Strain-level detection of
Fusobacterium nucleatum
in colorectal cancer specimens by targeting the CRISPR–Cas region
Authors: Shimomura Yumi , Sugi Yutaka , Kume Aiko , Tanaka Wataru , Yoshihara Tsutomu , Matsuura Tetsuya , Komiya Yasuhiko , Ogata Yusuke , Suda Wataru , Hattori Masahira , Higurashi Takuma , Nakajima Atsushi , Matsumoto Mitsuharu 
Published: October 2023

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.