Quantitative Profiling of Colorectal Cancer-Associated Bacteria Reveals Associations between Fusobacterium spp., Enterotoxigenic Bacteroides fragilis (ETBF) and Clinicopathological Features of Colorectal Cancer

Various studies have presented clinical or in vitro evidence linking bacteria to colorectal cancer, but these bacteria have not previously been concurrently quantified by qPCR in a single cohort. We quantify these bacteria (Fusobacterium spp., Streptococcus gallolyticus, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Enteropathogenic Escherichia coli (EPEC), and afaC- or pks-positive E. coli) in paired tumour and normal tissue samples from 55 colorectal cancer patients. We further investigate the relationship between a) the presence and b) the level of colonisation of each bacterial species with site and stage of disease, age, gender, ethnicity and MSI-status. With the exception of S. gallolyticus, we detected all bacteria profiled here in both tumour and normal samples at varying frequencies. ETBF (FDR = 0.001 and 0.002 for normal and tumour samples) and afaC-positive E. coli (FDR = 0.03, normal samples) were significantly enriched in the colon compared to the rectum. ETBF (FDR = 0.04 and 0.002 for normal and tumour samples, respectively) and Fusobacterium spp. (FDR = 0.03 tumour samples) levels were significantly higher in late stage (III/IV) colorectal cancers. Fusobacterium was by far the most common bacteria detected, occurring in 82% and 81% of paired tumour and normal samples. Fusobacterium was also the only bacterium that was significantly higher in tumour compared to normal samples (p = 6e-5). We also identified significant associations between high-level colonisation by Fusobacterium and MSI-H (FDR = 0.05), age (FDR = 0.03) or pks-positive E. coli (FDR = 0.01). Furthermore, we exclusively identified atypical EPEC in our cohort, which has not been previously reported in association with colorectal cancer. By quantifying colorectal cancer-associated bacteria across a single cohort, we uncovered inter- and intra-individual patterns of colonization not previously recognized, as well as important associations with clinicopathological features, especially in the case of Fusobacterium and ETBF.已有多项研究提出细菌与结直肠癌存在临床或体外关联，但此前尚未有研究在同一队列中通过定量聚合酶链反应（qPCR）对这些细菌进行同步定量分析。本研究对55名结直肠癌患者的配对肿瘤组织与正常组织样本中的以下细菌开展定量分析：具核梭杆菌属（Fusobacterium spp.）、解脲链球菌（Streptococcus gallolyticus）、粪肠球菌（Enterococcus faecalis）、产肠毒素脆弱拟杆菌（Enterotoxigenic Bacteroides fragilis, ETBF）、肠致病性大肠杆菌（Enteropathogenic Escherichia coli, EPEC）以及afaC阳性或pks阳性的大肠杆菌（afaC-或pks-positive E. coli）。本研究进一步探究了两类关联：其一为各细菌物种的存在情况与疾病部位、疾病分期、年龄、性别、种族及微卫星不稳定（MSI）状态的关联，其二为各细菌的定植水平与上述临床特征的关联。除解脲链球菌外，本研究在肿瘤与正常样本中均以不同检出频率检测到了本次分析涵盖的全部细菌。与直肠组织相比，结肠组织中产肠毒素脆弱拟杆菌（ETBF，正常样本与肿瘤样本的错误发现率（FDR）分别为0.001与0.002）以及afaC阳性大肠杆菌（正常样本FDR=0.03）的富集程度显著更高。产肠毒素脆弱拟杆菌（正常样本与肿瘤样本的FDR分别为0.04与0.002）与具核梭杆菌属（肿瘤样本FDR=0.03）的定植水平在III/IV期结直肠癌患者中显著升高。具核梭杆菌属是本次检测中最为普遍的细菌，在配对的肿瘤样本与正常样本中的检出率分别达82%与81%。具核梭杆菌属也是唯一一种在肿瘤组织中的定植水平显著高于正常组织的细菌（p=6e-5）。本研究还发现，具核梭杆菌属的高定植水平与微卫星不稳定高表型（MSI-H，FDR=0.05）、年龄（FDR=0.03）以及pks阳性大肠杆菌（FDR=0.01）存在显著关联。此外，本研究在本次队列中仅检出了非典型肠致病性大肠杆菌（atypical EPEC），此前尚无研究将该菌株与结直肠癌相关联。通过在同一队列中定量分析结直肠癌相关细菌，本研究揭示了此前未被认知的个体间与个体内定植模式，以及其与临床病理特征的重要关联，其中尤以具核梭杆菌属与产肠毒素脆弱拟杆菌的关联最为突出。