Sand bedding material is frequently used in dairy operations to reduce the occurrence of mastitis and enhance cow comfort. One objective of this work was to determine if sand-based bedding also supported the microbiologically based suppression of an introduced bacterial pathogen. Bedding samples were collected in summer, fall, and winter from various locations within a dairy operation and tested for their ability to suppress introduced populations of Escherichia coli O157:H7. All sources of bedding displayed a heat-sensitive suppressiveness to the pathogen. Differences in suppressiveness were also noted between different samples at room temperature. At just 1 day postinoculation (dpi), the recycled sand bedding catalyzed up to a 1,000-fold reduction in E. coli counts, typically 10-fold greater than the reduction achieved with other substrates, depending on the sampling date. All bedding substrates were able to reduce E. coli populations by over 10,000-fold within 7 to 15 dpi, regardless of sampling date. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to identify bacterial populations potentially associated with the noted suppression of E. coli O157:H7 in sand bedding. Eleven terminal restriction fragments (TRFs) were overrepresented in paired comparisons of suppressive and nonsuppressive specimens at multiple sampling points, indicating that they may represent environmentally stable populations of pathogen-suppressing bacteria. Cloning and sequencing of these TRFs indicated that they represent a diverse subset of bacteria, belonging to the CytophagaFlexibacter-Bacteroidetes, Gammaproteobacteria, and Firmicutes, only a few of which have previously been identified in livestock manure. Such data indicate that microbial suppression may be harnessed to develop new options for mitigating the risk and dispersal of zoonotic bacterial pathogens on dairy farms.Escherichia coli O157:H7 is a food-borne pathogen of global public health significance (39), so understanding the factors affecting its survival in the environment is critical to minimize its impact on human health. Cattle manure is a major reservoir of Escherichia coli O157:H7. However, the factors contributing to bovine colonization and shedding of E. coli O157:H7 are poorly understood, and there are still very few tools available to control these zoonotic bacteria in cattle populations (23). Early epidemiological studies in livestock populations showed that individual farms often tended to maintain either a high or a low prevalence of E. coli O157:H7 over time (18,45). This relative stability of pathogen prevalence on individual farms has been interpreted to be an indication for a role of stable farm management factors in governing the relative abundance of E. coli O157:H7 in cattle populations. In addition, the prevalence of E. coli O157:H7 is seasonally modulated. Most studies indicate a peak in prevalence in cattle that occurs in summer and is coincident with the seasonal peak in human illnesses (3,5,11,12,17).Interestingly, there...