Comparative shotgun metagenomic analysis of the feline fecal microbiome in response to fresh, dry, and wet diets in a randomized crossover trial

Fresh (mildly cooked) cat foods are an increasingly popular alternative to conventional extruded dry and retorted wet diets, differing in their processing and formulation. While the gut microbiota play an important role in feline health and physiological functions, characterization of the gut microbiome in cats fed fresh diets and direct comparisons with dry and wet diets have not yet been performed. In this 42-day, 3 × 3 Latin square crossover trial, 15 healthy adult cats received commercial, chicken-based, complete fresh, dry, and wet diets in one of three randomized diet sequences. Each diet was fed for 14 days, with fecal samples collected on days 12–14 for shotgun metagenomic sequencing, followed by taxonomic and functional annotation. Shannon and Simpson indices were lower with the dry diet than with the other two diets ( p < 0.001 for fresh vs. dry, p < 0.01 for wet vs. dry for both indices), while Chao1 was higher for the wet diet than for the dry ( p < 0.05) and fresh diets ( p < 0.001). PERMANOVA using Bray-Curtis distances showed significant differences in species, pathway, and CAZy term compositions among diets (all p < 0.001). The five most abundant phyla with the fresh and wet diets were Bacteroidota, Bacillota_A, Actinomycetota, Pseudomonadota, and Bacillota, whereas Bacillota_C replaced Pseudomonadota when cats were fed the dry diet. Differential abundance analysis of taxonomic and functional data using MaAsLin2 identified diet-associated changes (FDR-adjusted p < 0.10). Collinsella, Bacteroides , and Blautia species were enriched and Prevotella species were depleted with the fresh compared with the dry diet, while Prevotella species were depleted with the fresh compared with the wet diet. The dry diet was associated with amino acid biosynthesis, bacterial cell envelope biosynthesis, and polysaccharide degradation; the fresh diet with amino acid degradation, lipid biosynthesis, and utilization of starch/glycogen, fructans, and host-associated glycans; and the wet diet shared functional similarities with the other two diets. The current study demonstrates differences in the feline fecal microbiome at the species level and in predicted functional profiles among fresh, dry, and wet diets, however health implications remain unclear before they can inform feeding recommendations.