus nasoniae, Shigella flexneri, and Enterobacter cloacae were the unique species found HS fecal samples. In contrast, species found only BC fecal samples were Shigella dysentriae, Shigella sp.SF-2015, Yersinia enterocolitica, Escherichia fergusonii, Blautia obeum A2-162, Butyrivibrio fibrisolvens, Ralstonia solanacearum, Marinobacter adhaerens, Achromobacter xylosoxidans, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas aeruginosa, Staphylococcus xylosus, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus pettenkoferi, Solibacillus silvestris, Halobacillu shalophilus, Caulobacter vibrioides, Bacillus subtilis, Lactobacillus salivarius, Lactobacillus johnsonii, Lactobacillus helveticus.
Genetic analysis of E.coli found in both HS and BC fecal samples was carried out against the reference strain E.coli K-12 substr. MG1655 (NC 000913.3) on the basis of sequence similarity. E.coli dominated in BC fecal microbial profile was found to be 99.48% similar to the reference E.coli strain. Conversely, E.coli dominated in HS fecal samples was found to be 98.2% similar to the reference strain. Upon comparing the sequence similarity of E.coli dominated in the HS and BC fecal samples with respect to each other, the similarity was found to be 98% (Figure 8).