On- and off-tumour bacterial diversity between right and left colon
A total of 4.4 million reads (109,122 reads/sample) were obtained following quality control, with a sampling depth of 8,000 reads/sample. Comparison of alpha diversity metrics shows that the off-tumour microbiota of patients with right-sided colorectal cancer showed significantly greater bacterial abundance (ACE), species richness (Observed OTUs) and bacterial diversity (Chao1), compared to the off-tumour microbiota of patients with left-sided colorectal cancer (Figure 1; p<0.05). Consistent with this Jaccard similarity assessed beta diversity, showing that the off-tumour microbiota of right-sided and left-sided colorectal cancer patients formed significantly distinct bacterial community clusters (Figure 2; p<0.05).
In contrast the on-tumour microbiota showed no differences in alpha diversity between right- and left-sided colorectal cancer patients (Figure 1; ns). Furthermore, beta diversity analysis showed that right- and left-sided tumours showed no significant differences in Jaccard similarity (Figure 2; ns). Collectively this suggest that bacterial diversity is greater in the right colon compared to the left, however, the presence of a colonic tumour leads to a more consistent bacterial diversity between locations.
Gut phylogenetic profiles between right- and left-sided colorectal cancer patients
Firmicutes, Bacteroides, Proteobacteria and Fusobacteria constitute >95% of bacteria phyla in each group, showing largely consistent phylum relative abundance between locations (Figure 3a). The 3 dominant bacterial families across all locations are Lachnospiraceae, Ruminococcaceae and Bacteroidaceae. However, there is variation in the subsequent most abundant families between locations. Within the off-tumour microbiota the next most abundant family was Fusobacteriaceae, followed by Enterobacteriaceae in the right colon and Prevotellaceae in the left colon. While in the on-tumour microbiota the next most abundant family is Prevotellaceae, followed by Streptococcaceae in the right-sided tumour and Rikenellaceae in the left-sided tumours (Figure 3b).
Comparison of bacterial taxa between right and left colon
Differences in gut bacterial populations between the right- and left-sided colorectal cancer patients were assessed using LEfSe to determine bacterial taxa that are significantly enriched between locations. The off-tumour microbiota of right-sided colorectal cancer patients showed greater abundances of species of the Lachnoclostridium, Selenomonas and Ruminococcus genera. Whereas, the off-tumour microbiota of left-sided colorectal cancer patients is enriched with Epsilonbacteraeota phylum, Campylobacteria class, Pasteurellales and Campylobacterales orders, Campylobacteraceae, Bacillales Family XI, Clostridiales Family XI, Peptostreptococcaceae and Pasteurellaceae families and Campylobacter, Gemella, Granulicatella, Parvimonas, Anaerosporobacter, Lachnospiraceae (UCG010), Peptostreptococcus, Selenomonas and Haemophilus genera (Figure 4a, b).
The on-tumour microbiota of left-sided cancer patients showed greater abundances of Methylophilaceae and Vadin BE97 families and Alloprevotella, Intestinibacter, Romboutsia and Ruminococcus 2 genera, compared to the on-tumour microbiota of right-sided colorectal cancer patients (Figure 4c, d). These results support the diversity data, with the off-tumour microbiota showing large differences in bacterial populations between the right and left colon. In contrast, the on-tumour microbiota seems less affected by location, supporting a cancer defined microbiota that is more constant between the right and left colon.
Difference in paired on- and off-tumour bacterial taxa in right- and left-sided colorectal cancer patients
In order to assess the differences in bacterial taxa between the tumour-associated and tumour adjacent microbiotas in right- and left-sided colorectal cancer patients, we performed an LEfSe comparing paired on- and off-tumour bacterial taxa in each location. In the right-sided colorectal cancer patients there were 24 bacterial taxa that were differentially enriched between the on- and off-tumour microbiota. These include the Lachnoclostridium genus which was enriched in the on-tumour microbiota and the Cyanobacteria phylum, Melainabacteria class, Gastranaerophilales and Corynebacteriales orders, Dietziaceae, Corynebacteriaceae, Eggerthellaceae. Rikenellaceae and Clostridiales vadin BB60 group families, Dietzia, Paraprevotella, Prevotella 9, Alistipes, Lachnospira, Ruminococcus torques group, Paeniclostridium, Eubacterium coprostanoligenes group, Acidaminococcus and Aquabacterium genera which were enriched in the off-tumour microbiota (Figure 5c, d). In the left-sided colorectal cancer patients there were 3 bacterial taxa differentially enriched between the on- and off-tumour microbiota. These include the Porphyromonadaceae family and Lachnospira and Porphyromonas genera, which were more abundant in the on-tumour compared to the off-tumour microbiota (Figure 5a, b). Collectively this suggests that patients with right-sided colorectal cancer have an on- and off-tumour microbiota that is relatively consistent, showing only small differences in bacterial taxa lower taxonomic levels. In contrast, patients with left-sided colorectal cancer have an on- and off-tumour microbiota that show distinct bacterial populations, showing differences at the phylum, class, and order taxonomic levels.