Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR–Cas region

Abstract

ABSTRACT Fusobacterium nucleatum is associated with colorectal cancer (CRC), and identical F. nucleatum strains seemed to be detected in 75% of patients who exhibited the presence of F. nucleatum in both CRC and saliva samples in our previous study; however, the validation of strain identity and the development of a method for strain-level discrimination are required for etiological studies. We confirmed that F. nucleatum isolates obtained from CRC and saliva samples derived from patients with CRC originated from their identical strains using whole-genome sequencing. We evaluated the hypervariable region of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR–Cas) system in F. nucleatum strains isolated from the CRC and saliva specimens of CRC patients to develop a method for genotyping this bacterium at the strain level. The developed method consisted of two simple PCR steps to amplify the different lengths and sequences of the CRISPR–Cas regions from F. nucleatum strains using specific but common primers. This method could successfully detect identical strains present in both cryopreserved CRC samples and saliva obtained from the same CRC patient. Dynamic monitoring of F. nucleatum strains in saliva obtained from patients with colorectal adenoma before and after oral care showed interindividual variability in F. nucleatum strains during oral care. This study provided a simple and rapid method for comprehensively identifying F. nucleatum at the strain level in clinical samples, leading to a paradigm shift in CRC research, such as the investigation of pathogenic F. nucleatum strains and monitoring of pathogenic strains in clinical trials for preventing CRC recurrence. (This study was registered in the UMIN Clinical Trials Registry under ID UMIN000016229.) IMPORTANCE Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

Keywords

• Fusobacterium nucleatum
• colorectal cancer
• CRISPR–Cas system
• polymerase chain reaction
• strain level
• saliva