Longitudinal gut microbiome changes during the chemoradiotherapy component of total neoadjuvant therapy for rectal cancer

Total neoadjuvant therapy (TNT), including fractionated external-beam radiotherapy (EBRT), may alter the gut microbiome, yet treatment-associated temporal patterns remain incompletely characterised in rectal cancer (RC). Thirteen patients with locally advanced RC were prospectively followed during the chemoradiotherapy component of TNT, with faecal samples collected before treatment (Fx0), midway through radiotherapy (Fx14), and immediately after the final fraction (Fx28). Aggregate relative frequency was lower in patients than in controls and declined across treatment, whereas Shannon diversity followed a V-shaped pattern, decreasing at Fx14 and increasing again at Fx28. Low-abundance taxa accounted for most detected species despite contributing only a small proportion of cumulative relative abundance. The relative bacterial signal declined transiently at Fx14, accompanied by an increased proportional representation of eukaryotic reads. The rectal cancer-associated taxon Fusobacterium nucleatum declined by Fx28, while the relative representation of SCFA-associated taxa shifted toward acetate-associated and away from butyrate-associated groups, particularly in samples from patients with diarrhoea. Resistome composition also changed over time, with Bacteroides fragilis identified as a prominent inferred ARG carrier. Among the three sampled time points, Fx14 showed the greatest concurrent deviation across several microbiome features, suggesting that mid-treatment may represent a period of increased ecological instability.