BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) remains a significant public health concern in beef production, regardless of feeding systems. While consumer interest in grass-fed beef has increased due to climate change concerns and social media trends, the safety implications of different feeding practices on STEC prevalence and populations are not fully understood. Therefore, the objectives of this study were to evaluate the effects of grain-fed and grass-fed feeding systems on STEC prevalence, population, and its interaction with the fecal microbiota in beef cattle. Post-weaning steers were assigned to four feeding systems: Conventional grain-fed (CON, n = 21), 20-month grass-fed (20GF, n = 18), 25-month grass-fed (25GF, n = 16), and 45-day grain-fed after 20-month grass-fed (GR45, n = 13). Rectal fecal samples were collected at 14 months of age as baseline and pre-harvest for STEC enumeration, prevalence, and microbial analysis. The microbial DNA was extracted and sequenced for 16 S rRNA gene for microbiota analysis. RESULTS: Data demonstrated that cattle in grain-fed feeding system had a higher (P < 0.05) fecal STEC population than the grass-fed feeding system. However, the fecal prevalence of STEC was lower (P < 0.05) only in the GR45 compared to the grass-fed groups, while the CON group did not differ (P > 0.05) in STEC prevalence. In terms of STEC population, GR45 was more similar to the grain-fed group. Alpha diversity was greater (P < 0.05) in CON, followed by 25GF, with GR45 being the only system where alpha diversity decreased (P < 0.05) from baseline to harvest. Beta diversity showed a notable difference (R = 0.913, P = 0.001) in fecal microbial composition between CON and GR45. Firmicutes and Bacteroidetes were the dominant phyla across all feeding systems. At harvest, GR45 had the highest (P < 0.0001) Firmicutes abundance, followed by 20GF, while the lowest levels were observed in 25GF and CON. Among bacterial families, Peptostreptococcacea 44 bmcmicr BMC Microbiology BMC Microbiol BMC PMC12142967 12142967 12142967 40474080 10.1186/s12866-025-04073-6 Effect of conventional grain-fed and grass-fed feeding systems on fecal microbiota and shiga toxin-producing Escherichia coli in beef cattle Talukder Sudipta 1 Yang Frederick 1 Klopatek Sarah 1 Oltjen James 1 Yang Xiang 1 ✉ 1 Department of Animal Science, University of California-Davis, 2237 Meyer Hall, Davis, CA 95616 USA ✉ Corresponding author. Methods Study design The study was performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of California, Davis (UCD; protocol #20560). The steers used in this study were owned by the UCD and managed under IACUC-UCD-approved research and husbandry protocols. Details of the study design were previously described by Klopatek et al. [ 16 ]. 2 Principal Component Analysis of fecal microbiota of cattle raised under different feeding systems: Conventional grain-fed (CON, n = 21), 20-months-grass-fed (20GF, n = 18), 20-months-grass-fed and 45-days-grain-fed (GR45, n = 13), 25-months-grass-fed (25GF, n = 16) At harvest, fecal microbial composition was revealed to be different among cattle raised in different feeding systems ( R = 0.43, P = 0.001) (Fig. 3 ). Fig. 3 Principal Component Analysis of fecal microbiota at harvest of cattle raised under different feeding systems: Conventional grain-fed (CON, n = 21), 20-months-grass-fed (20GF, n = 18), 20-months-grass-fed and 45-days-grain-fed No change was observed in the relative abundances of bacteria at the family level in feces of the steers from 20GF. Fig. 6 Stacked histograms indicating relative abundance of families in fecal microbiome of cattle raised under different feeding systems at baseline and harvest. Feeding system included: Conventional grain-fed (CON, n = 21), 20-months-grass-fed (20GF, n = 18), 20-months-grass-fed and 45-days-grain-fed (GR45, n = 13), 25-months-grass-fed (25GF, n = 16). 14 primary families are shown in the figure. Family with a relative abundance of less than 1% are grouped as ‘Others’ Due to the significant prevalence of Firmicutes and the presence of Proteobacteria, which includes E. coli , a critical indicator bacteria for food safety, further analysis was conducted to better understand the role of these two phyla. The relative abundances of bacterial families of these two phyla are shown in Fig. 7 . The Clostridiaceae abundance was the highest ( P < 0.05) in the GR45 group, followed by 20GF, with both being higher ( P < 0.001) than in the 25GF and CON groups. Steers from GR45 also had a higher ( P < 0.05) abundance of Erysipelotrichaceae compared to the ones in 25GF and CON groups. Peptostreptococcaceae abundance was higher ( P < 0.05) in 20GF compared to 25GF and CON, while Ruminococcaceae was more ( P < 0.05) abundant in CON than in 20GF and GR45. For Proteobacteria, the abundance of Burkholderiaceae and Succinivibrionaceae families was highest ( P < 0.05) in GR45, compared to all other feeding systems whereas Enterobacteriaceae abundance was higher ( P < 0.05) in 25GF than in CON. Fig. 7 Stacked histograms indicating relative abundance of families of phyla (A) Firmicutes and (B) Proteobacteria in fecal microbiome of cattle raised under different feeding systems at harvest. Overall, the distinct bacterial compositions in cattle raised under different grass-fed and grain-fed feeding systems do not necessarily negate the influence of other factors, such as animal management practices, animal age, season, and expected external stresses, which can interfere with the fecal microbial population. Thus, to accurately evaluate the risk of foodborne pathogens transmission through animal fecal materials, it is essential to take into account a comprehensive assessment of all relevant factors that impact the safety of end products. Acknowledgements Not applicable. Author contributions JO, SK, and XY conceptualized and designed the experiments.