Fecal virome addition promotes biofilm biomass and produces distinct bacterial responses in biofilm and planktonic communities in an in vitro colon model

The gut microbiota is spatially organized, but microorganisms at the intestinal mucus surface remain understudied in virome microbiota research. Here, we investigated how fecal virome addition affects biofilm biomass and bacterial composition in surface-attached and planktonic communities using a biofilm-adapted in vitro colon model, CoMiniGut. Fecal samples were collected from three healthy donors at two time points approximately one year apart. Fecal communities from both collection time points were fermented for 48 h in CoMiniGut reactors containing mucin-coated glass beads. Each community was exposed separately to virome fractions from all three donors, testing autologous and heterologous combinations alongside matched microbiome-only controls. Two parallel CoMiniGut systems provided two reactor replicates for each donor treatment combination at each collection time point. Biofilm biomass was quantified by crystal violet staining, and biofilm-associated and planktonic communities from the same reactors were characterized by 16S rRNA gene sequencing. Across 36 reactor-level observations representing combinations of bacterial donor, virome donor, fecal collection time point, and CoMiniGut system, biomass was classified as increased in 18, neutral in 11, and decreased in 7. Each observation represented the mean of 5 - 6 beads normalized to the matched microbiome-only control. Among non-neutral observations, increases were more frequent than decreases (18 versus 7; two-sided exact binomial test, P = 0.043), whereas the magnitude of the relative change did not differ significantly between Increase and Decrease observations (P = 0.220). Virome addition also explained a small but significant proportion of variation in bacterial community composition (R2 = 0.024, P = 0.029), without substantially altering alpha diversity. No interaction between virome treatment and growth mode was detected for the overall community-level response, while separate differential-abundance analyses identified different sets of virome-associated taxa in biofilm-associated and planktonic samples. Thus, fecal virome addition was associated with a directional bias towards increased biofilm biomass and with distinct taxonomic patterns in biofilm-associated and planktonic communities, without extensive community restructuring.