Detection of colorectal-cancer-associated bacterial taxa in fecal samples using next-generation sequencing and 19 newly established qPCR assays.

We have previously identified increased levels of distinct bacterial taxa within mucosal biopsies from colorectal cancer (CRC) patients. Following prior research, the aim of this study was to investigate the detection of the same CRC-associated bacteria in fecal samples and to evaluate the suitability of fecal samples as a non-invasive material for the detection of CRC-associated bacteria. Next-generation sequencing (NGS) of the 16S ribosomal RNA (rRNA) V4 region was performed to evaluate the detection of the CRC-associated bacteria in the fecal microbiota of cancer patients, patients with adenomatous polyp and healthy controls. Furthermore, 19 novel species-specific quantitative PCR (qPCR) assays were established to detect the CRC-associated bacteria. Approximately, 75% of the bacterial taxa identified in biopsies were reflected in fecal samples. NGS failed to detect low-abundance CRC-associated taxa in fecal samples, whereas qPCR exhibited high sensitivity and specificity in identifying all targeted taxa. Comparison of fecal microbial composition between the different patient groups showed enrichment of Fusobacterium nucleatum, Parvimonas micra, and Gemella morbillorum in cancer patients. Our findings suggest that low-abundance mucosa-associated bacteria can be detected in fecal samples using sensitive qPCR assays.

Microbial dynamics with CRC progression: a study of the mucosal microbiota at multiple sites in cancers, adenomatous polyps, and healthy controls.

Accumulating evidence has related the gut microbiota to colorectal cancer (CRC). Fusobacterium nucleatum has repeatedly been linked to colorectal tumorigenesis. The aim of this study was to investigate microbial composition in different sampling sites, in order to profile the microbial dynamics with CRC progression. Further, we characterized the tumor-associated F. nucleatum subspecies. Here, we conducted Illumina Miseq next-generation sequencing of the 16S rRNA V4 region in biopsy samples, to investigate microbiota alterations in cancer patients, patients with adenomatous polyp, and healthy controls in Norway. Further, Fusobacterium positive tumor biopsies were subjected to MinION nanopore sequencing of Fusobacterium-specific amplicons to characterize the Fusobacterium species and subspecies. We found enrichment of oral biofilm-associated bacteria, Fusobacterium, Gemella, Parvimonas, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Selenomonas, Porphyromonas, and Prevotella in cancer patients compared to adenomatous polyp patients and control patients. Higher abundance of amplicon sequence variants (ASVs) classified as Phascolarctobacterium, Bacteroides vulgatus, Bacteroides plebeius, Bacteroides eggerthii, Tyzzerella, Desulfovibrio, Frisingicoccus, Eubacterium coprostanoligenes group, and Lachnospiraceae were identified in cancer and adenomatous polyp patients compared to healthy controls. F. nucleatum ssp. animalis was the dominating subspecies. F. nucleatum ssp. nucleatum, F. nucleatum ssp. vincentii, Fusobacterium pseudoperiodonticum, Fusobacterium necrophorum, and Fusobacterium gonidiaformans were identified in five samples. Several biofilm-associated bacteria were enriched at multiple sites in cancer patients. Another group of bacteria was enriched in both cancer and polyps, suggesting that they may have a role in polyp development and possibly early stages of CRC.