AimsThe objective of this study was to determine if the faecal microbiome has an association with Escherichia coli O157:H7 prevalence and enumeration. Methods and ResultsPyrosequencing analysis of faecal microbiome was performed from feedlot cattle fed one of three diets: (i) 94 heifers fed low concentrate (LC) diet, (ii) 142 steers fed moderate concentrate (MC) diet, and (iii) 132 steers fed high concentrate (HC) diet. A total of 322585 OTUs were calculated from 2,411,122 high-quality sequences obtained from 368 faecal samples. In the LC diet group, OTUs assigned to the orders Clostridiales and RF39 (placed within the class Mollicutes) were positively correlated with both E.coli O157:H7 prevalence and enumeration. In the MC diet group, OTUs assigned to Prevotella copri were positively correlated with both E.coli O157:H7 prevalence and enumeration, whereas OTUs assigned to Prevotella stercorea were negatively correlated with both E.coli O157:H7 prevalence and enumeration. In both the MC diet group and the HC diet group, OTUs assigned to taxa placed within Clostridiales were both positively and negatively correlated with both E.coli O157:H7 prevalence and enumeration. However, all correlations were weak. In both the MC diet group and the HC diet group, stepwise linear regression through backward elimination analyses indicated that these OTUs were significantly correlated (P<0001) with prevalence or enumeration, explaining as much as 50% of variability in E.coli O157:H7 prevalence or enumeration. ConclusionsIndividual colonic bacterial species have little impact on E.coli O157:H7 shedding but collectively groups of bacteria were strongly associated with pathogen shedding. Significance and Impact of the StudyBacterial groups in the bovine colon may impact faecal shedding of the zoonotic pathogen E.coli O157:H7, and manipulation of the intestinal microbiota to alter these bacteria may reduce shedding of this pathogen and foodborne illnesses.