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Microbiologynpj Biofilms and Microbiomes
Peer-reviewed journal article

Fusobacterium nucleatum is enriched in invasive biofilms in colorectal cancer

Jessica QueeniD, Zam Cing, Hana B. MinskyiD, Asmita Nandi, Taylor SouthwardiD, Jacqueline FerriiD, Madison McManniD, Thevambiga IyadoraiiD, Jamuna VadiveluiD, April Camilla RoslaniiD, Mun Fai LokeiD, Jane W. Wanyiri, James Robert WhiteiD, Julia L. DrewesiD, Cynthia L. SearsiD

Abstract

Fusobacterium nucleatum is an oral bacterium known to colonize colorectal tumors, where it is thought to play an important role in cancer progression. Recent advances in sequencing and phenotyping of F. nucleatum have revealed important differences at the subspecies level, but whether these differences impact the overall tumor ecology, and tumorigenesis itself, remain poorly understood. In this study, we sought to characterize Fusobacteria in the tumor microbiome of a cohort of individuals with CRC through a combination of molecular, spatial, and microbiologic analyses. We assessed for relative abundance of F. nucleatum in tumors compared to paired normal tissue, and correlated abundance with clinical and pathological features. We demonstrate striking enrichment of F. nucleatum and the recently discovered subspecies animalis clade 2 (Fna C2) specifically in colon tumors that have biofilms, highlighting the importance of complex community partnerships in the pathogenesis of this important organism.

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✦ Multi-agent verified full-text synthesis

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (1 Jan 2025) · CC BY 4.0

Multi-probe imaging and 16S rRNA profiling of Malaysian colorectal cancers show that Fusobacterium nucleatum, dominated by subspecies animalis clade 2, is enriched specifically within mucus-invasive tumor biofilms.

Although Fusobacterium nucleatum is frequently linked to colorectal cancer, strain heterogeneity and community context have obscured how specific subspecies colonize tumors and promote disease progression. Linking F. nucleatum subspecies animalis clade 2 enrichment specifically to polymicrobial mucus-invasive biofilms and late-stage tumors clarifies the ecological niche and polymicrobial partnerships driving colonization.

Key results

  1. ShownMucus-invasive bacterial biofilms occur on most colorectal tumors, are more frequent on right-sided than left-sided tumors, and associate with advanced cancer stage when Fusobacterium is present.

    FISH detected biofilms on 68.8% (77/111) of tumors and 79.0% (60/76) of paired normal tissues (vs 0/34 normal tissues from biofilm-negative tumors), with higher prevalence on right-sided (91.7%, 33/36) than left-sided (58.7%, 44/75; p=0.0003) tumors; 94.3% (66/70) of multi-probe biofilms were polymicrobial, 68.2% (45/65) had Fusobacterium spp., and Fusobacterium+ biofilms associated with later tumor stage (p=0.0212). · Colon tumors frequently have polymicrobial…; Figure legends

  2. ShownFusobacterium nucleatum and co-occurring oral bacteria are significantly enriched in colorectal tumors compared to paired normal mucosa, driven specifically by biofilm-positive tumors in stages II-IV.

    Across 108 tumor-normal pairs, F. nucleatum was detected in 86.6% (97/112) of tumors (0.01-40% of reads) and enriched vs normal tissue (p<0.0001), but stratification showed enrichment only in biofilm+ tumors (p<0.0001 overall; p=0.0012, p=0.0084, p<0.0001 in stages II-IV) and not biofilm- tumors; F. nucleatum correlated with P. micra (p<0.0001), P. stomatis (p<0.0001), G. morbillorum (p=0.005), & L. orale (p=0.006). · Fusobacteria are enriched in the colon tumor…; Fusobacterium nucleatum and other oral organisms…; Figure legends

  3. ShownFusobacterium nucleatum subspecies animalis clade 2 (Fna C2) is the dominant clade enriched across all stages of biofilm-positive colorectal tumors.

    Both Fna C1 (p=0.0046) and Fna C2 (p<0.0001) were enriched in tumors vs paired normal tissue, but Fna C2 was far more abundant in both tissues (p<0.0001) and enriched across all stages of biofilm-positive tumors (whereas Fna C1 was only enriched in stage III); subspecies animalis also predominated in biofilm-positive tumors by PCR (25/34 F. nucleatum-positive) and selective culture (50% of 6 F. nucleatum isolates). · F. nucleatum subspecies animalis clade 2 is the…; Figure legends

  4. ShownHigh tumor abundance of Fna C2 (>=1%) is associated with 83 predicted microbial metabolic pathway shifts, including elevated L-1,2-propanediol and amino acid degradation.

    PICRUSt2 functional inference across 112 tumors identified 83 differentially abundant pathways between >=1% and <1% Fna C2 tumors, with positive associations for L-1,2-propanediol degradation (PWY-7013), L-glutamate degradation V (P162-PWY), L-lysine fermentation (P163-PWY), and protein N-glycosylation (PWY-7031), and negative associations for fucose/rhamnose (FUC_RHAMCAT-PWY) and sucrose (PWY-5384) degradation. · Fna C2 abundance correlates with predicted…; Figure legends

Methods, data and reproducibility

The authors analyzed paired colorectal tumor/adenoma and distant normal colon tissues from 116 Malaysian surgical patients using multi-probe fluorescence in situ hybridization (FISH) for biofilm spatial profiling, V3-V4 16S rRNA amplicon sequencing with Resphera Insight species/clade assignment and PICRUSt2 metabolic inference, subspecies-specific PCR, and anaerobic selective culture.

  • Malaysian CRC cohort subset MAL3 V3-V4 16S rRNA amplicon sequencing of colon tumors, adenomas, and paired normal tissues (pooled with MAL1 and MAL2 to total 116 patients / 108 unique CRC pairs) · human · n = 79 tumors, 3 adenomas, 71 paired normals in MAL3 (116 patients across MAL1-3) · PRJNA1195962
  • Previously sequenced Malaysian CRC 16S rRNA cohort subset MAL1 · human · n = 22 tumors, 1 adenoma, and 20 paired normal samples · PRJNA325649
  • Previously sequenced Malaysian CRC 16S rRNA cohort subset MAL2 · human · n = 24 tumors and 22 paired normal samples · PRJNA325650
  • Methacarn-fixed colon tumor and paired normal sections analyzed by FISH, punch biopsies screened by subspecies PCR, and flash-frozen tumor biopsies used for anaerobic selective culture · bacteria (other) · n = 111 tumors by FISH (70 multi-probe); 34 by PCR; 13 Fusobacterium isolates
Methods & tools
Trimmomatic, FLASH, QIIME, BLASTN, Bowtie2, RDP classifier, Resphera Insight, PICRUSt2, REDCap
Reproducibility gaps
Open source R code used for meta-analyses and primary sequencing data are available from the authors upon request rather than a public code repository.
Limitations (3)
  • Low anaerobic culture yield from tumor biopsies stored for 5-10 years produced only 13 Fusobacterium isolates (6 F. nucleatum) across 112 individuals, precluding strain-level genomic comparisons. · stated by the authors
  • Only 4 individuals with surgical adenomatous polyps were available in the cohort, which was too small for comparative statistical analysis of adenoma biofilm enrichment. · stated by the authors
  • The Fusobacterium fluorescence in situ hybridization (FISH) probe is genus-specific rather than species- or clade-specific, and bulk 16S rRNA sequencing can underappreciate localized Fusobacterium blooms within specific tumor niches. · stated by the authors

Who should read:Read this if you research the colorectal cancer microbiome, polymicrobial mucosal biofilms, or the role of Fusobacterium nucleatum clades in tumor progression.

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